CA2029353C - Stabilization of specimens for microbial analysis - Google Patents

Abstract

The improvement of specimen quality for microbial analysis is addressed by the present invention which discloses a chemical composition for use in a method and apparatus for transporting a specimen suspected to contain microorganisms of interest to a laboratory for analysis and improved methods of analysis. A device and method for taking, storing, and preserving fluid samples is disclosed comprising a container and a composition designed to maintain the level of microorganisms present in a specimen during transportation of the specimen to a testing facility. The method and apparatus can be utilized on all types of aqueous specimens and specimens which may be extracted in aqueous solution for analysis of microorganisms therein.

Description

Stabilization of Specimens for Microbial Anal~sis TECHNICAL FIBLD
This invention relates to the field of analysis of microorganisms in a specimen. In particular, this invention relates to maintaining the quality or microbial integrity of a specimen from the time of collection to the time laboratory analysis is initiated.

2 2029~53 BACKGROUND ART
Accurate laboratory analysis of speclmens suspected of containing microorganisms is of utmost importance in the fields of medicine and food technology and safety, among others. While techniques have been developed for lmproving the rapidity and sensitlvity of microbiological identificatlon, drugs have been developed for fighting lnfection in patients, and sanitary condltions for food processing have become mandated by law, it is evident that problems remain.
For example, septlcemla, which is the presence of pathogenic microorganlsms ln the blood, is one of the most serious types of lnfectlons encountered. There ls unanimous agreement ln the medlcal profession that septicemia is second only to menlngitls ln terms of serlous lnfectlons. Even though modern medlclne has provlded an armament of antiblotlcs and antlfungal drugs, the mortallty rate from septlcemla ls approximately twenty-flve percent. Also, when shock accompanies septicemla, the mortallty rate lncreases to over slxty percent. Debllltatlng dlseases, ma~or surgery, a~m~ n ~ stratlon of lmmunosuppresslve drugs or anticancer medications cause patlents to be partlcularly prone to septlcemla. Early dlagnosls of the causatlve 2~ agent ln con~unctlon wlth the use of the approprlate antlblotlc therapy ls essentlal ln flghtlng septlcemia.
Consequently, lt ls lmperatlve that the physlclan know as rapldly as posslble, not only that the patlent has septlcemla, but also the ldentlty and/or antlblotlc susceptlblllty of the mlcroorganlsms lnvolved. Thus, proper and tlmely dlagnosls of septlcemla depends upon very rapld and efflclent analysls of the mlcroorganlsms ln patlent's blood. Further, lt ls necessary durlng the analysls of the mlcroorganlsms ln the patlent's blood that the blood sample not be contaminated with microorganisms from the hospital environment.
Another example of a disorder caused by microorganisms is the presence of pathogenic microorganisms in the urine, which occurs most commonly in infants, pregnant women, patients with obstructlve lesions, following the use of instrumentatlon ln the urlnary tract ~such as catheters), or with urologic diseases affecting micturitlon. Thls dlsorder can result ln a localized infection within the bladder or kidneys. When confined to the bladder, the infection ls usually well controlled by antimicrobial therapy. Once the kidneys are infected, however, lesions may contlnue to progress desplte treatment leading to chronlc 1~ pyelonephrltis and septicemia.
In the field of food technology, contamination occasionally becomes a problem that endan~ers human health. Contamination of milk, for example, has been known to occur even where a processing step to kill harmful microorganisms ls employed because equipment malfunctions, human error, and sometimes mysterious clrcumstances contrlbute to processlng lneffectlveness.
In such cases, rapid and accurate analysis of specimens from the food processlng apparatus and the food itself are important in establishlng the cause of the contamlnation so that the process may be remedied.
Various technlques are utllized for analysis of microorganlsms. Slmple quantitative analysls lnvolves determlning the number of microorganlsms in a given specimen regardless of mlcroorganism identity.
Quantitation may be accomplished by introducing a known volume of specimen (perhaps diluted by a known amount ln a nutrlent broth) onto a nutrlent agar and allowing formation of colonies. It may be deslrable to determlne 4 ~0293~3 the identity and/or antiblotlc susceptlbllity of the mlcroorganlsms found. Analysls to establlsh microorganism identity and/or susceptiblllty ls usually accomplished by sub~ectlng indivldual colonies to dlfferentiatlng medla.
In some instances, accurate quantitatlon as well as ldentlficatlon of particular microorganlsms, rather than mere determlnation whether that particular mlcroorganism is or is not present is highly important. Thus a determlnation that a specimen is ~posltlve" for mlcroorganlsms or ~negative~ for microorganlsms may be insufficient. Rather if the specimen is posltlve, lt may be necessary to know how many microorganisms of a partlcular species are present ln the speclmen. It ls normal for certaln microorganlsms to be present in the human mouth and throat at all tlmes, for example. These normal microorganlsms, referred to as normal flora, do not generally cause dlsease in the numbers normally present. However, lt is possible for an organlsm that may be part of the normal flora to prollferate to such an extent that lt becomes a dlsease-causlng organlsm (pathogen). It can be dlscerned, therefore, that the dlfference between the normal state of a human throat, for example, and a dlseased human throat may be not in the identity of a partlcular organlsm that may survive to the time of analysls, but ln the numbers of that organlsm present ln the patlent's throat. Generally, I the bloodstream ls sterile. However, translent bacteremia may occur where a few organisms enter the bloodstream through a cut or sore, for example, which is not usually a cause for alarm. Quantltation of microbes in a blood specimen is highly important to distinguish translent bacteremia from septlcemla and, perhaps, specimen contamination. While quantitation is of utmost importance ln analyzing blood speclmens, determinlng the ldentity of the microbial pathogen present is also important. Although it may not be necessary to identlfy a microorganism taxonomically to treat a patient, it may be important to determine microorganism susceptibility to antibiotics so that proper drug therapy may be chosen. This may be done by identifying the organism by genus and species since drug manufacturers often have pre-determined the effectiveness of a drug on particular taxomonic groups. Alternately, testing for drug effect (antibiotic susceptibility) may be accomplished.
In some fluids, microorganism concentration may be so low in the specimen that using conventional methods a tested portlon wlll not reveal microbial presence.
Recently, improvements useful for detecting low concentrations of microorganisms have been disclosed whlch have greatly improved detection of septicemia in blood before microorganisms have prollferated to such an extent that the patient is in a severe disease state.
Recently developed method and apparatus for concentratlng and detecting microorganisms from a sample fluid are dlsclosed in United States Patent No.
4,131,S12 entltled ~Method of Detecting Microbial Pathogens Employing a Cushioning Agent~ and its division, U.S. Patent No. 4,212,948 entitled "Apparatus For Detectlng Microblal Pathogens Employing A Cushioning Agent~. The technique disclosed in the above patents involves (when analyzing a blood sample) pre-lysis of corpuscular compounds followed by centrifugation to concentrate the mlcroorganisms away from the other constltuents including antimicroblal factors present in the blood. The concentrated microorganisms are then placed upon a nutrient media such that substances lnhlbitory to mlcroblal growth present in the sample is diluted a minimum of sixty-fold. It has been previously documented that this technlque ylelded more positive cultures than the conventlonal llquld broth culture, the pour plate method, or the flltratlon method uslng the solld matrix fllter. Gordon Dorn, Geoffrey A. Land, and George E. Wllson, ~Improved Blood ~ulture Technlque sased on Centrlfugatlon: Cllnlcal Evaluation,~ 9 J.
Cllnlcal Mlcrobloloq~ 391-396 (1979).
A problem remalns ln the field of mlcroblal analysls desplte the lncreaslng sophlstlcatlon ln technlques for detectlng and determlnlng the identlty of mlcroorganlsms wlthln a speclmen because the accuracy of the technlques ls llmlted by the microblal lntegrlty of the sample analyzed. By ~mlcroblal lntegrity" lt ls meant that a speclmen taken at one polnt ln tlme (to) and analyzed at another polnt ln tlme (tl) wlll provlde an accurate representatlon of the mlcroblal populatlon of lnterest ln the patlent, food supply or other source from whlch the speclmen was taken, when the speclmen ls analyzed.
At least three ma~or factors exlst whlch contrlbute to the lac~ of mlcroblal lntegrity of speclmens at tl.
The flrst ls that speclmens often contaln antlmlcroblal factors whlch may klll mlcroorganlsms of lnterest before tl. A second factor ls mlcroorganlsms of lnterest may not survlve ln the speclmen untll tl even lf no antimlcrobial factors are present. Thlrd, certaln mlcroorganisms may reproduce much more rapldly ln a speclmen than, for example, ln the patlent from whom the speclmen was taken. Fast-growlng but relatlvely harmless or lrrelevant mlcroorganlsms may overwhelm the speclmen so that more harmful species of lnterest are not detected by the analyzing laboratory. Fallure to detect the lmportant organlsm causes mlslnterpretatlon of the contamination problem even though the laboratory may correctly identlfy the organlsms that have prollferated. In each case, the sample analyzed at t will not glve an accurate plcture of the microblal problem in the patlent or other source. Slnce drug therapy prescribed by a physlclan may be dependent on laboratory determlnatlons of type of lnfectlng microorganlsms and degree of lnfectlon, solvlng the problem of mlcrobial lntegrlty may be vltal to the recovery of the patlent. False negatlves wlth respect to food processlng equipment or food ltself may be detrimental to public health. In additlon, misidentification of contaminatlon ln the food-related area may prevent dlscoverlng the source of contamlnatlon or cause the needless dlsposal of products. Dlscoverlng the source ls often necessary to prevent future incidents of contamlnatlon.
Where antlmicroblal factors, such as antlblotlc drugs, are present ln a speclmen several problems arlse.
For example, a patlent glven antiblotlcs by his or her physician may have a level of such drugs in the blood or urine. At to~ when a urine specimen ls taken (for example), the urlne may contain llvlng microorganisms and some antibiotic. The antibiotlc may continue to 2~ work to kill the microorganisms ln the specimen so that at tl, no living microorganisms remain. The laboratQry may test the urine speclmen and conclude that the patient no longer has a microbial problem. However, this may be inaccurate. Unlike the speclmen, the patlent's system may continue to be seeded wlth microorganisms from the source of lnfectlon. Whlle the level of antlbiotics ln the specimen might be sufficient to kill microorganlsms thereln, this does not necessarily reflect the status of the infectlon wlthln 8 202935~
the patlent. Additionally, living organisms are required for ldentification and antibiotic susceptibillty testlng of microorganisms. If the specimen arrlving at the laboratory has no livlng microorganlsms, the laboratory cannot usually accurately identlfy the organlsms nor determlne antibiotic susceptlbillty. Drugs whlch may be more effective in eliminating particular organisms may not be prescribed if a less effective drug is taken by a patient and is effective enough to destroy the microbial lntegrity of the specimen taken from that patient, even though it ls not effective enough in the patient's system to destroy the infecting mlcroorganisms. Natural bacterlocidal substances found in some speclmens, such 1~ as blood, may also change the microblal lntegrlty of the speclmen before lt ls analyzed causlng lnaccurate results.
Even lf no antimlcroblal factors are present in a speclmen, a mlcroblal lntegrity problem remains. If llving microorganisms are contained ln a specimen at to, but fail to survlve to tl, no microorganlsms wlll llkely be detected by the laboratory because detectlon technlques are chlefly based on mlcroorganism reproductlon. Such a sltuatlon will lead to false 2~ negatlve reports and potentlally harmful consequences if mlcroblal lnfectlons or contamlnations go untreated.~-Organlsms may reproduce so well ln a speclmen that laboratory analysls wlll falsely indicate that the patlent, foodstuff, or food processlng equlpment ls highly contamlnated. Incorrect drug therapy may be admlnlstered that is both unnecessary and potentially harmful by itself to some patients. Also, the rapldly-reproducing organlsm may cause other more harmful mlcroorganisms ln the specimen to die ln the specimen, although they may be reproducing rapidly in the patient. Since appropriate drug therapy may dlffer depending on the identity of the problem organlsm, the patient may not be treated properly for eliminatlng the more virulent, undetected mlcroorganism and will thus be harmed. In the case of food analysls, misidentlflcation of the source of contamlnatlon may result and thus the source which lntroduced the vlrulent mlcroorganlsm may not be dlscovered.
The problem of lack of mlcroblal lntegrlty ln specimens may be lncreased because of hospltal inefflclency ln transportlng the speclmen to the laboratory and backlogs occurring in the laboratory of samples to be analyzed. Although most textbooks and handbooks of mlcrobiologlcal technlque mandate a speclmen hold tlme of less than two hours, lt ls often lmpractlcal to comply wlth this standard of efflciency.
The problem may be even worse when the speclmen must be transported from a remote slte such PS a doctor's offlce, a food processlng plant, or a sewage-treatment plant to a central laboratory. The accuracy of analysls decreases the longer it takes to transport the specimen to the laboratory because of the deterloratlon of mlcroblal lntegrlty of the speclmen.
Thus, ldeally, when testlng fluids for dlffering levels of organisms, the level of organlsms present ln the sample should not change between the tlme a sample ls taken, and the tlme the sample is tested. However, the envlronment of a test sample is usually dlfferent from the envlronment of the tested fluid due to varying temperature, llght, avallablllty of nutrlents, etc.
Thls can cause large dlfferences in the number of organlsms present at the time of testing. For example, the number of llve bacteria in a urine sample whlch has 2029~3 been out of the donor's body for a few hours can lncrease due to reproduction, or they can decrease due to the action of antlbiotics which the donor may be taking. Likewise, organlsms present in water can quickly consume all avallable nutrlents ln a sample container and die before a test can be conducted. In any case, the longer the time between taking the sample and testing the sample, the higher probability of an incorrect test result there will be.
While the specimen quallty problem has been addressed by the art, no known approach has been entirely effective and some have introduced further problems.
The simplest approach dlsclosed by the prior art is rapid transfer from the point of specimen collection to the polnt of analysis. For organisms partlcularly sensitive to transport, lmmediate streaklng on nutrlent plates has been suggested literally at the bedslde of the patient. As pointed out, it is often difflcult to make sure that a specimen has been transported wlthin a recommended time frame. Even if it has, if the specimen contains antlbiotlcs, up to 50~ of the microorganisms of lnterest may be killed within 15-20 minutes. Thus, lt can be seen that transport to a lab in two hours or less may be insufficient. Immedlate streaking at bedside may cause loss of aseptic technique and the remalning ~-problem of transport of the plate to the laboratory.
Antibiotic presence may still present a problem.
The transport of specimens in the past has often been undertaken in initially sterile contalners ln an attempt to improve specimen quallty. Even if a specimen is collected ln a sterlle container, however, the mlcrobial lntegrlty of the specimen may deterlorate during transport because lnltlal contalner sterility 2~2~3 neither prevents death nor overgrowth of microbes in the specimen. Additlonally, sterlllty of contalners could be lost where such specimens as urlne, for example, are collected as soon as the closure means is removed for micturitlon.
In U.S. Patent No. 4,145,30~ ('304) and U.S. Patent No. 4,174,277 ('277), a method and structures for the removal of antimlcrobial factors were disclosed. A
mlxed resln bed adsorbs the antlblotics to prevent cidal effects on the microorganlsms of interest. Multiple physlcal entrles lnto the speclmen are required ln the resln bed system ln that the speclmen must be collected from the-patlent, transferred to the resln bed for adsorptlon of antlblotlcs, and removed from the resln bed. The more physical entries a specimen is sub~ected to, the hlgher the rlsk of mlcroblal contamlnatlon from the skln of the operator or the environment. The resin bed ls insoluble and therefore requlres physical manipulatlons before the speclmen may be analyzed. Loss of mlcroorganlsms may result from some non-selectlve adsorptlon. Addltionally, the mlxed resln system fails to address the malntenance of mlcroblal cells ln a viable conditlon without replication.
Certain systems are taught for use ln urlne speclmens whlch address the problem of uncontrolled growth of partlcular species of lnterest which could~
skew analysis. However, most of these systems focus on killlng bacteria that may be present since the speclmen will be assayed for general chemical levels, such as glucose, billrubln etc. In systems taught for preservlng mlcroblal lntegrity, antibiotlc blockage ls generally not addressed. Thus, no means of preservlng the actual count of mlcroorganlsms in the presence or 20293~3 absence of bactericidal agents ls addressed by known urine speclmen-treatlng agents.
Malntalning a speclmen at about 4C from the tlme of collectlon to the tlme of analysls ls another known approach to attemptlng to malntaln speclmen quallty.
Since low temperature may slow microblal growth, antlblotlcs whlch act on only repllcatlng organlsms may lose effectiveness. However, thls approach ls lmpractlcal ln the fleld, and the low temperature may detrlmentally affect the vlability of certain mlcroorganlsms whlle belng an lneffectlve control on the growth of others. Addltlonally, the actlon of antlblotics is not necessarlly controlled by the low-temperature approach. An example of a microorganlsm whlch may be kllled by the cold ls Streptococcus pneumonlae, one that a physlclan would be lnterested ln detectlng as it ls an etlologlcal agent of lobar pneumonla dlsease. Thus, lt is preferable to malntain I the sample at room temperature of about 21-25-C.
Other methods for lmprovlng speclmen quallty include Amles (C. Amles and F. Path, 58 Canadlan J.
Publlc Health 296 (1967)) and Stuarts (R. Stuart et.
al., ~The Problem of Transport of Speclmens For Culture of Gonococcl," 45 Canadlan J. Public Health 73 (1954)).
These methods may provlde some lmprovement of speclmen quallty for some mlcroorganlsms of lnterest, however-these systems fall to address the posslble presence of antiblotlcs ln a speclmen, the dlfferlng nutrltlonal needs of dlfferent mlcroorganisms, and the effect of speclmen hold tlme on accurate mlcroorganlsm quantltatlon.
Another problem left unaddressed by prevlous approaches to mlcroblal detectlon ls the posslblllty that addltlonal mlcroorganlsms wlll be introduced to a 13 202935~
specimen from an external source. Thls "contaminatlon~
of the specimen will cause lnaccurate results slnce, for example, a patlent may be deemed to have a microorganism in the blood that ln fact ls not present. Contamination of speclmens becomes more llkely the more the specimen is transferred from contalner to contalner and the more lt undergoes physlcal manlpulations. For example, a commerclally avallable system for urlne speclmen transport (Becton-Dlckenson) requlres manipulatlon from the urlne collectlon vessel to the contalner wlth the preservatlve therein. It ls therefore desirable to provide collection vessels which reduce the manipulations required, provide a means to instantly preserve the microbial integrlty of a sample, and ln a most preferred embodlment can be utlllzed for other processing steps ln the analysls of mlcroorganlsms of interest.
Therefore, a method and means ls needed for recelvlng a fluld sample suspected of contalnlng mlcrobial pathogens and antlmlcrobial factors which will minim1ze the risk of contamlnatlon, reduce or ellmlnate the requirement of sterllity of the collection vessel for some specimens, provide for deactivation of antlmicrobial factors during the time that the sample is transported so that once the sample is removed from the collection and/or processing vessel and placed on gr~owth media, the microorganisms of interest present in the sample including the fastidious mlcroorganisms of interest wlll proliferate and become ldentifiable, and which will maintain the viablllty of at least some of the microorganisms of interest, preferably so that the microbial integrity of the sample is maintained from time of specimen collection ~to) to the time of specimen analysis (tl ) .

It has now been found that mlcroblal lntegrlty of patient specimens and other speclmens may be preserved so that analysis at a tl up to about 72 hours after to will result ln a much more accurate representatlon of the microbial population in that sample than has prevlously been posslble. Thls has been done by provlding an admlxture of individual chemlcals whlch solublllze in an aqueous speclmen to form a unlque mlxture whlch acts synerglstlcally as a preservatlve of mlcroblal lntegrlty of the specimen. By "preservative"
it ls meant that the unlque mlxture prevents repllcatlon of microorganisms of interest, allows lmproved survival of said mlcroorganisms until the lnception of laboratory analysis, and blocks the action of antimicrobial factors that may be present in the specimen. By ~mlcroorganlsms of lnterest~ it ls meant the microorganisms to be tested for in the laboratory protocol. It may not be necessary or desirable to preserve the vlabllity, for example, of every possible microorganism that may be present in a given speclmen. In the food lndustry, for example, non-harmful or even beneficial microorganisms may be present in food which a laboratory would not be lnterested ln ldentifying. However, the laboratory would be lnterested in testing for microorganisms potentially harmful to human health. Therefore, preservation of the latter ~microorganisms of lnterest~
would be addressed by the present lnventlon. In addition, the growth of the microorganisms which are not of interest must be kept ln check to prevent masking of the harmful mlcroorganlsm ln the analysls procedure, and to prevent the rapidly producing non-harmful organisms from depletlng the nutrlents and causlng death of other microbes. The present inventlon ls effective ln inhibiting repllcation of such potentially lnterferlng 20293~

organisms. The present lnvention thus allows a longer time to elapse between speclmen collection and specimen analysis than has prevlously been posslble wlthout sacrlficing accuracy. It also allows for more accurate analysis even if a sample ls analyzed within a short time period because it blocks the actlon of antlmlcrobial factors whlch may destroy microorganisms of lnterest even wlthln the two hour processlng tlme perlod recommended in the prlor art.
In addltion, no reason ls known why the disclosed specimen transport system would not be advantageous for improving the accuracy of analysis of specimens for perlods exceeding 72 hours. If the vlablllty of even a few mlcroorganisms of interest ls maintalned, the mlcroblal lntegrity of specimen analyzed wlll be lmproved over that posslble accordlng to the prior art, resultlng in improved laboratory analysis.
Disclosed ls a novel method, article and compositlons for detectlng mlcroblal pathogens. In another aspect, thls invention relates to a novel technlque and means for selectlvely separatlng mlcroorganlsms from a sample fluld whlch contaln antlmlcroblal factors. In stlll another aspect, thls inventlon relates to a method and means for use ln the detectlon of mlcroblal pathogens whlch provldes improved recovery of mlcroorganlsms. In yet another aspect, thls inventlon relates to a method and means for accurately quantltatlng the number of mlcroorganlsms present ln a sample fluld st a glven tlme when quantltated at a later tlme.
An artlcle for recelvlng speclmens ls dlsclosed whlch lncludes a means for preservlng the mlcrobial lntegrlty of the specimen.

-16- 20293a3 SUMM~RY OF THE INVENTION
According to the invention, there is provided an apparatus for stabilizing the level of microorganisms in a sample comprising a container and a composition effective for preserving the microbial integrity of the specimen. The composition is located in the container whose structure provides for admixing of the specimen and the composition as the specimen enters the container.
In one embodiment there is provided an apparatus for the collection and treatment of microorganisms in a specimen comprising: (a) a container of generally elongated shape having a first end and a second end; (b) a piston slidably mounted in said container; (c) a shaft attached to said piston and extending through said first end, said shaft includes a narrow portion causing said shaft to break at said narrow portion when said shaft is bent; (d) a detachable cap removably attached to said second end; said cap, said container and said piston forming a sealed chamber, in which the pressure in the chamber may be decreased by moving said piston toward said first end; said cap being puncturable for insertion of said specimen, and; (e) a water-soluble specimen transport system deposited in said container, said specimen transport system comprising a water-soluble additive at a concentration effective to prevent replication of microorganisms present in said specimen, when said specimen is mixed with said water additive therein to form a solution, and reducing the cidal activity toward said microorganisms of antimicrobial factors present in said specimen so that at least some microorganisms will be capable of replication upon dilution of said solution on medium capable of supporting replication of said microorganisms.
In a second embodiment there is provided an apparatus for the collection and treatment of microorganisms in a specimen comprising: (a) a container of generally elongated shape having a first end and a second end; (b) a piston slidably mounted in said container: (c) a shaft attached to said piston and extending through said first end, said shaft being -16a-detachably connected to said piston by complementary thread means; (d) a detachable cap removably attached to said second end; said cap, said container and said piston forming a sealed chamber, in which the pressure in the chamber may be decreased by moving said piston toward said first end; said cap being puncturable for insertion of said specimen, and; (e) a water-soluble specimen transport system deposited in said container, said specimen transport system comprising a water-soluble additive at a concentration effective to prevent replication of microorganisms present in said specimen, when said specimen is mixed with said water additive therein to form a solution, and reducing the cidal activity toward said microorganisms of antimicrobial factors present in said specimen so that at least some microorganisms will be capable of replication upon dilution of said solution on medium capable of supporting replication of said microorganisms.
Also according to the invention, there is provided a method for collection and transportation of fluid specimens comprising the admixing of a composition effective for preserving the microbial integrity of the specimen with the specimen in a container; the container's structure providing for the admixing of the specimen and the composition as the specimen enters the container.
In a third embodiment there is provided a method of collecting and transporting a fluid specimen suspected to contain microorganisms comprising the steps of: depositing the specimen in a container in which there is residing a water-soluble specimen transport system effective for preserving the microbial integrity of the specimen; said container being of generally elongated shape having a first end and a second end, said container further comprising: a piston slidably mounted in said container; a shaft attached to said piston and extending through said first end, said shaft includes a narrow portion causing said shaft to break at said narrow portion when said shaft is bent; a detachable cap removably attached to said second end; said cap, said container and said piston -16b- 2029353 forming a sealed chamber, in which the pressure in the chamber may be decreased by moving said piston toward said first end;
said cap being puncturable for insertion of said specimen, and; wherein said specimen transport system comprises a water-soluble additive at a concentration effective to preventreplication of microorganisms present in said specimen, when said specimen is mixed with said water additive therein to form a solution, and reducing the cidal activity toward said microorganisms of antimicrobial factors present in said specimen so that at least some microorganisms will be capable of replication upon dilution of said solution on medium capable of supporting replication of said microorganisms.
In a fourth embodiment there is provided a method of collecting and transporting a fluid specimen suspected to contain microorganisms comprising the steps of: depositing the specimen in a container in which there is residing a water-soluble specimen transport system effective for preserving the microbial integrity of the specimen; said container being of generally elongated shape having a first end and a second end, said container further comprising: a piston slidably mounted in said container; a shaft attached to said piston and extending through said first end, said shaft being detachably connected to said piston by complementary thread means; a detachable cap removably attached to said second end; said cap, said container and said piston forming a sealed chamber, in which the pressure in the chamber may be decreased by moving said piston toward said first end; said cap being puncturable for insertion of said specimen, and; wherein said specimen transport system comprises a water-soluble additive at a concentration effective to prevent replication of microorganisms present in said specimen, when said specimen is mixed with said water additive therein to form a solution, and reducing the cidal activity toward said microorganisms of antimicrobial factors present in said specimen so that at least some microorganisms will be capable of replication upon dilution of said solution on medium capable of supporting replication of said microorganisms.

-16c-Further, in accordance with the invention, compositions and methods for deactivating antimicrobial factors and maintaining the microbial integrity within a specimen after it has been collected and before the microorganisms of interest are analyzed are also disclosed.
According to a preferred embodiment of the invention, admixing of the composition and specimen takes place in a container comprising a screw on cap (thereby reducing the chances of contamination of the test personnel or the specimen when the cap is removed and replaced as may occur when a rubber stopper is used), a slidable piston within the container, and a detachable piston rod wherein the rod may be removed from the piston (leaving no portion of the rod outside of the container and allowing the container to be placed in a test tube rack, stood on a table, or mounted in a centrifugation device).

Also, according to a preferred embodiment of the sub~ect lnventlon, a partlcular class of composltions, effectlve for preservlng the microbal integrlty of the specimen, (hereafter, the speclmen transport system) soluble in aqueous solution effective for deactivating antimicrobial factors withln a speclmen contalnlng said antimicrobial factors and microorganisms and method of use thereof, is provided which serves the following purposes:
(1) immediate blockage of the cidal action of penicillins, cephalosporlns, and amlnoglycosides, and antibiotics which require mlcroblal growth for effectlveness;
(2) lnltlatlon of anaeroblc condltions to allow maintenance of the llfe of fastldious organisms susceptlble to the lethal actlon of oxygen;
3) complete neutralizatlon of the cldal action of normal human blood and cldal components lnherent ln other specimens;
(4) to hold stable the viable count of microorganisms over a period of time; and (5) provlde for the optimal nutritional needs of the microorganisms of lnterest.
The procedure can be utlllzed on all types of body flulds such as blood, bone marrow, splnal and pleural fluids, body secretlons, urine and the llke as well as non-fluld speclmens from a patlent from whlch mlcroorganlsms may be extracted ln aqueous solutlon.
The mlcroblal lntegrity of water supply specimens, food specimens and samples of surface contamination of food preparatlon or processlng equlpment and other speclmens are also appropriately preserved wlth the present invention. Generally, when employed in connectlon wlth 20293~3 a blood sample, a lysing agent wlll be employed. A
mucolytic agent may be advantageously employed wlth sputum. An example of an effectlve lysing or mucolytic agent is detoxified saponln which ls disclosed in U.S.
Patent 4,053,363 to Dorn, et al. The novel composltlon of the sub~ect lnventlon can be utlllzed ln a sample collectlon or transportlng contalner and allowed to be admixed with the sample after lt has been collected but before microbial pathogens thereln are analyzed by a method such as, for example, deposltlng them upon a growth medla for mlcroblal pathogens. The novel composltion of the sub~ect lnventlon can be ln the form of an aqueous solutlon contalned withln sald sample collectlon and transportlng contalner. However, the novel compositlon for speclmen transport ls preferably posltloned ln said contalner ln the form of solld partlcles whlch are soluble ln the sample fluld or the aqueous extract of the speclmen as the case may be.
It ls envlsloned that the sub~ect lnventlon can be utlllzed wlthln the lysls-centrlfugatlon devlces such as dlsclosed in U.S. Patent 4,212,948 lssued July 15, 1980 ; and entltled ~Apparatus For Detectlng Mlcroblal Pathogens Employlng A Cushlonlng Agent~, whlch employs the baslc method dlsclosed ln U.S. Patent 4,131,512 lssued December 26, 1978 entltled ~Method For Detectlng Microblal Pathogens Employlng A Cushlonlng Agentn.
Also, ln accordance wlth one embodlment of the sub~ect lnventlon, a novel method of assembllng and sterlllzlng a lysls-centrlfugatlon devlce ls provlded whlch includes:
a) deposlting a liquid cushioning agent such as disclosed in said '948 patent, and a specimen transport system ln the form of solid particles withln a lysls-centrlfugatlon tube;

(b) creating a vacuum in said tube and heating said tube to the vaporlzatlon temperature of said liquid cushloning agent, e.g., about 120C for a sufficient time, e.g., about 30 mlnutes to sterllize the lnterlor of sald tube and thereafter coollng sald tube to room temperature.
In additlon, the system of the sub~ect lnventlon can be utlllzed ln practlclng the lysls-centrlfugatlon technique as disclosed ln U.S. Patent 4,164,449 lssued August 14, 1979 and entltled ~Surface Separatlon Technique For The Detectlon Of Mlcroblal Pathogens N .
As an example, a speclmen mlght be held ln a contalner such as the lysls-centrlfugatlon tube descrlbed above whlle the tube ls belng held for processlng.
Surprlslngly the novel system of the sub~ect lnventlon will lnhlblt repllcatlon of mlcroorganlsms which are contalned wlthln the speclmen for a perlod of tlme up to about 72 hours after speclmen collectlon. It ls belleved that repllcatlon may be lnhlblted for even longer perlods when the sub~ect lnventlon ls utlllzed, dependlng on the ldentlty of the mlcroorganisms.
The speclmen transport system of the sub~ect lnventlon contalns extremely hlgh concentratlons of speclflc chemlcal compounds whlch serve to neutrallze antlbiotlcs and/or normal human serum factors. These elevated concentratlons cannot readlly be lncorporated in conventlonal broth systems currently used by many laboratorles to test for mlcroorganlsms because the high concentrations of chemlcals requlred would prove lnhibltory to many potentlally pathogenlc organlsms.
However, where the specimen of lnterest has a high concentration of microorganlsms, such as a urine specimen, the lnventlon may be usable ln con~unctlon 202~3~3 with a conventlonal broth system, wherein the transport vessel contains the speclmen and the composltion of instant invention, this being diluted lnto the broth system when analysis ls lnitiated. The speclmen transport compositlon of the sub~ect lnventlon wlll effectively deactivate most antibiotics and other antimicroblal factors where a sample fluid is mixed therewith and will stabllize the viability of microorganisms of lnterest.
It ls usually necessary that the resultlng admixture of specimen and the disclosed composltion be diluted on growth media at the tlme analysls ls lnltiated ln order that the concentratlons of the deactlvatlng chemlcals be reduced to a concentratlon lS nonlnhibitory to mlcroorganlsms of interest. Thus, the lnventlon is partlcularly useful and advantageously employed in a method ln whlch dilutlon ls necessary prlor to mlcroblal analysls. For example, swabs, sputums, urlnes, blood processed by and the lysls-centrlfugatlon systems dlsclosed ln U.S. Patents 4,164,449; 4,131,512; and 4,221,948 descrlbed above generally requlre a hlgh dilutlon factor and therefore are sultably preserved by the present lnventlon.
As an lllustratlon of the beneflts of the lnstant lnventlon, the lysls-centrlfugatlon system as descrlbed above ls an approprlate example. If the speclmen transport system ls lncluded wlthln the centrlfugatlon tube for treatlng the blood sample prlor to centrifugatlon and deposlt of the concentrated mlcroorganlsms on the medla, the mlcroorganlsms of lnterest wlll be protected from attack by antl-mlcroblal factors whlch are present ln the llquld sample such as antlblotics and serum factors which are cidal ln nature.
In contrast, wlthout the lnstant invention, microblal 20293~3 pathogens may be destroyed within the centrlfugation tube prior to processlng resulting ln undeslrable false negative analysls results of cultures or lnaccurate quantitatlon. The baslc beneflt of use of the sub~ect lnventlon can be more graphlcally lllustrated by the followlng theory. Septlcemla, mlcroorganlsms ln the bloodstream wlth cllnical signs of shock, disseminated lntravascular coagulatlon (clotting) and elevated temperature (fever), hypotension, etc., does not lmply that the blood-stream ltself ls lnfected. In thls theoretical model, there is primary lnfectlon elsewhere such as the kldneys, a lung, or the llke, and the mlcro-organlsms are belng seeded at a given rate into the bloodstream. The immune system and/or antlblotlcs are eliminatlng the mlcroorganisms at a flxed rate. A
patlent survlves a septlc crlsls lf and only lf the seedlng rate ls less than the rate of clearance. Thus, based upon thls theoretlcal model, conventlonal blood culture systems wlll yleld a slgnlflcant number of false negatlve cultures because once the speclmen ls drawn, mlcroblal seedlng from the prlmary source ceases to the speclmen, but the antimlcrobial factors present ln the patlent's blood are stlll actlve. Hence, durlng transport to a laboratory for processlng, these factors may klll the vlable organlsms that were present at the tlme of draw, and therefore, render the test negativé.
Thls concept becomes especlally lmportant for lmmuno-loglcally competent patients and those who are on a broad spectrum of antiblotlcs. Thus, the practlce of the improvement of the sub~ect inventlon ln con~unction wlth the lysis-centrifugation system ls to llterally preserve the microblal status of the blood sample by lnstantly blocking the known deleterlous action of the lmmune system and bacterlcidal antibiotlcs prlor to 202~3.53 dilution of these factors on agar plates which ls an inherent feature in the lysis-centrifugation method.
The employment of the specimen transport system in urine analysis will involve the presence of the speclmen transport system in the micturition receptacle from which a clinically appropriate aliquot of urine may be removed for direct microbial analysis. Thus, the practice of the sub~ect invention ls to literally preserve the mlcroblal status of the urlne sample by lnstantly blocklng the known deleterlous actlon of bacterlcldal antlblotlcs and by actlng as a bacterlostatic agent even ln the absence of antimlcrob$al agents.
The employment of the instant lnvention with throat culture swabs, vaglnal swabs, tissue, bone-marrow and other specimens similarly advantageously preserves the mlcrobial lntegrlty of the speclmen.
i 23 2~29353 BRIEF DESCRIPTION OF DRAWINGS
Thls invention can be more easily understood from the study of the drawlngs in whlch:
FIGURE 1 ls cross-sectional view of a centrifugation artlcle which can be used to practice the sub~ect invention;
FIGURES 2-9 depict steps of a method for detectlng microbial pathogens which can employ the sub~ect invention; and FIGURE 10 depicts another embodiment of the sub~ect invention which comprises a devlce for collecting and transporting body secretion samples.
FIGURE 11 graphlcally depicts the preservatlon of microbial integrity of a specimen in the presence of an antibiotic in the first hour with the sub~ect lnvention as compared to conventlonal systems (detailed in Example V) .
FIGURES 12-16 graphically depicts the preservation of microblal lntegrity of a specimen in the presence of an antibiotic over a four hour time period as compared to conventional systems (detailed in Example V).
FIGURE 17 ls a cross section of one embodiment for collecting and transportlng samples.
FIGURE 18 is a cross section of an embodiment of FIGURE 17 after a sample has been drawn.
FIGURE 19 is a cross section of an embodiment o~
the invention incorporatlng a puncturable closure member, useful with the embodiment of FIGURE 17.
FIGURE 20 is a cross section of an embodiment for collecting and transporting samples showing an alternative piston surface and shaft detachment.
FIGURE 21 ls a cross section of the embodiment of FIGURE 17 as 1t mlght be used ln drawlng a fluid sample from a sample reservoir.

FIGURE 22 iS a cross sectlon of another embodlment for collecting and transporting samples as lt mlght be used as a vacuum draw sampler.
FIGURE 23 lS a cross sectlon of another embodlment for collecting and transportlng samples showlng the specimen transport system ln a powder state.
FIGURE 24 lS a cross sectlon of the embodiment of FIGURE 20 showing an alternatlve locklng method.
FIGURE 25 ls a cross sectlon through llne A-A ln FIGURE 24.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Initially, it should be noted that as used herein, the unit designation "ug~ signifies micrograms.
Although not the only use for the invention, by way of explanation, embodiments of the invention will be described as they would typically be used in collecting, preserving, and transporting urine or blood samples.
Referring to FIGURE 17, an embodiment of the invention may be described as an apparatus simllar in shape to a typical hospital syringe having a container 210, a piston 230, and a shaft 220. Closure member 250 may take the form of a screw on cap (FIGURES 17, 18, 19, and 21), or a puncturable stopper (EIGURE 23) . An advantage of twisting the cap on, rather than using a stopper, is that a twisted or screwed cap does not have to be popped off. This reduces the chance of contamination of the testing personnel or the sample, or I creating aerosols and other potential hazards in the air. When closure member 250 does take the form of a cap, it has a sealable aperture 270 contained therein.
Composition 260 is a composition, effective for preserving the microbial integrity of the specimen, and may be designed to retard the increase or decrease in the number of organisms present in the sampled fluid.
Composition 260 may be in any state such as a tabletc a powder, or a liquid; and a preferred composition, called a specimen transport system herein, is described in more detail below.
Referring to FIGURE 21, when taking a urine sample, a tube 285 is placed over aperture 270 and inserted into specimen cup 282 which contains the urine to be sampled.
Shaft 220 ls drawn back, bringing piston 230 along and pulling urine into the cylinder. Composition 260 dissolves into the urine in the cylinder. Once a , -sufficient sample has been drawn, tube 285 may be removed and dropped into the specimen cup, thereby lessening the chance that urine from tube 285 will contact any people. Next, cap 290 may be placed over aperture 270 to close container 210 (FIGURE 18) .
In FIGURES 17, 18 and 21, a means for removlng shaft 220 is shown in the form of a narrow portlon 240 of shaft 220, and as shown ln FIGURE 18, lt allows the sample taker to break shaft 220 lnslde container 210.
The remover means allows the entlre apparatus to stand on a table or be placed ln a rack. In FIGURE 20 and FIGURE 22 the shaft remover takes the form of a threaded connectlon 241 between plston 230 and shaft 220.
Removal of shaft 220 allows the apparatus to be used ln appllcatlons normally requlrlng test tubes or other contalners whlch may be stood in a rack or used ln a centrifugation devlce.
Now referrlng to FIGURE 19 and FIGURE 22, an embodlment for blood sampllng ls shown. Puncturable membrane 100 may be provided for seallng aperture 270.
Piston 230 is drawn back, thus creatlng a low pressure area in the cylinder. A lock 215 may be provlded which anchors piston 230 and preserve the low pressure area.
Once plston 230 ls ln place, shaft 220 may be removed and dlscarded. Lock 215 may also take the form of snapplng members 252 and 254 as shown ln FIGURES 24 and 25.
Again referrlng to FIGURE 22, needle 277 lS
connected to a fluld source to be sampled. Examples of sources would lnclude an lntravenous connectlon to a patient's vein or artery, a traditional syrlnge whlch has drawn a sample, or a tube extendlng lnto a speclmen cup contalnlng fluid. Needle 277 may puncture membrane 275, and fluid will be drawn into the cyllnder by the 2 0 2 9 3 ~ 3 low pressure. Needle 277 may then be removed, and membrane 275 wlll again seal. Cap 290 may then be placed over aperture 270 for protection of the membrane.
Composition 260 dissolves lnto the fluid, and the specimen may be transported to the lab for tests. Once in the lab, concave surface 280 allows the specimen to be centrifuged ln the apparatus.
Now referring to FIGURE 23, an embodiment for taking samples through a needle 277 ls shown whereln closure member 250 takes the form of a pliable stopper, the composition 260 is in a powder state, and container 210 ls made from glass. The pressure ln contalner 210 ls lower than the outslde pressure. Needle 277 punctures pllable stopper 250, fluid enters container 210, and mixes with composltion 260. Needle 277 is then withdrawn, and pliable stopper 250 seals the puncture.
The device may then be taken to a lab for testing.
Choice of materlal for the container ls not I critlcal; for example, glass or plastic may be used.
Glass holds a better vacuum than plastic, but plastic does not break as easily. Other containers which may be used include: general syrlnges, special purpose syringes such as described in U.S. Patent Number 4,459,997 to Sarstedt, and glass vacuum tubes adapted to be punctured by a needle through a pliable stopper.
In embodiments using ~ powder or li~uid state composition, a one way valve may be used to prevent the composition (or fluid which has come in contact with the compositlon) from flowlng out of the aperture. In the case of a powder, a screen may be used to keep the powder in the cyllnder, whlle still allowing the fluid to pass lnto the contalner.

DETAILED DESCRIPTION OF THE SPECIMEN TRANSPORT SYSTEM
In a preferred embodlment of the lnvention, the composition effective for preserving the mlcrobial integrity of the specimen comprlses the novel specimen transport system of the sub~ect inventlon, lncluding specific chemical agents at relatively high concentrations which will deactivate antimicroblal factors such as antlblotlcs and the cldal agents wlthin a specimen such as normal human blood, among others.
The specimen suspected of contalnlng mlcroorganlsms of interest may be a fluid such as blood or urine or a semi-solid or solid from whlch mlcroorganlsms are ! collected and suspended ln an aqueous solution. Thls may be done, for example, by wiping a sterlle swab 1~ against a solid surface of lnterest, retaining the swab and placing the swab in a suitable solution effective to sustain vlabillty of microorganisms of lnterest. As another example, muscle tissue may be transported to the j laboratory for later analysis for microorganisms of interest by taking a portion of said tissue and placing it in a aqueous receiving solutlon which will allow permeation and diffusion into the tissue to preserve any microorganisms in said tissue. Effective nutrients to sustain viability of microorganisms of interest are to be present in the transporting media. ~Effective nutrients" to be added to a specimen may be anything~
from sterile, distilled, deionlzed water to a complete commercially available broth for microorganism growth dependlng on the nature of the specimen and the ldentlty of the mlcroorganlsm of interest. The criteria for belng ~effectlve~ is the ability to sustaln the viability of the microorganism of interest from the time of speclmen collection (to) to lnltlatlon of speclmen analysis (tl) sufficiently, in the presence of the 20293~;3 bacteriostatic agents added as a part of the specimen transport system of the present invention, so at least some of the microorganisms of interest alive in the specimen at to wlll be able to replicate at tl. In the s ma~ority of instances, the survival of microorganisms from to to tl will be at least so% and often over 80%
wlth the use of the present lnvention. However, advantages are provided by the instant invention over the art even if survival rate is not high since the survival of microbial species to tl is improved by this invention, leading to better identification and antibiotic susceptibillty testing than ever before possible.
In some cases, the effective amount of nutrients will be only pure water, for example where the specimen is not inherently aqueous. What will comprise an effectlve amount of nutrients to be added depends not only on the nature of the specimen but the identity of the microorganism of interest. In addition a proper balance must be achieved between supplying nutrients effective for microbial repllcation and preventing the replication of the mlcrobes during specimen transport wlth bacterio-statlc agents. Different mlcroorganisms have different nutritional needs. The nutrients supplied ln connectlon wlth the lnstant lnventlon should allow the microorganisms of interest to survive untll tl, so that when the specimen is diluted upon growth media (such as an agar plate) so that the factors in the lnstant invention lnhlbltory of repllcatlon of said microorganisms of interest are no longer effective, the survlving microorganisms of interest will be able to repllcate so that testlng and identification may proceed.

202g3~3 For example, neither blood nor urine will generally require addition of nutrients to accomplish the results described above as each lnherently contains sufflclent nutrlents which microorganisms of general interest need over transport time perlods. However, when microorganisms of lnterest have been collected by means of a tool to which microorganisms become attached, such as for example a swab, effective nutrltional components must be supplied in con~unction with the bacteriocidal agents. A swab ls commonly used to collect specimens from patient's throats, for example. In addition, lt may be deslrable for certain microorganlsms of interest to add nutrlents even to specimens such as blood and urlne to prolong vlablllty. Speciflc examples below indlcate the use of effectlve nutrients in the speclmen transport system of the lnstant inventlon.
A growth base effective for supporting general nutrltlonal needs of mlcroorganlsms of lnterest without i inhlblting them ls deslrably added lf the speclmen itself does not lnherently contaln thls effectlve nutrltlon. One effectlve growth base ls Mueller-Hlnton Broth (avallable from BBL Mlcroblology Systems, Cockeysvllle, Md 21030). Thls conslsts of Beef extract ~3 g/l) Acld Hydrolysate of Caseln (7.5 g/l) and starch (1.5 g/l). Another effective growth base ls Tryptlc Soy Broth (avallable from BBL Mlcroblology, Cockeysvllle,~ MD
21030). The compositlon of the growth base chosen should be noted so that lf such growth base contalns a portion of effective nutrients that would otherwise be added separately, the amounts wlll be ad~usted so that the total concentratlon of the particular-nutrlent wlll be known. For example, lt may be desirable to add starch to the nutrlent medlum especially if Haemophilis ls an organlsm of lnterest. Mueller-Hlnton Broth contains starch, so the amount added will take the Mueller-Hinton contribution lnto account.
In the specimen transport system of the lnstant inventlon, a combinatlon of effectlve nutrlents and repllcatlon lnhibitors ls achieved which provides nutrlents to mlcroorganlsms of interest, yet lnhlblts repllcatlon of all mlcroorganlsms ln the speclmen to preserve the mlcrobial lntegrlty of the speclmen. In comblnatlon wlth approprlate repllcatlon lnhlbltors, lt has been found that about 0 to about 10% (w/v of growth base per total volume of speclmen plus transport system is effectlve where lt ls necessary to add nutrlents. A
preferred range ls 0.1% to 5.0%. Even more preferred ls from about 1% to about 3%.
Starch ls preferably employed ln connectlon wlth throat cultures, where Haemophllls ls a mlcroorganlsm of lnterest, slnce starch appears to aid Haemophllls survlval, however starch ls not consldered necessary for all speclmens or mlcroorganlsms of lnterest. When starch ls deslrable, lt has been found effectlve from about 0.005% to about 2.0% (w/v of growth base per total volume of speclmen plus transport system). More preferred ls 0.01% a range from about to about 1.5%.
Most preferred ls a range from about 0.1% to about 1.0%.
Agar ls also a deslrable, but not necessary, nutrient. It provldes a surface for growth and keeps mlcroorganlsms dispersed ln a fluld medlum. The range of agar employable ls from about 0 to about 5% (welght per volume of speclmen and speclmen transport system total), preferably 0.5% to about 2% and most preferably 0.1% to 1.0%.
The effective nutrlents for a speclmen suspected to contaln Haemophllls lncludes hemoglobln. Hemoglobln also lmproves Streptococcus pneumonlae and so is desirable when this is the organlsm is of interest.
Surprislngly, when hemoglobln ls utllized for the transport system of the instant lnvention, no source of NADP (nicotinamide adenlne dlnucleotlde phosphate) need S be added to support Haemophllls. It ls known that some Haemophllls strains requlre a so-called ~x~ factor and a so-called "v" factor (NADP). Hemoglobln supplles the ~x" factor, but the need for addlng an exogenous source of NADP ls not evident when the instant invention admixture is employed.
Deactivatlon of antlmlcroblal factors is also part of the functlon of the instant lnvention. For example, in accordance with one embodlment of the invention, blocking agents for amlnoglycoslde antibiotics and polymyxln B are included within the specimen transport system. Typical amlnoglycoside antibiotics include gentamicin, tobramycin and amlkacin. The amlnoglycosides and polymyxin B all have net posltlve charges. when this charge is blocked, these compounds lose thelr potency. Therefore, in accordance with one embodiment of this invention, a blocker for this posltive charge is included withln the specimen transport system. A preferred compound is sodlum polyanetholsulfonate. The sodium polyanetholsulfonate 2S will inhibit the action of amlnoglycosldes and polymyxln B in direct proportion to its concentration.
Surprisingly, it has been found that the concentration I needed to completely inhlbit these antlbiotics ls a concentration of at least approxlmately 0.06~
weight/volume of specimen of sodlum polyanethol-sulfonate, a concentratlon taught to be toxlc by the prlor art. Another such blocker compound is sodlum amylosulfate. The speclmen transport system of the sub~ect lnventlon contalns sufflcient sodlum 33 20293~
-polyanetholsulfonate to result in between about 0.06% to about 6.0~ and preferably from about 0.1% to about 2.0%
(by weight of the SPS based upon the total welght of sample fluld and specimen transport system compositlon).
- Most preferably, SPS is added ln the range of from about 0.3% to about 1.0~ (by weight of SPS based upon the total welght of sample fluld and speclmen transport system compositlon). The ~toxlc~ effect of sodium polyanetholsulfonate to certain mlcroorganisms has been eliminated ln the lnstant lnventlon by employlng lt ln a method where subsequent dllutlon on growth media to an approxlmate flnal concentratlon of 0.03% or less (by weight sodium polyanetholsulfonate on the medlum).
The concentratlon of SPS employed ln the instant invention ls one sufficient to block the actlon of aminoglycosides, streptomycln and polymyxln B, as prevlously discussed. SPS at high concentration ls also effectlve ln controlling the repllcation of some microorganisms from to to tl and as a result, lowerlng the effectiveness of antlblotics which require microbial replication for activity. It is surprislng that SPS
can be used in a system involving the detectlon and ldentification of microorganisms slnce the prlor art teaches that SPS is toxlc to mlcroorganlsms at concentratlons exceedlng 0.03~. Such low concentratlons of SPS as are taught to be nontoxlc ln the prlor art~
would be lneffectlve ln the lnstant system to accompllsh the desired results.
The speclmen transport system of the sub~ect inventlon preferably contains a water-soluble component effective for blocking the action of peniclllln and cephalosporins, and whlch ln comblnation wlth other components of the speclmen transport system will exert a bacteriostatic effect on the replicatlon of 20293~3 microorganisms in the speclmen without exerting a cldal effect on the microorganlsms of interest. Sulfhydryl-containing compounds such as L-cystelne, N-acetyl-cysteine, thioglycolate, glutathione and mercaptoethanol are suitable antibiotlc lnhibitors for the penicillin and cephalosporin classes. However, it has now surprisingly been found that the concentrations used in the past are suboptimal to achieve the desired goal of antibiotic blockage, and that higher concentrations, taught to be toxic to microorganisms in the prior art, may be used in a method for preservlng the microbial integrity of a specimen wlth the advantage of both ! blocking antiblotic action and acting as a bacteriostatic agent in combination with other specimen transport system components. Another effectlve antiblotlc blocker that may be employed ln the specimen transport system of the sub~ect lnvention ls an enzyme speciflc for the antibiotic. If utilized, enzyme ls ! employed ln con~unction wlth a sulfhydryl-contalnlng compounds in the present lnventlon as lt has been found that the combinatlon of enzyme with the other specimen transport system components exerts an effect not possible with enzyme alone.
It ls preferred that the component effectlve for blocking the actlon of penlcllllns and cephalosporins be available ln a dry form, such as a salt or a freeze-drled form so that lt may be used ln a dry admlxture.
However, llquid blocklng components such as mercapto-ethanol may be utillzed lf desired in a llquld version of a specimen transport system, or as part of liquid specimen diluent supplled in con~unction wlth a dry admlxture.

One or more sulfhydryl-contalning compounds may be used ln comblnatlon, particular comblnatlons belng preferred.
L-cystelne ls the preferred lnhlbltor of penicillins and cephalosporlns present ln the specimen transport system in an amount to result ln from about 8.2 uM to about 8.25 mM ln the combined sample fluld and specimen transport system. The most preferred amount differs accordlng to the speclmen. Wlth urlne speclmens, lt ls preferred to employ a range from about .82 mM to about 41.3 mM, most preferably 4.1 mM to 24.8 mM. with throat cultures and other specimens, the preferred range ls from about .82 mM to about 24.8 mM
and most preferably from about .82 mM to about 8.3 mM.
In a partlcularly preferred embodlment, the speclmen transport system of the sub~ect lnventlon contalns a synerglstlc mlxture of thloglycolate and cystelne wlth cystelne contalned thereln ln an amount from about 8.2 uM to about 82.5 mM and thioglycolate contalned thereln ln an amount from about 0 to about 42.5 mM (molar equlvalents based on the molecular welght of thloglycollc acld as the actlve ingredient). Thls comblnatlon wlll deactlvate the penlcilllns, cephalosporins and some amlnoglycosldes very effectlvely and also reduce the vlscoslty of the thus formed system and increase shelf life of the dry admlxture.
Thioglycolate and simllar compounds by themselves cause an undeslrable lncrease ln vlscoslty of the transported speclmen. It has been found, however, that the above-descrlbed comblnation of cystelne and thloglycolate results in much lower viscoslty after lyslng of blood, for example. In addltlon, the combination allows proportlons of thloglycolate that are less toxlc to the fastldlous mlcroorganlsms. Another advantage ls that cystelne ls easlly oxldizable and the presence of thioglycolate helps malntaln the cystelne ln a reduced state ln the course of preservlng the mlcroblal lntegrlty of the speclmen, for example, durlng the preparation of the lysls centrlfugatlon tube and for shelf life stabillty of the speclmen transport system admixture. An example of the combination of the cystelne and thloglycolate that can be used in a centrifugation tube as set forth ln U.S. Patent No.
4,212,948 lncludes an lnitlal concentratlon of cystelne of 1.2~ and thloglycolate of 0.1% by welght ln the sample fluid and specimen transport system and once finally diluted on growth media as disclosed in said patent a final concentration of cysteine of about 0.018 and thioglycolate of about 0.002% by welght. It ls noted that because of the propensity of the cystelne to oxldize, it is desirable to add the cysteine during the manufacture of a centrifugation tube durlng the last step prior to tube evacuation and autoclaving. The purity of the cysteine ls lmportant. Because of the hlgh concentration of cysteine requlred ln the speclmen transport system, this compound should have a purlty of greater than 95~. If one uses cystelne whlch ls contamlnated wlth cystlne, the cystlne wlll preclpltate out durlng the processlng of blood. Slnce, the cystine preclpltate resembles small colonles of mlcroorganlsms on the agar plate, thls ls an undeslrable property. The lncluslon of the thloglycolate and cystelne comblnatlon has a secondary effect ln that lt will protect anaeroblc mlcroorganlsms, e.g., clostrldlal specles, from belng polsoned by the oxygen present ln the blood speclmen durlng transport of the speclmen to the laboratory.
Thls ls due to the fact that the thloglycolate and other sulfhydryl compounds are excellent oxygen scavengers.

20293~3 Cystelne ls much more effectlve than other sulfhydryl compounds in blocking the cldal action of penlcllllns, cephalosporlns and some amlnoglycosides on a gram or molar basis, and as mentloned, an additlonal benefit of the presence of the cystelne is that it will reduce the viscoslty of lysed blood whlch improves the sedimentatlon of the mlcroorganlsms in a centrifugation tube. Preferably, the free base form of cysteine is utilized to prevent the necesslty for addltlon of high concentrations of pH ad~uster such as would be required wlth cysteine-HCl. However, the latter may be used.
If lt is desired to utlllze another sulfhydryl compound rather than cystelne, and not in con~unctlon wlth cysteine, appropriate concentrations to achleve an effect to simulate cysteine's effect as closely as posslble may be utllized.
Thioglycolate may be used ln a blood specimen in the range of from about 4.4 mM to about 43.8 mM, preferably from about 8.8 mM to about 35.1 mM and most preferably from about 17.5 mM to about 30.7 mM.
Glutathione ls effectlvely used ln blood from about 1.63 mM to about 16.3 mM, preferably from about 3.25 mM
to about 13.0 mM and most preferably from about 6.5 mM
to about 11. 4 mM.
For speclmens other than blood, lt ls preferred to use hlgher amounts of thloglycolate or glutathlone.
Thloglycolate ls effectively employed from about 4.4 mM
to about 52.6 mM, preferably 8.76 to 43.8, and most preferably from about 17.5 mM to about 35Ø
Glutathlone is preferably employed from about 1. 6 mM to about 19.5 mM, more preferably from about 3.25 mM to about 16.3 mM and most preferably 6.51 mM to about 13.0 mM.

202935~

In accordance wlth another embodiment of the sub~ect lnvention, deactlvators for sulfa compounds are present in the speclmen transport system. It ls belleved that the sulfa compounds exert thelr antlmicrobial actlon by lnterferlng wlth the follc acld pathway of bacteria. This pathway ls essentlal for the synthesis of the nucleic aclds whlch are the prlmary compounds of mlcroblal DNA. Accordlngly, preferably para-amlnobenzolc acld (pAsA) may be added to the specimen transport system as a competltlve lnhibitor of sulfa compounds. The preferred concentratlon of PABA
ls ln the range of from 5 micrograms per milllllter to about 500 mlcrograms per millillter and the most preferred range is ln the range of from about 10 micrograms per mllllllter to about 100 mlcrograms per mllllllter of the comblned sample and speclmen transport system. However, the lnhibltion of repllcatlon provlded by the comblnatlon of the other speclmen transport system components may make the addltlon of PAsA
necessary only ln clrcumstances where very hlgh sulfa compound concentratlons are present or where it ls deslred to extend the hold tlme of speclmens to the extent that the sulfa drugs begln to exert a cldal effect on the mlcroorganlsms of lnterest.
The speclmen transport system of the sub~ect lnventlon can also contaln enzymes whlch react wlth and deactlvate certaln antlblotlcs, for example, beta-lactamase, and penlcllllnase. Usually from about 1 to about 20 unlts of actlvlty of such enzymes wlll be effectlve ln the system to provlde the blocking effect ln comblnation wlth the other components of the speclmen transport system. Example XV shows the synerglsm achieved wlth employment of enzyme with other specimen transport system components.

39 202935~
The specimen transport system of the sub~ect invention can include other compounds, depending upon the usage of the system, for example, the system can contain lysing agents such as purlfied saponin dlsclosed in U.S. Patent 3,883,425 lssued May 13, 1975 and entitled "Detoxiflcatlon Of Saponlns~ when lt ls deslred to process blood. The composltion can also contain anticoagulant such as cltrate or ethylenedlamlne-tetraacetlcacld (EDTA).
The antlblotlc blockers of the lnstant lnventlon, ln comblnatlon, serve as bacterlostatlc agents. In addltion, lt may be deslrable to add addltlonal bacterlostatlc agents to prevent the repllcatlon of all mlcroorganlsms ln the speclmen. The bacterlostatlc agent chosen should be noncldal to mlcroorganlsms of lnterest, as prevlously defined. The choice of bacterlostatlc agents wlll be dependent on the type of speclmen and the ldentlty of the mlcroorganlsm of interest. Also, lt may be lmposslble or hlghly unllkely that certaln mlcroorganlsms could exlst ln partlcular speclmens so that there would be no need to employ a particular bacterlostatlc agent dlrected toward controlllng the growth of that certaln mlcroorganlsm ln the partlcular system. Thus a carbohydrate, a sugar or salt such as sodlum chlorlde or lts equlvalent ls deslrably employed to lncrease the hypertonlclty of the aqueous speclmen or speclmen recelvlng fluld wlth respect to urlne speclmens, swab collected speclmens, and other speclmens ln order to control the replicatlon of the more rapidly growlng organisms, for example Enterobacteraclae and Proteus. Suitable salts lnclude sodlum or potasslum chlorlde, ammonlum salts such as (NH4)2S04 and NH4NO3 and other salts of nltrates, sulfates, acetates and admixtures thereof. Sultable 20293~3 sugars include sorbltols, mannitols, glucose and the like. Preferably, a sodium or potassium chloride ls utilized in the range of 0-171.1 mM, preferably from about 8.5 mM to about 136.9 mM, and most preferably from about 17.1 mM to about 85.5 mM ln the speclmen and specimen transport system admixture combined.
It may be desirable to add a substance effective for inhlbiting the replication of gram posltive mlcroorganisms without being cidal to microorganisms of lnterest. For example, Streptococcus faecalis and Streptococcus aqalactiae may mask the presence of Streptococcus pyoqenes because the two former organisms are fast-growers. Slnce S. faecalis, S. aqalactae and S. pyoqenes are all gram positive, it ls not desirable to employ a substance cidal toward the gram positive class in the specimen transport system as S pyoqenes would be killed along with the other gram posltive organisms and thus could not be isolated. It has now been found that effective growth inhibition of microorganlsms without death can be achieved by the combination of the speclmen transport system components plus a dye such as brllllant green or malachlte green.
Also effective in combination with the other system components is oxgall (dehydrated fresh blle). Brllllant Green is utlllzed ln the range of 0 to 4.1 uM. It ls preferred that lt be added from about 100 nM to about 3.3 uM. Most preferred is a range of about 200 nM to about 2.1 uM. If Malachlte Green ls employed, the concentratlon ln the final specimen solutlon should be from about 0 to 5.5 uM, preferably 100 nM - 4.4 uM and most preferred 2.7 uM - 27.4 uM. Other dyes may be employable at concentratlons lnhlbltory to gram posltlves without belng cidal, the lnhlbltlon reverslble upon ade~uate dllutlons.

202~3~3 Oxgall ls utilized, in an amount from about o w/v to about .002~ w/v, preferably .00005~ w/v to about .0016% w/v, and most preferably from about .0001% w/v to about ~001% w/v. Slnce oxgall ls llterally dehydrated fresh bile from oxen gall bladders, no certain molecular weight or consistency between preparations ls posslble.
Therefore, the amounts given are estlmated based on preparations purchased from Difco, catalogue # 0128-02.
In some speclmens, lt may be deslrable to add addltlonal bacterlostatlc agents. It has been found that calclum proplonate, methyl paraben, potasslum sorbate, sodlum nltrate, and sodlum benzoate appropriately work ln the transport system as a bacterlostat prlmarlly for E. coll, Klebslella, and Enterobacterlaceae. These agents are generally effectlve from about 0.1~ to about 10% w/v preferably 0.01~ to about 8.0~ w/v, and most preferred 0.1~ to about 5~ w/v. Calclum proplonate ls most preferred.
Based on the molar equlvalents of proplonlc acld as the actlve ingredlent, lt is utllized from about 0 to about 42.1 mM, preferably from about 42 uM to about 33.7 mM, and most preferably 421.4 uM to about 21.2 mM.
It is desirable to keep the pH of the system at about 6.5-7.5. Therefore, it may be appropriate to buffer the speclmen with an effectlve pH buffer after ad~usting spec~mens whlch are markedly acidic or baslc.
The pH of the urine is one indication of the body's natural defense mechanlsm. Thus, extremes of pH
(acldlc) may klll mlcroorganlsms of lnterest in the speclmen before analysls. Extremes of pH may lndicate rapid replicatlon of mlcroorganlsms whlch may mask the microbial lntegrity at time tl. However, the pH buffer must be compatible with the system. A preferred pH
buffer is sodium blcarbonate. For urlne, lt may be 20293~3 present in the range from about 1.2 mM to about 238.0 mM, preferably from about 2.4 mM to about 59.5 mM. The concentration may be modified to achieve the desired buffering result. For other specimens, not lncluding blood which does not generally requlre a buffer, the concentration may range from about 0 mM to about 60 mM, preferably from about 0.6 mM to about 24.0 mM dependlng on the needs of pH ad~ustment.
The specimen transport system chemical component is preferably a dry admixture which ls employable in a specimen collection vessel for aqueous specimens, and whlch will dissolve in said aqueous specimen when the specimen is introduced into the collection vessel. It is most preferable if the collection vessel ls utilized for specimen transport and perhaps other processing steps to reduce manipulation of the sample and risk of contamination. An example of collection/processing vessel can been seen in Example XVI. An example of use of the dry admixture in connection with urlne may be seen in Example XI. It is more convenient to employ a dry, water-soluble, admixture in a collection vessel for most of the specimen transport system components. It is hlghly deslrable to employ L-cysteine or any sulfhydryl-contalnlng substance employed in a dry admixture to increase shelf-llfe of the specimen transport system admixture. Where a llquid sulfhydryl compound, such~as mercaptoethanol, is employed lt ls desirable to provide a closed contalner with an inert atmosphere, such as N2 gas, to prevent oxidation.
In speclmens which are not inherently aqueous, or whlch are collected using an absorption devlce such as a swab, lt ls necessary to employ an aqueous fluid as part of the speclmen transport system. Thls aqueous fluld comprises an effective dlluent which in comblnatlon with 20293~

the dry admixture components wlll preserve the microbial integrity of the specimen. All the specimen transport system components may be put in the dry admlxture with the exceptlon of agar, an optlonal nutrlent whlch may be desirable for some microorganisms of interest and lnherently llquid substances such as mercaptoethanol.
Agar must be pre-dlssolved wlth adequate heat ln an aqueous solutlon. In one embodlment of a swab-collected speclmen transport system, nutrlents comprlslng growth-supportlng broth and agar wlll be employed so that an approprlate volume of aqueous solutlon for recelvlng the swab will contaln effective amounts of the broth and agar. In thls embodiment, a compartment in the devlce for recelvlng the swab will contain an aqueous recelving fluld, the compartment belng breakable by the swab to release the llquld so that the dry admlxture of speclmen transport system components will be mixed with, and dissolved in, the aqueous broth-agar solution near the time the swab/specimen is collected and placed ln the speclmen transport devlce. It may be practical to add certaln components of the specimen transport system to an aqueous receiving solution rather than a dry admixture because of the low concentrations of the components required. An aliquot of a concentrated stock solutlon of the component might be added to the aqueous recelving solution rather than .
admixing a small amount of dry component with the dry admixture.
Thus, ln one embodiment of a speclmen transport system for speclmens collected by swab, an aqueous recelvlng solutlon ls prepared accordlng to the followlng method.

DILUENI PREPARATION
Preparation of stock solutlon a. Preparation of diluent wlthout Brllllant Green:
- Mueller-Hlnton Broth (MHB) 4.4 g (BBL; Cockeysvllle Md) Agar .2 g Starch .8 g 100 ml H20 Autoclave for 15 mlnutes at 121-C
Store 25 ml ln 50 ml sterlle plastlc conlcal tubes in 4C cold room b. Preparatlon of dlluent wlth Brllllant Green:
Mueller-Hlnton Broth 4.4 g Agar .2 g Starch .8 g 75 ml H20 Boll the mlxture, then add 25 ml 20 ug/ml Brllllant Green (2 mg/100 ml H2O). Autoclave ln 100 ml allquots for 15 mlnutes at 121-C. The color should be lime green as lt cools to room temperature.
Store 25 ml ln 50 ml sterlle plastlc conlcal tubes ln 4-C cold room.
c. Preparatlon of 1:100 hemoglobln solutlon:
1. Put .1575 g hemoglobln* powder lnto beaker.
2. Put ln 100 ml Delonlzed H20.
3. Put stlr bar into beaker.
4. Stlr solutlon slowly for at least 30 mln.
wlthout heat.
5. Using a spatula work ln any floatlng powder on the foam or glass back lnto the solutlon untll completely dlssolved. Keep dolng thls untll all powder ls dlssolved.

20293~3 6. Using two fllter papers (Whatman 934 AH -glass flber), prefllter the solution, wash fllterlng unlt after fllterlng 100-300 ml. Do not fllter more than 300 ml at a tlme.
7. Autoclave ln 100 ml aliquots for 15 mlnutes at 121C.
8. Store 50 ml ln 50 ml sterlle plastlc conlcal tubes ln 4-C cold room.
d. The stock solution is 1 part of dlluent mixed wlth 1 part of hemoglobin solutlon.
Final concentratlon of stock aqueous receiving solution:
1:200 hemoglobin - .07875%
MHB full-strength - 2.2~
Starch - 0.55% (0.15% is from MHB full-strength) Agar - 0.1%
Brilllant Green - 0.00025~ (2.5 ug/ml) * G B CO Dri-Form Hemoglobln. Catalog $ M00230.

A dry admlxture of L-cystelne, SPS, thioglycolate, sodium chloride and calcium propionate to provide the following concentration in the transport system aqueous receivlng solution ls then made:
L-Cystelne 0.25% (2.06 mM) SPS 0.6%
Thloglycolate 0.01% (108.6 mM) Sodium chloride 2.0% (34.33 mM) Calcium Propionate 3.0% (20.52 mM).
The dry admixture is added to the approprlate volume of the aqueous receiving solution, preferably at the tlme of speclmen collectlon.
Other embodlments will be evldent from the above dlsclosure. It ls envlsioned that a fully dry admixture will be more appropriate for aqueous specimens such as 46 20293~3 urine and blood. A dry admixture and a separate aqueous receiving solution might be more preferable for a swab-absorbed specimen. Stlll another embodiment is a fully liquid system where the ingredients normally in the dry admixture are pre-dissolved ln an aqueous receiving solutlon and stored ln a non-oxldlzlng environment.
As an example of a device for collection of aqueous specimens, a urine collectlon/transport device incorporating a dry admixture such as disclosed above with an additional pH bufferlng substance ls disclosed so that a patient may micturate directly lnto the collection/transport device, the dry admixture immediately mixing with and dissolving in the urine specimen. The devlce ls then closed and transported to the laboratory. The volume of the speclmen ls standardlzed by the devlce so that the concentration of the water-soluble dry admixture once solublllzed wlll be approprlate to preserve the microbial integrlty of the specimen.

EXAMPLE I
PRESERVATION OF MICROBIAL INTEGRITY OF A
RECONSTRVCTED SPECIMEN IN THE ABSEN OE OF ANTIBIOTICS
A reconstructlon specimen was prepared by lnoculatlng a sterlle cotton swab with 0.1 ml of a suspension of Pseudomonas aeruqinosa (1 x 104 organisms per ml) (isolated and identified from a cllnical specimen according to known procedures approved by the American Society of Microbiology). The swab was placed in either 5 mls of Mueller-Hinton Broth Mix [hereinafter MHBM] 2.26 Mueller-Hinton Broth (MHB) (BBL Microbiology Systems, Cockeysville, Md. 21030); .55% starch (.15%
from MHB); .10% Agar and .079% hemoglobin or the 202935~

Specimen Transport System composltion described in the following table. The results indicate that the speclmen transport system was effective in maintainlng the microbial integrity of the specimen. The survlval rate was determined by inoculating three chocolate agar plates with .01 ml of treated (specimen transport system) or untreated (MHBM alone) specimen at varlous time polnts. The number of colonles whlch grew on each plate were counted and an average of the three plates taken. A survival rate of 1.00 lndicates 100% survival, values greater than 1.00 indicate growth and values less than one indicate death.
It ls evident that the speclmen transport system used ln the above example preserved the mlcrobial integrity of the sample so that quantitatlon of the number of microorganisms of interest at 72 hours after specimen collection would be possible. Wlthout use of the specimen transport system of the sub~ect lnvention, uncontrolled growth of the organism occurred. For example, at 24 hours, the sample without the sub~ect invention exhiblted over a 58 fold lncrease from time of specimen collection to time of analysis. It is predictable that false positive results as to the microbial population present ln the speclmen at the tlme of collection would be obtained by a laboratory analyzing the specimen to which no specimen transport system was added. Even early as 4 hours past the time of collectlon, the results would be skewed.

48 202~3~
Survlval Rate Over Tlme Tlme ln Hours 0 ~ 6-8 24 ~8 72 With Transport System 1.00 1.02 0.791.08 1.02 0.66 5~ posltlan*
wit ~ t Transport System1.002.91 3.8058.1358.1358.13 O~osltlon *296 NaCl; 3% calclum proplonate; .2596 cysteine; 2.5 x 10-496 Brllliant Green; 0.696 SPS; 0.01% thloglycolate;
2.2% Mueller-Hlnton Broth; O. 55% starch (0.15%
contributed by Mueller-Hlnton,Broth); 0.1% agar;
0.07875% Hemoglobin (All % in welght per total volume).

EXAMPLE II
PRESERVATION OF THE MICROBIAL INTEGRITY OF A
RECONSTRUCTED SPECIMEN IN THE PRESENCE OF ANTIBIOTICS
The reconstructed specimens were prepared as described in Example I. The same specimen transport system composition was tested. Antlblotlcs were added at a concentratlon of the antlclpated average maxlmum serum level. A value of 1.00 - 10096 survlval.
Wlthout the dlsclosed inventlon, the mlcroblal integrity of the specimen clearly began to deteriorate even 4 hours after the speclmen was taken. In the table below, it can be discerned that false negatlve cultures would be hlghly probable. Quantltatlon wlthout use of the dlsclosed composltlon would be hlghly lnaccurate.

20293~3 Survlval Rate Over Tlme Time in Hburs ~mikacin (2/ug/ml)& Transport 1.00 1.081.11 1.01 1.10 1.14 System ~acin (2/uq/ml) alone 1.000* 0 0 0 0 PiperAc~ n (60 ug/ml) 1.00 0.991.07 1.13 0.91 0.95 + Transport System Piper~rlllln (60 uq/ml) alone 1.000.76 0.21 0.01 0 0 Ticarc~llin (150 ug/ml) 1.00 0.971.06 1.12 1.02 1.01 + Transport System Ticarcillin (150 uq/ml) alone 1.000.68 0.30 0 0 0 * O - no growth ~cPrn~hle EXAMPLE III
SYNERGISTIC ~ OF COMBINED SPECIMEN TRANSPORT SYSTEM
COMPONENTS ON PRESERVATION OF THE MICROBIAL
INTEGRITY OF A RECONSTRUCTED SPECIMEN
Reconstructed speclmens were prepared as described ln Example I with the indicated microorganisms llsted in each table below rather than P. aeruqinosa.
It can be seen in the Survlval Rate results that the comblned components of the speclmen transport system exert a synergistic effect compared with lndividual , components. For example, in Table III-4, the specimen transport system held the survival over tlme at a relatlvely constant level. Growth occurred with the other lndividual treatments, ln some cases the overgrowth of the mlcroorganlsm dominating the plate (TNTC values). If multlple organlsms were present as would be the case in an actual speclmen, thls overgrowth would be especially ~nsatisfactory. In Table III-l, it 2029~S3 can be seen that SPS, NaCl or MHB when used alone did not allow quantitative survival at 24 hours.
The following were tested alone or in combination with other components:

Mueller-Hinton Broth (MHB) Beef Extract 0.3%
Acid Hydrolysate of Casein 1.75%

10 Starch 0.15%

Mueller-Hinton Broth Mix (MHBM) 15 Mueller-Hinton Broth 2.0%
Starch 0.55% ~0.15 % from MHB) Agar 0.10%
Hemoglobin 0.07875%

Brilliant Green Mueller-Hinton Broth Mix Mueller-Hinton Broth 2.2%
Starch 0.55% (0.15% from MHB) 25 Agar 0.10%
Hemoglobin 0.07875%
Brilliant Green 0.00025%

TRANSPORT SYSTEM
Brilliant Green Mueller-Hinton Broth Mix + 0.25%
Cysteine + 0.6% SPS +2% NaCl + 0.1% Thioglycolate All numbers following organism identity indicate the culture number from the American Type Culture Collection Rockville, Maryland. SPS = sodium polyanethol sulfonate.

~`

Survival Rate of Haemophilus influenzae 19418 Transport Time in Hours 5 Individual Components Q 4 6-8 24 0.5% Cysteine 1.00 1.02 0.98 2.05 0.6% SPS 1.00 1.10 1.07 0.08 2% NaCl 1.00 0.23 0.21 0.006 Mueller-Hinton Broth 1.00 0.87 0.84 0.04 10 Mueller-Hinton Broth Mix 1.00 1.64 3.60 7.85 Brilliant Green Mueller- 1.00 1.32 1.41 1.02 Hinton Broth Mix Combined Components 15 Transport System 1.00 1.01 0.97 0.76 Survival Rate of Streptococcus pneumoniae 6301 20Transport Time in Hours Individual Components 0 4 6-8 24 0.5% Cysteine 1.00 0.65 0.19 0.03 0.6% SPS 1.00 1.11 1.02 2.72 2% NaCl 1.00 0.94 1.21 0.72 25 Mueller-Hinton Broth 1.00 1.09 1.62 TNTC
Mueller-Hinton Broth Mix 1.00 1.36 3.33 8.10 Brilliant Green Mueller- 1.00 1.04 0.98 0.69 Hinton Broth Mix Combined Components Transport System 1.00 0.90 0.85 0.66 TNTC = Too Numerous To Count ~r _ 2029353 Survival Rate of Streptococcus pyogenes 19615 Transport Time in Hours Individual Components . 0 4 6-8 24 0.5% Cysteine 1.00 1.08 1.21 3.61 0.6% SPS 1.00 1.05 1.54 2.60 2% NaCl 1.00 1.11 1.12 1.82 Mueller-Hinton Broth 1.00 1.34 1.61 6.90 Mueller-Hinton Broth Mix 1.00 1.56 1.95 4.32 Brilliant Green Mueller- 1.00 1.53 1.86 1.54 Hinton Broth Mix Combined Components Transport System 1.00 0.94 0.91 0.68 Survival Rate of Staphylococcus aureus 25923 20Transport Time in Hours Individual Components 0 4 6-8 24 0.5% Cysteine 1.00 1.30 1.46 4.08 0.6% SPS 1.00 1.65 3.73 TNTC
2% NaCl 1.00 1.44 2.32 TNTC
Mueller-Hinton Broth 1.00 2.80 4.57 TNTC
Mueller-Hinton Broth Mix 1.00 1.98 14.11 48.43 Brilliant Green Mueller- 1.00 0.94 0.93 0.35 Hinton Broth Mix Combined Components Transport System 1.00 0.94 0.86 0.80 TNTC = Too Numerous To Count ~' Survival Rate of Streptococcus faecalis 2492-2 Transport Time in Hours Individual Components 0 4 6-8 24 0.5% Cysteine 1.001.40 2.00 TNTC
0.6% SPS 1.002.31 3.98 TNTC
2% NaCl 1.001.60 3.08 TNTC
Mueller-Hinton Broth 1.002.99 7.18 TNTC
Mueller-Hinton Broth Mix 1.0011.14 24.83 60.56 Brilliant Green Mueller- 1.001.59 2.01 2.83 Hinton Broth Mix Combined Components Transport System 1.000.92 0.85 2.49 TNTC = Too Numerous To Count Survival Rate of Escherichia coli 25922 Transport Time in Hours -Individual Components O 4 6-8 24 0.5% Cysteine 1.003.04 6.63 TNTC
0.6% SPS 1.004.02 18.20 TNTC
2% Salt 1.002.33 6.29 TNTC
Mueller-Hinton Broth 1.003.66 15.40 TNTC
Mueller-Hinton Broth Mix 1.004.40 28.44 59.87 Brilliant Green Mueller- 1.002.62 6.16 72.46 Hinton Broth Mix Combined Components Transport System 1.000.98 1.03 0.67 TNTC = Too Numerous To Count ~r .

Survival Rate of ~lebsiella pneumoniae 632-2 Transport Time in Hours Individual Components 0 4 6-8 24 0.5~ Cysteine 1.00 2.715.68 TNTC
0.6% SPS 1.00 4.87TNTC TNTC
2% NaCl 1.00 3.417.20 TNTC
Mueller-Hinton Broth 1.00 4.90TNTC TNTC
Mueller-Hinton Broth Mix 1.00 5.3353.09 96.41 Brilliant Green Mueller- 1.00 6.8731.98 59.17 Hinton Broth Mix Combined Components Transport System 1.00 1.081.03 0.70 TNTC = Too Numerous To Count EXAMPLE IV
PRESERVATION OF MICROBIAL INTEGRITY OF A THROAT

PATHOGEN IS ADDED
The effectiveness of the disclosed specimen transport system on preserving the microbial integrity of a throat swab specimen containing a known amount of a pathogenic microorganisms along with the normal flora found in the throat is shown in the following table. It was demonstrated that overgrowth of normal flora could mask a pathogenic microorganism in a specimen for analysis.
Normal throat flora were collected from 20 healthy donors (three swabs per donor). Each swab was then inoculated with between 104 - 106 of a human pathogen.
The microorganisms present on each swab were subsequently extracted at time zero by vigorous i~
~; ~

20293~3 agitatlon into 5 ml of a selected transport system. The swabs were discarded, and the liquid portions were held at 24C for subsequent quantitative analysis at 0, 4, 6, 24 hours in order to determine the relative survival of the pathogen versus overgrowth by normal flora present on the swab. The following organlsms were tested: E. coll, P. aeruqinosa, S. aqalactiae, H.
influenzae, S. pyoqenes, E. cloacae, K. pneumoniae, S.
aureus, and S. faecalis.
With the Stuarts transport system, overgrowth by the normal flora rendered the sample dlfficult to interpret wlthin six (6) hours. The low survival observed at 24 hours (0.39) could either reflect death of the pathogen or masklng of the organism by excessive 1~ normal flora. Similar results were obtalned with the Amies transport system. The amount of overgrowth varied dependlng on the pathogen under analysis. The more fastidious organlsms (e.g., Haemophllls lnfluenzae) were more susceptlble to overgrowth. Excesslve growth of normal throat flora was effectlvely suppressed wlth the disclosed specimen transport system, whlch prevented overgrowth of the pathogen by normal flora ln the absence of antlblotlc over 24 hours.

20293~3 _ Detectability of a Pathogen in the Presence of Normal Flora rSurvival 1.00 = 100%l Time in Hours Normal Flora + Streptococcus agalactiae Plus Specimen Transport System1 1.00 1.00 0.87 0.83 Normal Flora + Streptococcus 1.00 1.09 -3 _3 agalactiae With Stuart's system2 1Specimen Transport System utilized in this Example comprised an admixture of 1.5% NaCl, 2.0% cysteine, 0.6% SPS, and 0.01% thioglycolate.
2Stuarts system as disclosed in Stuart et al, "The Problem of Transport of Specimens for Culture of Gonococci," 45 Canadian J. of Public Health 73 (1954)-30vergrowth of normal flora making accurate count difficult.
EXAMPLE V
PRESERVATION OF MICROBIAL INTEGRITY IN A
RECONSTRUCTED SPECIMEN FROM To = O TO Tl =

It is recommended in most manuals that a specimen be analyzed within 2 hours after collection. However, this assumed safe time period is not valid in all situations.
The disclosed invention is shown to be a significant improvement over prior art transport systems which do not prevent significant deterioration of microbial integrity even within as little as 15-20 minutes.
The Amies transport system C. Amies et al., 58 Canadian J. Public Health, 296 (1957) (available from ~`

Curtin Matheson Sclentific, Inc.) The formula (per llter of distilled water) is:
- sodlum chloride 3.0 g - potassium chloride 0.2 g - calcium chloride 0.1 g - magnesium chloride 0.1 g - mono potasslum phosphate 0.2 g - disodium phosphate 1.15 g - sodlum thloglycollate 1.0 g - agar 7.37 g Stuart's Transport Medlum, 45 Canadlan J. Publlc Health 73, 75 (1956) ls the followlng: 6 g Bacto Agar in 1900 mls dlstilled water, 2 ml thloglycolllc acid (Dlfco) brought to pH 7.2 wlth lN NaOH. 100 ml 20% (w/v lS ln water) Na glycerophosphate and 20 ml CaC12 (1% w/v ln water) ls then added. 20 ml 1% w/v CaC12 ls added and the solutlon brought to pH 7.4 wlth lN HCl. 4 ml 0.1%
methylene blue ls then added.
The speclmen transport system of the lnstant lnventlon deplcted ln the followlng charts was of the formula:
2% Na Cl .25% L-cystelne (free base) 3% Calclum proplonate 2.5X10-4% Brllllant Green 0.6% SPS
0.01% Thloglycolate 1 2.2% Mueller-Hlnton Broth 0.55% Starch 0.1% Agar 0.7875% Hemoglobln Antlblotlcs were added at the average maxlmum serum level as determlned by publlshed reports. These values are set out ln Example VI, Table VI-2.

20293~3 ~8 The organism/ml level was tested at each time point indicated on the graphs (FIGURE ll-FIGURE 16).

In FIGURE 11, lt can be seen that the mlcrobial integrlty of the reconstructed speclmen containlng Enterobacter cloacae uslng conventlonal transport systems deteriorates wlthln 20-30 mlnutes ln the presence of the antlblotlc Tobramycln at 40 ug/ml. The specimen transport system ln contrast held the count constant over tlme.

In FIGURE 12, lt can be seen that the speclmen transport system exhlblts superlorlty 4 hours after speclmen collection, thus surpasslng the two-hour recommendatlon for speclmen analysls ln the prlor art.

In FIGURE 13, an Escherlcha coli reconstructed specimen is tested. The specimen transport system exhibits superiorlty in malntainlng the mlcroblal integrlty of the specimen ln the presence of Amlkacln at 21 ug/ml.

FIGURE 14 deplcts the preservation of the mlcrobial integrlty of Streptococcus pneumoniae wlth the sub~ect invention compared to conventional systems ln the presence of Amplclllln at 21 ug/ml. A somewhat higher recovery ln organism/ml ls demonstrated.

60 20293~3 FIGURE 15 deplcts the effect of Moxalactam loo ug/ml in a reconstructed E. coll specimen. The specimen transport system was able to preserve microbial integrity beyond a two-hour transport time.

FIGURE 16 depicts the effect of the specimen transport system on Klebsiella pneumoniae in the presence of Cephalothin. The Amles and Stuarts Systems received a sllghtly higher lnoculum than the Specimen Transport System, however the former two systems still show dramatlc drops ln organlsms/ml at 3 hours.

202g35~

EXAMPLE VI
COMPARATIVE AVERAGE MICROBIAL INTEGRITY (SWABS~
Specimen transport was tested by obtaining microbial pathogens from the American Type Culture Collectlon (ATCC), Rockville, Md and inoculatlng multlple sterlle cotton swabs with 1 x 104 of a slngle pathogen. Each lnoculated swab was placed ln an aqueous preparatlon comprising 0.25% (2.06 mM) L-cystelne (free base), 0.6% SPS, 0.01~ (108.6 mM) thioglycolate, 2.0 (34.22 mM sodium chlorlde)~ 3.0% (20.52 mM) calcium propionate, 2.2% Mueller-Hlnton Broth, 0.55% starch;
0.1% agar; 0.7875% (1.2 uM) hemoglobln, and 2.5 X10-4%
Brllllant Green (0.5 uM) or the transport medlum disclosed in 45 Canadian J. of Public Health 73 (1954) 1~ or Amies's Transport Medium (without charcoal) (58 Canadian J. of Public Health 296 (1967)).
Elther a speciflc concentratlon of a selected antlbiotic or no antlbiotlc was added to each lndlvldual I aqueous preparatlon. The antlblotlc concentratlon was chosen accordlng to publlshed values of the average maxlmum serum levels that would be found ln patlents.
Thls level ls lndlcated for each antlbiotic in Table VI-2. (It should be noted that for urine speclmens, not tested ln thls example, 10X the antlbiotlc average maxlmum serum level was employed). The number of bacteria ln each speclmen were determlned at each of~4 tlme polnts ln the three transport solutlon preparations by transferrlng 0.01 ml to each of three chocolate agar plates, lncubating at 37-C for 24 hours, counting the number of colonles, and calculating the number of microorganlsms survlving per ml.
In the chart below, a value of 1.00 ~ 100~
survlval. Thus at o hours, all test speclmens show a value of 1.00. A value greater than 1.00 indicates repllcation of the organism occurred in the transport period by the factor tlmes 1.00 whlch yields that value.
A value less than 1 indlcates that the numbers of organlsm were reduced durlng transport (death occurred).
Thus a value of .5 indlcates a loss of half the origlnal number of organisms. The values ln the chart are averaged for the gram negative organlsms tested (see chart below) and the gram positive organisms tested (see chart below) for the antibiotic classes given.

TABLE VI-l LIST OF ORGANISMS USED FOR SPECIMEN
TRANSPORT SYSTEM COMPARISONS

G~AM NEGATIVE ATCC tl CLINICAL STRAIN2 Enterobacter cloacae 3118-1 Escherichia coli 25922 Haemophilus influenzae19418 Haemophilus influenzae9795 (Type B) Haemophllus lnfluenzae9133 Haemophllus influenzae8149 Klebsiella pneumoniae 632-2 Pseudomonas aeruqinosa 277 Staphylococcus aureus25923 lAmerican Type Culture Collection, Rockville, Md.
2Clinical isolate identifled according to methods approved by the American Society of Microbiology.
GRAM POSITIVE
Streptococcus aqalactiae624 Streptococcus faecalis 2942-2 Streptococcus pneumoniae6301 Streptococcus pneumoniae9163 Streptococcus pneumoniae10813 _: 2029353 Streptococcus pneumoniae 27336 Streptococcus pyoqenes 19615 Streptococcus pyogenes 12344 (Type 1) Streptococcus pyogenes 12383 (Type 3) Streptococcus pyogenes 12385 (Type 4) ANTIBIOTICS USED FOR EXPERIMENTS
DRUG-MANUFACTURER AVERAGE MAXIMUM
SERUM LEVELS (u~/ml) I. AMINOGLYCOSIDES
AMIKACIN BASE - Bristol Laboratories 21 GENTAMICIN SULFATE - Schering Corporation 6 TOBRAMYCIN - Eli Lilly & Company 4 II. CEPHALOSPORINS
CEFAMANDOLE LITHIUM - Eli Lilly & Company 20 CETRIAXONE - Hoffman-La Roche, Inc. 90 CEFOTAXIME SODIUM - Hoechst-Roussel Pharmaceuticals, Inc. 20 CEFOXITIN SODIUM - Merck, Sharp, & Dohme 25 CEPHALOTHIN SODIUM NEUTRAL - Eli Lilly & Company20 MOXALACTAM DIAMMONIUM - Eli Lilly & Company 100 III. PENICILLINS
AMPICILLIN TRIHYDRATE - Bristol Laboratories 21 CARBENICILLIN DISODIUM - Beecham Laboratories 71 (20 E.Coli) 30 METHICILLIN SODIUM - Bristol Laboratories 9 MEZLOCILLIN SODIUM - Miles Pharmaceuticals 4 PENICILLIN G POTASSIUM BUFFERED - Eli Lilly & Company 20 PIPERACILLIN SODIUM - Lederle Piperacillin, Inc.60 TICARCILLIN DISODIUM - Beecham Laboratories 150 IV. OTHERS
BACTRIM (Sulfamethoxazole-Trimethoprim) - Hoffmann-3 La Roche, Inc.
40 CHLORAMPHENICOL - Parke-Davis 18 ERYTHROMYCIN GLUCEPTATE - Eli Lilly & Company 8 GANTRISIN (Sulfamethoxazole) - Hoffman-La Roche, Inc. 100 POLYMYXIN B SULFATE - Pfizer, Inc. 2 TETRACYCLINE HCl - Lederle Laboratories Division 9 VANCOMYCIN HYDROCHLORIDE - Eli Lilly & Company 8 , .

i ``~
'1, AVERAGED RECOVERY VALUESl FINAL DEVICE COMPARISONS: TRANSPORT TIME IN HOURS

S.T.S.1 STUART' S3 AMIES' S4 S . T.S. STUARTS AMIES S.T.S. STUARTS AMIES S.T.S. STUARTS AMIES
GRAM-NEGATIVES
I. AMINOGLYCOSIDES 1.00 .93 .002 .003 .73 0 0 .44 0 0 II. CEPHALOSPORINS 1.00 .69 .20 .12 .45 .08 .04 .28 .03 .02 III. PENICILLINS 1.00 .95 .36 .40 .80 .11 .08 .57 .06 .03 IV. OTHERS 1.00 .89 .38 .65 .91 .21 .32 .78 .10 .16 V. NO ANTIBIOTIC 1.00 .97 8.91 14.67 .84 41.28 46.04 55 33.65 39.73 GRAM-POSITIVES
I. AMINOGLYCOSIDES 1.00 .88 .30 .38 1.06 .15 .32 1.49 .02 .12 II. CEPHALOSPORINS 1.00 .72 .65 .62 .58 .28 .32 .41 .12 .13 III. PENICILLINS 1.00 .96 .39 .37 .96 .08 .09 1.45 .03 .03 15 IV. OTHERS 1.00 .93 .76 .77 .77 .52 .51 .62 .33 .24 V. NO ANTIBIOTIC 1.00 .91 1.15 1.12 1.23 7.56 17.78 2.48 7.79 18.94 TOTAL ANTIBIOTICS 1.00 .87 .38 .41 .78 .18 .21 .76 .09 .09 TOTAL
WITHOUT ANTIBIOTICS 1.00 .94 5.03 7.90 1.04 24.42 31.91 1.52 20.72 29.34 NUMBER OF SPECIFIC DEVICE RECONSTRUCTIONS
S.T.S. STUARTS AMIES
WITH ANTIBIOTICS 419 272 284 t~
WITHOUT ANTIBIOTICS 119 131 68 CL~
538 403 352 = 1293 Total Reconstructions C~
lThe above data the generated using 19 pathogens - listed in Table VI-A. Data from all gram negative organisms was C~
averaged separately from gram positive organisms.
2S.T.S. = Specimen Transport System 3Stuart, 45 Canadian J. Public Health 73 (1954) 4Amies, 58 Canadian J. Public Health 296 (1967) (without charcoal) ~029353 Now referring to FIGURE 1, centrifugation article 20 is depicted which is disclosed in the above-described U.S. Patent 4,131,512 and its division U.S. Patent 4,212,948. These patents are directed to a method and apparatus whlch provides for improved rapid quantltatlve analysls of a blood sample for the presence of microbial pathogens.
The blood sample is lysed and deposlted on a hlgh density water lmmlsclble, hydrophoblc, nontoxlc, llquid cushioning agent and sub~ected to centrifugation. The microbial pathogens contalned in the lysed blood sample will collect ln a layer ad~acent the lnterface of the cushioning agent and the blood sample residue, and, in this concentrated form, can easily be separated from the residual portion of the blood sample for culturlng and quantitative counting. As shown, the artlcle 20 comprises an elongated tubular centrifugation vessel 22 having a conventlonal in~ectable closure member 24 which sealably closes the upper end thereof, and an in~ectable closure member 26 whlch sealably closes the lower end thereof. Article 20 contalns an effectlve amount of cushionlng agent 28. The speclmen transport system when utilized in elongated tubular centrifugation vessel 22 ls deposited as layer 30 of particulate solld on cushlonlng agent 28. The speclmen transport system can be contalned wlthln an aqueous solutlon wlthln artlcle 20, e.g., about one-half milliliter, but it is preferred that said system be ln the form of solld partlculate powder 30. Solid partlculate powder 30 is not soluble within the llquld cushloning agent 28 and has a higher shelf stabillty than the liquid solutlon formed of the lngredlents. In addltlon, the use of the partlculate solld speclmen transport system allows a novel .". , ., 20293~3 sterlllzation technlque to be carried out within the interlor of article 20 whlch will be hereln descrlbed below. In the preferred embodlment of the sub;ect invention, the specimen transport system ls present whether ln aqueous solutlon or layer 30 sufflclent so that when a sample fiuld such as blood ls deposited thereln, the comblnatlon of speclmen transport system and blood will contaln from about 0.1 to about 6% by welght thereof of sodlum polyanetholsulfonate; from about 0.5 to about 2.5% by welght of cystelne; from about 0.1 to about 1.6% by welght thereof of thloglycolate; and from about 5 mlcrograms per mllllllter to about 500 mlcrograms per mllllllter of para-amlnobenzolc acld. In addltlon, slnce thls partlcular embodlment ls used for processlng blood samples, the resultlng total volume wlll also lnclude from about 0.02 to about 1% by welght of purlfled saponln and from about 0.01 to about 0.5% by weight of EDTA. When the specimen transport system ls ln the form of an aqueous solutlon, the centrlfugatlon vessel 22 will draw approximately 7.5 milliliters of blood. It is preferred that sald speclmen transport system be at least 3% by volume of the total llquld ln centrlfugatlon vessel 22 lncludlng the total quantlty of the specimen transport system, the sample fluld and the cushlonlng agent and preferably from about 5% to about 30% by volume thereof. When the speclmen transport system ls ln the form of partlculate layer 30, the elongated tubular centrlfugatlon vessel 22 wlll draw about 8 mllllllters of blood. In the most preferred embodlment of the sub~ect lnventlon, layer 30 will contain 0.096 grams of cystelne; 0.008 grams of thloglycolate; 0.048 grams of sodium polyanethol sulfonate; 0.018 grams purifled saponln; and 0.008 grams of EDTA. It ls noted 67 20293~3 that the EDTA ls not necessary to prevent blood clot formatlon so long as adequate amounts of sodium polyanetholsulfonate are present. For example, another satisfactory blood treating system (layer 30) contains 0.048 grams sodium polyanetholsulfonate, .08 grams cysteine, .oog grams thloglycolate and 0.019 grams purlfied saponln.
The comblnatlon of speclmen transport system and urine will preferably contaln from about 0.6 percent to lo about 2.0 percent by welght thereof sodlum ~ polyanetholsulfonate; from about 0.5 percent to about 2.5 percent by welght thereof, free-based cysteine;
about 0.1 percent by welght thereof, thloglycolate;
about 2.0 percent by welght thereof, sodlum blcarbonate;
and from about 2.5 percent to about 4.0 percent by weight thereof, sodlum chlorlde. The sodlum blcarbonate was added to the urlne speclmen transport system ln order to ad~ust for the normal acldlty of urlne and thus attaln a neutral pH. The added salt, ln the form of, for example, sodlum chlorlde, lncreases the bacterlostatlc effect of the system ln the absence of antlblotlcs ln the urlne. A free-based L-cystelne, such as ICN cystelne, ls preferably substltuted for the prevlously employed L-cystelne-HCl as the former does not produce a gaseous reactlon when comblned wlth the sodium blcarbonate buffer as seen prevlously in the , L-cystelne-sodlum blcarbonate mixture.
Centrifugation vessel 22 can be made of siliconlzed glass or hard plastlc such as polycarbonate or polypropylene. In~ectable closure members 24 and 26 can comprlse rubber seallng stoppers. In~ectable closure members 24 and 26 both carry lndentatlons 24a and 26a, respectively, to enhance the ease of ln~ection by common types of in~ectlon needles. Evacuated space 32 ls 202935~

maintained at a lower than atmospheric pressure at a predetermined value so that the centrlfugatlon vessel can receive a known amount of liquld by in~ectlon through in~ectable enclosure member 24 without excessive pressure belng bullt up wlthin the interior thereof which would cause ln~ectable closure members 24 and 26 to become dislodged from the openlngs withln the centrifugatlon vessel 22.
Referrlng especlally to ln~ectable closure member 26 at the lower end of centrlfugation vessel 22, lt ls noted that lnner surface 34 of ln~ectable closure member 26 is positloned at an angle wlth respect to the walls of centrlfugatlon vessel 22.
It ls noted that artlcle 20 ls especlally deslgned to be utlllzed wlthin an angle rotor centrifuge and that the angled lnner surface 34 is a complement of the angle of the rotor. It should be noted, however, that the device of the sub~ect lnventlon can be utlllzed ln a conventlonal swlnglng bucket-type centrifuge. In the latter instance, surface 34 should be perpendlcular to the bottom of artlcle 20 and ls otherwlse utlllzed ln the same general manner as wlll be described herein below for the article 20 illustrated in FIGURE 1.
Surface 34 should be smooth and substantially free of lnterstltial spaces and crevlces ln whlch mlcroblal , pathogens could be entrapped. Further, the clrcular sealing area around surface 34 where the materlal of ln~ectable closure member 26 meets the walls of the centrlfugation vessel 22 should be tlghtly sealed so that the lnterface does not provlde a large clrcular crevice in which microbial pathogens could become lodged The angle of lncline of smooth surface 34 wlth respect to the walls of centrifugatlon vessel 22 ls determined accordlng to the centrlfugatlon apparatus ln which artlcle 20 ls to be centrlfuged.
As dlscussed above, when a swlnglng bucket-type centrlfuge ls utllized, surface 34 wlll be posltloned perpendlcular to the bottom of the artlcle 20. However, when an angle rotor centrlfuge ls utlllzed, surface 34 wlll carry the complement of the angle of the rotor.
Therefore, ln general, when the rotor angle ranges from about 60 to 10, the angle of surface 34, or angle of incllne 36 wlthln the centrlfugation vessel will range correspondlngly from 30- to 80. Thus, the angle of lncllne, depicted by arc 36, will generally be the complement of the angle at whlch devlce 20 rests wlthln the centrlfuge durlng centrlfugatlon. For example, the angle of lncllne 36 deplcted ln FIGURE 1 ls approxlmately 34-. Thus, for example, when article 20 is placed in an angle rotor centrifuge in which centrifugation occurs at approximately 56-, fluids contained withln artlcle 20 wlll be forced agalnst surface 34 at a substantlal perpendicular angle.
The amount of cushionlng agent 28 employed should be sufficlent to completely cover surface 34 upon centrlfugation. The amount of cushloning agent utilized can vary wlth the parameter of the particular system chosen, for example, the stopper design, volume of resldual blood and volatlllty of the cushioning agent utilized. A preferred amount of cushioning agent can comprlse from about 3.3% to about 40% by volume based on the volume of the cushlonlng agent-resldual blood sample mlxture whlch ls removed from artlcle 20 and tested for the presence of mlcroblal pathogens.
Generally, the cushioning agent of the sub~ect lnventlon can comprlse a hlgh denslty, hydrophobic, water lmmlsclble llquld. As noted prevlously, the term 70 20~9353 ~high density" as used hereln refers to a liquid which will not be supported by the mixture of blood and blood treating fluid or any other sample fluld suspected of containing microbial pathogens in the presence of centrifugal force. In addition, the cushioning agent should be nontoxic to microbial pathogens and relatively inert with respect to butyl rubber, silicone rubber and other types of elastomers employed in the manufacture of the in~ectable closure members described above. The density of the cushioning agent can be in the range of from about 1.2 grams per cubic centimeter to about 2.0 grams per cubic centimeter. Generally, fluorinated hydrocarbons having the above descrlbed characteristics and having molecular weights in the range of from about 300 to about 850 are preferred. Furthermore, fluorinated hydrocarbons havlng the above quallties which have a vapor pressure at 77-F and 1 atmosphere from 0.06 psi (0.3 mm Hg) to about 0.58 psl (30 mm Hg) and preferably a vapor pressure approximately equal to that of water. Therefore, cushlonlng agents havlng the above descrlbed qualltles and bolllng polnts of about 200F to about 420-F ~93C - 216-C) and preferably of about 225F to about 280-F (106-C to 138C) can be utillzed. The cushioning agents preferably have speclfic heat at least equal to or greater than 0.2 g-cal/gc at 77F and 1 atmosphere, and most preferably specific heat at least equal to or greater than water.
The cushlonlng agent should also have a vapor pressure whlch will not disrupt the in~ectable closure means from the tube during manufacturing steps such as autoclaving, for example. Fluorinated hydrocarbons sold under the trade mark FLUORINERT by 3M Company of Minneapolis, Minnesota, have been found to perform well as cushioning agents. Specifically, types FC-75, FC-48, and FC-43 of 202g353 the FLUORINERT series have been found to be especially useful.
Although the exact function which such cushioning agents perform ls not fully known, lt ls belleved that they improve collectlon of mlcroblal pathogens whlch have passed from suspenslon ln a centrlfuged blood sample ln at least two ways. Flrst, the cushloning agent serves to seal lnterstltlal spaces, cracks and crevlces both on the smooth surface 34 of the centrlfugatlon vessel 22 and the lnterface between the walls of the centrlfugatlon vessel 22 and ln~ectable closure member 26. Thus, mlcroblal pathogens whlch mlght otherwlse become entrapped ln such lnterstltlal spaces, and therefore not recovered, are recovered wlth the cushlonlng agent 28 when lt ls removed from artlcle 20. Secondly, it ls belleved that the cushlonlng agent does act to cushlon the lmpact of mlcroblal pathogens whlch are forced out of suspenslon ln a blood sample durlng centrlfugatlon. Thls cushlonlng effect reduces the danger of ln~ury to mlcroblal pathogens which mlght otherwlse occur upon lmpact. Further, whlle some of the mlcroblal pathogens may actually pass lnto the cushlonlng agent, substantlally none wlll pass completely through lt and a ma~orlty wlll form on lts surface at the lnterface between the cushlonlng agent 28 and the blood sample and collect ln a layer.
After the cushlonlng agent 28 has been deposlted wlthln centrlfugatlon artlcle 20, the speclmen transport system 30 for the blood may also be deposlted there.
Once the speclmen transport system 30 has been deposlted ln centrlfugatlon artlcle 20, ln~ectable closure member 24 can be put ln place and space 32 evacuated to the desired lower than atmospheric pressure, e.g., 25 to 30 lnches of mercury. In accordance with one embodiment, the lnterlor of centrlfugatlon vessel 20 is next sterlllzed by a novel technlque. It has been found that lf a centrlfugatlon vessel is heated to the vaporlzatlon point of the FLUORINERT material therewlthln, e.g., at least about 120C and held for a sufficient time, e.g., at least about 30 minutes, the lnterlor of the tube and the solld particulate specimen transport system 30 will become sterlllzed by the hot FLUORINERT vapors. Once thls ls done, the centrlfugation vessel 20 ls merely cooled to room temperature and packaged for sale, for example.
Now referrlng to FIGURES 2-9, an analysls sequence ls schematlcally depicted lllustratlng a preferred embodlment of the sub~ect lnvention. As an example, a procedure which ls carrled out ln accordance wlth one embodlment of thls lnventlon for detectlon of mlcroblal pathogens withln a blood sample can be carrled out conveniently wlth the followlng apparatus:
The above descrlbed centrlfugatlon article 20 contalning the cushionlng agent 28 and speclmen transport system 30. The vessel can be of 12-14 milliliters ln volume.
A sterlle glass syringe and one 1 1/2 inch 21 gauge disposable hypodermlc needle;
One sterlle glass syrlnge and one 1 inch 18 gauge dlsposable hypodermlc needle;
One 5/8 lnch 25 gauge hypodermlc needle with cotton lnserted at lts hub (used as a vent);
Two blood agar plates;
Two chocolate agar plates.
It ls noted that with the exceptlon of centrlfugation article 20 or some equlvalent article, varlous types of well-known laboratory apparatus and culture medla can be used to carry out the novel process of the sub~ect lnvention. It is particularly noted that the culture media set forth above are exemplary only and are generally preferred to be utilized for detecting the most commonly known microbial pathogens. The blood agar plates suggested are conventionally utilized blood agar plates which are basically sheep's blood and a base nutritional agent such as braln heart lnfuslon, whlch is held together with an agar solidlfylng agent on a petri plate. The chocolate agar plate is deslgned to grow certain factltious pathogens, e.g., Hemophilus.
Thus, whlle varlous apparatus can be utlllzed in the method of the sub~ect lnvention, the above 11st of apparatus and materlals can be conveniently utlllzed in the scope of this lnventlon ln a manner set forth below.
To utlllze centrlfugatlon artlcle 20 set forth ln FIGURE 1 ln the drawing, lt ls lnltially posltloned so that ln~ectable closure member 26 wlth its smooth angled surface 34 is at the lower end of article 20 so that the cushlonlng agent 28 speclmen transport system sollds 30 rest upon smooth angled surface 34. In practlce, a mixture of cushlonlng agent 28 and the solld partlcles of speclmen transport system 30 may occur due to handling so that two distinct layers may not always be present. Thls unstable mlxture of cushionlng agent 28 and speclmen transport system 30 ln no way adversely affects the method set forth hereln slnce the sollds formlng system 30 wlll rapldly dlssolve ln the aqueous sample (blood) and separatlon of the two resultlng llquld phases rapldly occurs upon centrifugation.
Next, a predetermlned amount of a blood sample 38 drawn from the patlent, for example, 8 mllliliters of blood, ls lnjected lnto the evacuated space or centrifugatlon artlcle 20 as deplcted ln FIGURE 3 uslng a common type of syrlnge 40. Alternately, the sample can be drawn directly into article 20 using a standard and double needle fixture supplied wlth conventlonal vacuum blood drawing devlces such as sold under the mark "vacutainer~ by Becton Dlckinson. Then, article 20 containlng the blood sample 38, the specimen transport system 30, and the cushloning agent 28 ls sub~ected to mixlng to lnsure that the anticoagulants, red cell lysing agent, and the specimen transport system 30 are completely admlxed wlth the blood sample 38. Thls mixing step ls deplcted schematlcally by FIGURE 4. The mlxing step will insure that the specimen transport system 30 containing the lyslng agent wlll be completely admixed with and solubillzed by the blood sample. Thls solubilizlng actlon wlll assure contact between antlmicroblal factors and the chemlcal components of the speclmen transport system 30 and thus assure that any pathogens contalned wlthln the blood sample 38 wlll be protected from antlmicroblal actlvlty.
After the blood sample 38 has been treated ln this manner, centrifugation artlcle 20 is centrifuged to cause the mlcroblal pathogens withln the treated blood sample 42 to pass out of suspension and collect ad~acent the interface of the high denslty cushloning agent 28 and the resldual of the sample fluid. Some microblal pathogens wlll actually be deposlted upon the sldewall of centrlfugation vessel 22 ad~acent the hlgh end of~
smooth surface 34 at polnt 22a. Thls centrlfugatlon step ls represented schematlcally by FIGURE 5. The speed and time of centrlfugatlon can vary widely depending upon the constructlon material of centrifugation artlcle 20 and the type of centrifugation apparatus. The centrifugatlon can be conveniently accompllshed by lmpartlng from between about lSoo to 6000 gravlties and preferably from about 1500 to 3000 75 202~3~3 gravities to the centrifugation artlcle 20 contalnlng the treated blood sample 42 and cushlonlng agent 28. As deplcted in FIGURE 5, an angle rotor centrlfuge ls employed whlch places the centrlfugation artlcle 20 at an angle of 56 for example, (deplcted by arc 43) durlng centrlfugatlon. Thus, lf smooth angled surface 34 ls at a 34 angle wlth respect to the lnterlor walls of centrlfugatlon artlcle 20, the treated blood sample 42 and cushlonlng agent 28 wlll be forced agalnst smooth angled surface 34 at a relatlvely perpendlcular angle durlng centrlfugatlon. It is noted that when a swlnglng bucket type of centrlfuge ls employed, centrlfugatlon artlcle 20 wlll be centrlfuged at substantlally 0~ wlth respect to a horlzontal surface. Thus, ln such a case, the angle of surface 34 wlll be approxlmately 90- and an ln~ectable rubber closure member havlng a flat lnner surface can be substltuted for ln~ectable closure member 26.
Once the centrlfugatlon step has been completed, centrlfugatlon artlcle 20 can be removed from the centrlfuge and the ma~or portlon of the treated blood sample 42 from whlch mlcroblal pathogens have been separated can be removed. It ls noted that, as used hereln, the term "resldual treated blood" or ~resldual blood n refers to a blood sample whlch has been centrlfuged such that the mlcroblal pathogens present thereln have collected at the bottom of the sample, hence, leavlng the ~resldual" portlon of the sample substantlally free of mlcroblal pathogens. Thls step ls deplcted ln FIGURE 6. To ald ln ease of removal, a vent needle 44 ln the form of a common hypodermlc needle wlth cotton ln lts hub, for example, ls ln~ected through in~ectable closure member 24. A second hypodermlc needle wlth syrlnge 45 attached can then be ln~ected through in~ectable closure member 26 to remove a ma~or portion of the resldual treated blood sample 42 from which mlcroblal pathogens have been separated. For example, when the centrlfugation vessel has a volume of from 12 to about 14 milllliters, a 1 1/2 inch 18 gauge needle can be employed to remove all but about 1.3 to 1.7 mllliliters of the treated blood sample 42. As shown, it is preferred that the ma~or portion of the residual blood sample to be wlthdrawn from the interior of centrifugation vessel 22 ls wlthdrawn at a polnt opposlte the sldewall ad~acent the upper bevel end of smooth surface 34 to avold dlsturblng the layer of mlcrobial pathogens whlch has formed on and wlthin the interface of the two liqulds and on the sldewall of centrifugatlon vessel 22 ad~acent the upper end of said beveled smooth surface 34. The ma~ority of the residual blood is removed ln this step; however, a small portion of the residual blood should be left in the centrifugation vessel 22 such that of the total fluid remalning, the cushioning agent comprises from about 3.3~ to about 40.0% by volume. It is preferred that no more than about 20% by volume shall be sald cushioning agent because greater quantltles of sald cushlonlng agent may deleterlously effect the morphology of 2s mlcroblal pathogen colonles ln subsequent pathogen growth steps used ln the process.
Once the ma~or portlon of the treated resldual blood sample has been removed, both needles may be withdrawn from ln~ectable closure members 24 and 26, and centrifugation article 20 is then sub~ected to a second mixing step depicted schematlcally by FIGURE 7.
However, if desired, vent needle 44 can be left in its positlon through in~ectable closure member 24 to assist in removal of the pathogen containing fluid in a later step. The second mixing step serves to resuspend microbial pathogens which have separated from the ma~or portion of residual treated blood sample 42 and whlch have formed the layer described above. Resuspension of the mlcrobial pathogens so collected in the remaining minor portion of the reslduaI treated blood sample 42 insures greater and more uniform recovery.
Once the mixing step has resuspended, the mlcroblal pathogens in a minor portlon of the residual treated blood sample 42, the mlxture of mlcroblal pathogens in the residual treated blood sample and the high density cushloning agent can be removed from centrlfugatlon article 20. Thls step ls depicted ln FIGURE 8. As noted above, if desired, the venting hypodermic needle 44 may be inserted through in~ectable closure member 24 to allow easler removal of the remainlng constituents.
The syrlnge 46 with attached hypodermic needle can then be in~ected through in~ectable closure member 26 to draw out the mlxture 48 of cushlonlng agent 28, minor remalnlng portion of resldual blood sample 42 and microbial pathogens present therein. It is noted that particularly good recovery can be obtalned lf the hypodermic needle used to remove these constituents is in~ected at the lower end of the angled smooth surface 34. It is believed that the angle of surface 34 acts, in part, as a funnel into which the remainlng fluld contalnlng the microbial pathogens flow. This mlxture 48 of hlgh denslty llquld cushlonlng agent 28, and the remalning mlnor portlon of the resldual treated blood sample 42 with the recovered microbial pathogens should be approximately 1 1/2 mllllllters of fluld. Thls fluld is then distributed on approprlate growth media. This step is then schematlcally illustrated in FIGURE 9 in 78 20293~3 the drawing. with the apparatus set forth above, the material can be distributed as follows:
Two blood agar plates can receive 0.4 mllllllters of the aqueous solutlon and can be lncubated at 36-C in an anaerobic envlronment. ~wo chocolate agar plates can receive 0.4 milliliters of the aqueous solutlon and can be lncubated at 36C ln a candle ~ar. The growth media should be checked dally for the presence of colonles.
Microbiological analysls technlques can be employed.
The number of microblal pathogens ln one mllllllter of the blood can be determlned by multlplylng the number of colonles by a correctlon factor. Thls correctlon factor takes lnto conslderatlon the recovery rate for a glven organlsm, the volumes of blood and high denslty cushionlng agent employed and the amount of flnal mlxture plated. In the general example set forth above, the correctlon factor ls 0.5.
The above procedure wlll result ln a dllutlon of the remalnlng mlnor portlon of the resldual treated blood sample 42 to at least about 1:60 on the growth medla. Thls wlll assure that any resldual quantlty of the chemlcals wlthln the speclmen transport system wlll be dlluted sufflclently so as to not lnhlblt the growth of mlcroblal pathogens therewlthln. The speclmen 2~ transport system of the sub~ect lnventlon wlll elther neutrallze or lnhlblt cldal drugs. For example, the sodlum polyanetholsulfonate wlll generally neutrallze and the cystelne wlll generally lnhlblt. Furthermore, the effect of oxygen on cystelne after removal of the sample from centrlfugatlon vessel 22 wlll destroy lts lnhlbltlng effect on mlcroorganlsms. The above descrlbed dllutlon procedure may be necessary to dllute drugs and/or component of the speclmen transport system to levels whlch are nelther cldal nor ~nhlbltory to the 79 2~129353 growth of mlcroorganlsms. In addition, for those antibiotics which may be present in the blood sample whlch exert only an lnhlbitory and not a cldal effect on mlcroorganlsms, the 1:60 dllutlon will generally prove adequate to reverse their lnhibitory effect on microorganisms. An example of this class of compound is gantrisin. Thus, in general, the 1:60 dllution will prevent the lnhlbitlng of growth for most mlcro-organisms/antlblotlc comblnatlons. Nevertheless, there are certain mlcroorganisms whlch are uniquely sensltive to the killing or inhibitory action of certaln classes of antiblotlcs. For example, lf one ls attemptlng the lsolatlon of a very sensltive strain of S. aureus (minimum lnhlbltory concentratlon of 0.2 mlcrograms per mllllllter) and the blood sample contained 20 micrograms per milllliter of antibiotic not blocked by sodium polyanetholsulfonate, para-aminobenzolc acld, or cysteine-thioglycolate, the organlsm would not grow on a conventional agar plate t20 milliliters of medla) ln accordance wlth the above-descrlbed method whlch normally deposits 0.4 mlcrograms per millillter of blood sample. This combination yields a flnal dllutlon of approxlmately 1:60. Thus, thls example would yield a flnal concentratlon of 0.33 mlcrograms per mllllliter of antibiotlc throughout the plate which would lndeed lnhibit the subsequent growth of the S. aureus straln.
Furthermore, the dllutlon of the antlblotic ls not instantaneous and lnitially the high levels of the antlbiotic on the surface of the agar plate might exert a lethal effect. To circumvent this problem and yet preserve the known advantages of the lysis-centrifugatlon technique improved with the novel speclmen transport system of the sub~ect inventlon, one further modiflcatlon is required: namely, a blg petri plate. Cllnlcal laboratorles concurrently use a 150 mm x 20 mm petrl plate for testlng antlblotlcs. Thls plate contains between 60 ml and 80 ml of medla and has 2.25 times the surface area of a conventional 100 mm x 20 mm petri plate. When one streaks the 0.4 ml blood sample uniformly on the surface of thls large plate, one achieves a 2.25 fold increase in the diffusion rate and a final dilution between about 1:200 to about 1:270. In the example used above, this will result in a final antlblotlc concentratlon of between 0.1 mlcrograms per mllllllter and 0.075 mlcrograms per mllllliter. When thls plate ls used, the flnal concentratlon of the antlblotlc ls well below the mlnlmum lnhibitory concentration and the organlsm should grow ln normal fashion. Thus, while the large plate need not be used ln each lnstance, it should be used when certaln fastldious organisms-antibiotlc comblnatlons are ¦ suspected, such as S. aureus-cephalothln.
Now referrlng to FIGURE 10, another embodlment of the sub~ect lnventlon ls deplcted whlch comprlses a device for collecting and transporting body secretlon samples. Devlce 100 comprlses an elongated flexlble tube 102 enclosed at one end 104 and open at lts opposlte end 106. Cap 108 encloses the open end.
Contained wlthin the tube near closed end 104 ls a crushable ampule 110 contalnlng a sultable llquld growth medla for microbial pathogens. Dlsposed ad~acent ampule 110 ls sorbent materlal 112 whlch can be any sultable sorbent such as cotton. Sorbent material 112 contalns dispersed therein speclmen transport system solids 114.
Disposed wlthln the open end of tube 106 ls swab member 116 which comprlses a handle 118 and an absorbent tlp 120 for recoverlng body secretlons from a leslon, for example.

Specimen transport system sollds 114 can be the same material dlsclosed for use ln the lysis-centrlfugatlon vessel descrlbed above and can be present ln the same relatlve quantltles based upon the amount of solids 114 and growth medla 110 and body secretlon collected on absorbent tlp 120 as the components descrlbed above ln relation to a blood sample. In operatlon, cap 106 ls removed and swab 116 ls removed from the lnterlor of tube 102. The swab contacts body secretlon from an open leslon, for example, and ls lnserted agaln withln the interior of tube 102 and cap 108 is placed over the open end 106 thereof.
Thereafter, the portlon of tube 102 ad~acent closed end 104 ls squeezed and ampule 110 is ruptured to cause the llquld growth to be released therefrom and saturate sorbent materlal 112 and solublllze speclmen transport system solids 114. The resultlng liquld contalnlng the dissolved speclmen transport system ls sorbed by the tlp of swab 120 and provldes a medla for sustalnlng mlcroblal pathogens present on the tlp and also an speclmen transport system for deactlvatlng antlmicroblal factors whlch mlght be present ln the body secretlons sorbed on the tip 120 of swab 116. The swab 116 ls later removed from contalner 102 and mlcroblologlcally 2~ analyzed ln a manner descrlbed above.
The followlng addltlonal examples are glven to ~
better facllltate the understandlng of thls lnventlon and are not lntended to llmlt the scope thereof. In Examples VII-XVI:
CFU - Colony-formlng unlts of a mlcroorganlsm/ml of blood lnltlally lnoculated lnto the tube. Seven and one-half ml of blood are processed per tube.

20293~3 % Recovery ~ Percentage of organism recovered in the gradient of all organisms found after processlng.
S-Factor ~ Survival lndex - Number of CFU
recovered from all contents ln tube/number of CFU lntroduced:
S - 1 means no klll; S - 0.1 means 10% survlval EXAMPLE VII
ACTION OF SODIUM POLYANETHOLSULFONATE
(SPS) ON GENTAMICIN
Tests were made comprlslng the orlglnal centrl-fugation artlcle dlsclosed and clalmed ln U.S. Patent 4,212,948. In the orlglnal verslon, each tube contalns lS 0.3 ml of FLUORINERT FC48 as cushlonlng agent and as a blood treatlng fluld 0.5 mllllllters of dlstllled water contalnlng O.005 mllllllters PPG, 0.008 grams of EDTA
and 0.0048 grams of SPS together wlth 0.018 grams of purifled saponln as a lyslng agent. The tube was sterlle wlth the aqueous solutlon havlng a pH of 7.4 and sufflclent vacuum to draw approxlmately 7.5 milllliters of human blood. A second type tube was prepared except sodlum polyanetholsulfonate was added to the aqueous solutlon ln an amount to equal 0.6% by welght of the flnal concentratlon of treatlng fluld and blood sample.
Next a serles of the above descrlbed orlglnal tubes and the orlglnal tubes plus the sodlum polyanetholsulfonate were tested by adding known quantltles of Staphylococcus aureus ln a blood sample contalnlng 6 micrograms per mllllllter of gentamlcln. Blood was lysed, the tubes were held at room temperature (approxlmately 72-F) for 2 hours prlor to centrlfugation to slmulate cllnlcal condltlons. Thereafter the tubes were centrlfuged as descrlbed above and the concentrated material plated on growth media and tested for recovery. The results are set forth below.

Table VII-l Staphylococcus aureus (ATCC 25923) Gentamicln ~6 ug/ml) S~ CFU RECOVERY FACTOR
Original 133 100 .06 Original + 0. 6~ SPS 203 80 .9 The orlglnal tube gave an overall recovery of 6%
while the SPS system gave a recovery of 72~ (11.0 fold improvement).

EXAMPLE VIII
DEACTIVATION OF AMPICILLIN BY THIOGLYCOLATE
Thls example was carried out in the same fashion as Example VI except the stated quantltles of thloglycolate were added to the second and thlrd serles of tubes.

Table VIII-l Staphylococcus aureus ~ATCC 25923) Amplclllln (21 ug/ml) S ~
SYSTEM CFU RECOVERY FACTOR
Orlglnal 196 33 .002 Origlnal + l~ thioglycolate* 490 89 .040 Original + 6~ thioglycolate 466 89 .13 ~Amount of thioglycolate based upon treating fluld and blood sample.
The orlglnal tube gave an overall recovery of 0.07%
recovery versus 12.5~ for the 6% thloglycolate system--a 179 fold lmprovement.

EXAMPLE IX
DEACTIVATION OF AMPICILLIN AND GENTAMICIN
BY A NOVEL CYSTEINE-THIOGLYCOLATE COMBINATION
The serles of runs set forth below were carried out in the same fashlon as Example VII above except with the quantlty and amount of antlblotlc and the stated quantltles of thloglycolate-cystelne whlch were added to the liquid blood treating material.

Table IX-l ! 10 Staphylococcus aureus (ATCC 25923) Ampiclllln (21 ug/ml) S
SYSTEM CFURECOVERY FACTOR
Original 196 33 .002 Orlginal ~ 0.5% thioglycolate* 245 98 0.80 + 0.2% cystelne*
Original + 0.1% thloglycolate* 696 99 1.1 + 1.2% cystelne*
*Amounts based upon total quantlty of treating fluids and blood sample.

Table IX-2 Staphylococcus aureus (ATCC 25923) Gentamlcln (6 ug/ml) S`
SYSTEM CFURECOVERY FACTOR
Orlglnal 133100 .1 Orlginal + .5% thloglycolate* 203 100 .2 Orlglnal + .5% thloglycolate* 287 86 .8 + .2% cysteine*
~Amounts based upon total quantity of treatlng fluld and blood sample.

20293~3 The comparative data ln Tables IX-l and IX-2 above clearly demonstrate the use of the th~oglycolate-cysteine combination.

EXAMPLE X
SYNERGISTIC ACTlON OF THIOGLYCOLATE-CYSTEINE
MIXTURE IN LOWERING VISCOSITY OF LYSED HUMAN BLOOD
In each instance, 7.5 millillters of human blood was treated with an aqueous solution contalning 2.5% by weight purified saponin and quantities, if any, of 10thioglycolate and cysteine as illustrated in the table (based upon the total quantity of treating fluid and blood). The viscosity of each sample was measured at the temperature between 23.5 and 24.8-C. The results are set forth below:

Table X-l TREATMENT VISCOSITY
(Saponin--2.5%) (Centistokes) 1. Saponin xl 4.04 2. nn + O .1% thioglycolate 7.28 3. nn + o . 5% n 7.77 4. nn + 1 . 0% n 8.56 5. nn + 2.0% n 8.51 6. nn + 3.0% n 8.46 7. nn + O .1% cystelne 4.56 8. nn + o . 5% n 3.46 9 . nn + 1 . 0% n 2.89 10 . nn + 1% thloglycolate + 5.14 0.1% cystelne 11 . nn + 1% thloglycolate + 4.30 0.5% cystelne 12. nn + 1% thloglycolate + 3.75 1.0% cystelne 13. nn + 1% thloglycolate + 3.43 2.0% cysteine 14. nn + 3% thioglycolate + 6.28 o.l~ cysteine 15. n~ + 3% thioglycolate + 4.58 0.5% cystelne I ~ . Nn + 3% thioglycolate + 3.44 1.0% cysteine 17 . nn ~ 3% thloglycolate + 3.82 1.5% cystelne Temperature of samples were between 23.5C - 24.8-C

EXAMPLE XI
EFFECT OF SPECIMEN TRANSPORT SYSTEM IN IMPROVING
BLOOD SPECIMEN MICROBIAL INTEGRITY
The data in the following tables lllustrate the followlng important aspects of the lnventlon, namely:
1. In the presence of average serum levels of dlfferent antlblotlcs the orlglnal system can lose up to 99.7% of the origlnal lnoculum Staphylococcus aureus wlth amplcillin). For S. aurels 13 of 19 antibiotics killed 50% or more of organlsm within two hours. For Escherichia coll thls excesslve cldal actlon occurred wlth nine of the antlblotlcs. (See Tables XI-l and XI-2).
2. Wlth the new system, the hlghest klll rate was 70% and a reductlon of the inoculum to 50% or less occurred with two antlbiotlcs for S.
aureus and two wlth E. coll. By addlng large plates to the new devlce, the cidal effect observed in these four cases can be vlrtually ellminated ~S - .8 versus .3; S - .9 versus ! . 5; S ~ .8 versus .5 and S - .S versus .3, where 5 e 1.00 ~ 100% survlval).
In summary, the new system ln con~unctlon wlth effectlve dllutlon (l.e. the use of large petrl plates) ls capable of effectlvely blocklng the cldal actlon of blood and therapeutic antlblotlcs upon the bacterla 20293~

present in a blood sample durlng transport and processing.
The data presented in Tables XI-3 through XI-10 below confirm the general effectlveness of thls inventlon on other pathogens commonly isolated from the blood of patlents sufferlng from septlcemla.
The procedure set forth below was followed for each pathogen, using various antibiotlcs. Concentrated residue from each tube was plated on both small and large plates to generate the data lllustrated.
A serles of orlglnal lysls-centrlfugatlon devlces were assembled as descrlbed ln Example VII. A second serles of lysls-centrlfugatlon devices were assembled as ln Example VII wlth the exception that the aqueous phase was modlfied as follows:
0.5 millillters of dlstllled water containing 0.005 mllllllters of polypropylene glycol was placed lnto the tubes.
A total of 0.17 grams of powdered mlxture was then added to each tube. The mlxture contalned the following components:
1.8 grams of purlfied saponln, 4.8 grams of sodlum polyanetholsulfonate, 0.8 grams of thloglycolate, and 9.6 grams of cystelne.
The tubes were sterlllzed by autoclaving and had a final pH of between 6.6 and 6.8.
Sufflcient vacuum was placed in the tube to draw 7.5 milliliters of blood.
In each instance, the stated amount of specific mlcroorganlsm as illustrated in tables below and antiblotlc was added to 7.5 mllllllters of blood. The blood was then deposited into the lysis-centrifugation tube and the tube was held at room temperature for 2 hours to slmulate clinlcal condltlons, and thereafter was sub~ected to centrlfugatlon as descrlbed ln this speciflcatlon. The concentrated resldue ln each tube was then plated ln equal allquots on five agar plates contalnlng appropriate growth medla whlch had dlmenslons of 100 milllllters x 20 mllllliters and growth was observed. One milllllter of the supernatant remalnlng after centrlfugatlon was also plated on the flve plates.

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20293~3 The above experlments were repeated except instead of the 100 milliliter by 20 milllliter petri plates containlng media, the concentrated resldue from each tu~e was plated on a 150 milliliter by 20 mllllllter petrl plate whlch contalns between 60 mllllllters and 80 milllllters of medla and had approxlmately 2.25 tlmes the surface area of 100 mllllllter by 20 mllllllter petrl plate descrlbed whlch were used to generate the data ln Table XI-l above. The results are set forth ln Table XI-2 below.

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n o ~ o _ 94 2029353 Table XI-3 ENTEROBACTER CLOACAE
#1344-2 - SMALL PLATES
% Recovery S-Factor OldNew Old New Ampicillin 99.595 1.5 + .5 .8 + .1 Carbenicillin 0 94 0 .6 + .3 Ticarcillin 100 86 .06 + .071.3 + .6 Tobramycin 100 75 .03 + .01.9 + .2 Chloramphenicol 98 80 .9 + .21.2 + .3 Tetracycline 98 88 .9 + .51.4 + .7 Gantrisin 97 99 .6 + .1.8 + .1 No Drug 97 98 1.4 + .21.2 + .1 Cefoxitin 95 97 .9 + .2.8 + .2 Cephalothin 99.688 1.0 + .2.9 + .1 Gentamicin 95 99 .04 + .031.1 + .1 LARGE PLATES
% Recovery S-Factor Old New Old New Tetracycline 97 96.7 + .1 .9 .2 Tobramycin 98 93.9 + .5 1.1 .2 Chloramphenicol + 97 + .9 .3 + Not tested because recovery is good on small plates.
Table XI-4 KLEBSIELLA PNEUMONIAE
#632-2 - SMALL PLATES
% Recovery S-Factor Old New Old New Ampicillin 97 93 .5 + .31.0 + .1 Carbenicillin 93 94 .1 + .1.8 + .2 Ticarcillin 99 89 1.0 + .1.9 + .1 Tobramycin 85 93 .3 + .31.1 + .2 Chloramphenicol99 93 1.3 + .21.0 + .4 Tetracycline 98 95 1.0 + .1.9 + .2 Gantrisin 95 98 1.0 + .1.9 + .1 Cefoxitin 49 97 .02 + .021.0 + .1 No Drug 92 93 1.1 + .3.7 + .1 Cephalothin 100 98 .2 + .1.5 + .2 Gentamicin 90 99 .02 + .01.9 + .2 ~i `
~. ~

2`0293~3 LARGE PLATES
% Recovery S-Factor Old New Old New Carbenicillin 92 88 .5 + .3 .7 .2 Table XI-5 PSEUDOMONAS AERUGINOSA
#27853 - SMALL PLATES
% Recovery S-Factor Old New Old New Ampicillin 97 94 .6 + .4.8 + .2 Carbenicillin 98 95 .9 + .3.9 + .1 Ticarcillin 93 91 .3 + .11.2 + .1 Tobramycin 98 90 1.0 i .1.9 + .2 Chloramphenicol 96 86 .7 + .21.1 + .2 Tetracycline 95 89 1.0 + .11.2 + .1 Gantrisin 99 98 1.2 + .2.9 + .1 No Drug 97 97 1.6 + .3.9 + .2 Cefotaxime 99 96 .9 + .21.1 + .3 Cefoxitin 97 86 1.4 + .2.9 + .4 Cephalothin 90 92 1.4 + .11.4 + .01 Gentamicin 98 56 1.0 + .41.2 + .2 Moxalactam 97 87 .6 + .1.9 + .1 Table XI-6 STREPTOCOCCUS PNEUMONIAE
#6301 - SMALL PLATES
% Recovery S-Factor Old New Old New Penicillin 76 63 .02 + .16.8 + .2 Ampicillin 43 65 .01 + .01.6 i .2 Methicillin 83 86 .003 + .004.4 + .2 Tobramycin 97 88 .8 + .4.6 + .4 Chloramphenicol99 93 .6 + .4.8 + .2 Tetracycline 100 99 .3 + .2.4 + .2 Erythromycin 97 98 .3 + .31.3 + .3 Cefoxitin 97 99 .4 + .1.5 + .2 No Drug 93 90 1.0 + .031.0 + .1 Gentamicin 99.8 100 1.0 + .11.1 + .1 *Incubation period -- 48 hours.

_ 96 20293~3 LARGE PLATES
% Recovery S-Factor Old New Old New Tetracycline 99 100 .5 _ .2.7 _ .2 Tobramycin 98 99 1.0 _ .31.1 + .2 Ampicillin + 82 + .9 _ .3 Cefoxitin 100 99 .2 + .1.8 _ .1 Methicillin + 97 + .6 _ .1 Penicillin + 63 .9 _ .1 + Not tested because recovery is good on small plates.

Table XI-7 STREPTOCOCCUS PYOGENES
#19615 - SMALL PLATES

% Recovery S-Factor Old New Old - New Penicillin 0.2 100 .02 _ .02.6 _ .2 Ampicillin 0 99 .0002 _ .0003.6 _ .1 Methicillin 95 90 .2 _ .1.8 _ .2 Tobramycin 98 100 .6 _ .1.5 _ .2 Chloramphenicol 98 97 .5 _ .1 .4 _ .2 Tetracycline 100 96 .3 + .1.1 _ .1 Erythromycin 100 100 .02 i .01.02 _ .01 Cefoxitin 98 100 .3 _ .1 .2 _ .03 No Drug 92 95 1.0 _ .5 .5 _ .1 Gentamicin 99 94 .6 _ .1 .9 + .1 LARGE PLATES
% Recovery S-Factor Old New Old New Methicillin 99 99.8.6 + .1 1.7 + .2 Tobramycin 99 1001.0 + .1 1.1 + .3 Chloramphenicol 99 99 .8 _ .3 .9 _ .3 Tetracycline 99.6 100.6 _ .1 .7 + .3 Erythromycin 100 100.1 _ .1 .1 _ .1 Cefoxitin 98 95.6 + .1 .7 _ .2 No Drug 98 97.8 + .1 1.0 _ .2 Ampicillin + 100 + 1.5 + .2 Gentamicin + 99.5 + 1.2 + .3 y Table XI-8 HAEMOPHILUS INFLUENZAE
#19418 - SMALL PLATES
% Recovery S-Factor Old New Old New No Drug 89 78 .6 + .2 .9 + .4 Ampicillin 33 95 .01 + .01.9 + .1 Cefoxitin 80 97 .l + .l .7 + .2 Clindamycin 96 99 1.2 + .2l.l + .2 Erythromycin 94 100 .4 + .l.7 + .2 Gentamicin 90 77 .4 + .l1.3 + .4 Kanamycin 94 99 .8 + .1.9 + .l Methicillin 94 99 .9 + .2.8 + .1 Penicillin 73 99 .1 + .1.6 + .l Tetracycline100 99 .2 + .1.6 + .1 Vancomycin 95 99 .7 + .2.8 + .2 LARGE PLATES
% Recovery S-Factor Old New Old New No Drug 95 99 .9 + .11.2 + .l Cefoxitin 93 95 .8 + .6.7 + .2 Gantrisin 92 98 1.2 + .7.6 + .l Penicillin 100 99 .3 + .2.6 + .2 Table XI-9 BACTEROIDES FRAGILIS
#23745 - SMALL PLATES
% Recovery S-Factor Old New Old New No Drug 88 51 .7 + .21.0 + .3 Carbenicillin96 82 .09 + .05.5 + .l Cefotaxime 97 100 .7 + .2.8 + .1 Cefoxitin 94 99 .5 + .3l.l + .5 Chloramphenicol 87 88 .9 + .2 .9 + .1 Erythromycin 98 64 .7 + .5.6 + .2 Penicillin 87 51 .7 + .1.7 + .1 Tetracycline 90 90 .5 + .1.5 i .1 Vancomycin 95 99 .7 + .2.8 + .2 CLOSTRIDIUM SPOROGENES
#19404 - SMALL PLATES
% Recovery S-Factor Old New Old New No Drug 97 93 .6 + .1 .6 .2 Carbenicillin 98 99 .3 _ .3 .8 _ .3 Cefotaxime 97 98 .5 _ .2 .5 _ .2 Chloramphenicol 96 97 .7 i .4 .6 _ .4 Clindamycin 99.7100 .4 + .2 .3 _ .2 Erythromycin 96 99 .5 + .2 .8 _ .2 Gantrisin 93 99 .5 _ .1 .7 _ .1 Gentamicin 98 98 .8 + .2 .6 + .4 Penicillin 100 96 .08 + .04 .8 + .3 Once again a new cocktail protected the microorganisms from the cidal effect of the antibiotics. As expected for those antibiotics which do not exert a cidal effect, both the original tube and the modified tube containing the specimen transport system yielded the same actual recovery of microorganisms. A large dilution is apparently needed (1:267) when dealing with a few specific organism-antibiotic combinations, e.g., S. aureus with cephalothin. These data suggest that large dilutions will only be required for a few antibiotics, e.g., cephalothin, tetracycline, erythromycin, and certain organisms, e.g., +cocci. The aminoglycosides, penicillin, ampicillin, and chloramphenicol are completely neutralized by the cocktail while the cephalothins are partially neutralized.

EXAMPLE XII
A series of the original tubes as described in Example XI and the tubes containing the specimen transport system as described in Example XI were ., 20293~3 utllized to process blood from patients suspected of havlng septicemia with confirmed positive blood cultures. In each case, blood from the patient was placed in the original tube and the modified tube containing the specimen transport system. The tubes were centrifuged and the concentrated residue plated on the small petri plates described in Example XI. The results of the tests are set forth in Table XII-1 below.

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20293~

CONCLUS IONS:
1. In 68% of the positlve samples the new tube ylelded more organisms/ml of blood. The dlfference ranged between a low of 5% and a hlgh of 614% lncreased count.
2. The new system mlssed three posltlves whlle the orlglnal system mlssed flve.
3. In four cases the orlglnal system gave a hlgher count. However, this level ls well withln expected experimental variabillty.
4. Thirty-six percent (36%) of the patlents were not on antlblotlcs at the time of blood collection.
The new system yielded higher counts ln 50% of the cases. The dlfference ranged from a low of 134%
and a hlgh of 614% lncrease.
5. Seventeen (17) cultures were slmultaneously posltlve at the same time. Two cultures (one Llsterla and one Escherlchla coll) were posltive one day earlier in the new system.

As shown in the table, ln 68% of the samples, the modlfled devlce contalnlng the speclmen transport system ylelded hlgher counts (whlch ranged between 5% and 614%
lncrease) than dld the orlglnal devlce. In flve instances, the orlglnal devlce was negative and the new devlce posltlve. Although the ma~orlty of samples were posltlve at the same tlme, there were two cases ln which the new devlce detected a positlve culture one day earller (E. coll and one Llsterla specimen).
Surprislngly, the new devlce appears to yield greater counts even when the patlent was not on antlbiotics (3 patients). Thls lndlcates that the new device contain~ng the specimen transport system more effectlvely blocked the patient's lmmune system than dld 202935~

the llquid blood treatlng solution of the orlginal device.

EXAMPLE XIII
A first serles of orlglnal lysis-centrlfugatlon devlces were assembled as descrlbed ln Example VII. A
second series of lysls-centrifugatlon devlces were assembled the same as the second serles of such devlces ln Example 5. A thlrd serles of lysls-centrlfugatlon devlces were assembled as follows:
To the artlcle as dlsclosed ln U.S. Patent 4,212,948 were added 0.3 mllliliters of FLUORINERT FC48 as cushlonlng agent along wlth the followlng compounds ln dry partlculate powder form:
0.008 grams of thloglycolate;
0.048 grams of sodlum polyanetholsulfonate; and 0.018 grams of purlfled saponln.
The tubes in the third serles were evacuated sufflclent to draw 8 mllllliters of blood. Thls serles of tubes was then heated to 121-C for 30 minutes and then allowed to cool to room temperature.
In each lnstance, the stated amount of speclflc mlcroorganlsms and antlblotlcs (lf any) as illustrated in Tables XII-l through XII-6 below was added to 7.5 milllllters of blood ln the flrst and second serles of tubes and 8 mlllillters of blood ln the thlrd serles of tubes. The blood was then deposlted into the respectlve lysls-centrlfugation tube and each tube was sub~ected to centrlfugatlon as descrlbed in thls speclflcatlon. Llke quantltles of each mlcroblal pathogen-antlblotlc comblnatlon were plated on both large and small petrl plates as descrlbed ln Example XI. The results are set forth ln Tables XIII-l through XIII-6 below:

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As can be seen from the data above, the lysis-centrifugatlon devices made ln accordance with the sub~ect invention that contaln the dry partlculate powdered specimen transport system of the sub~ect invention performed at least as well as the systems ln accordance with this inventlon containlng the specimen transport system in aqueous solutlon within the tube.
Both of the new systems clearly outperform the original system as set forth in the Examples.

lo EXAMPLE XIV
INCREASING HOLD TIME FOR BLOOD SPECIMENS
A flrst series of orlglnal lysis-centrlfugation devlces were assembled as described ln Example VII. A
second series of lysls-centrlfugatlon devices were assembled ln the same manner as the third series of lysis-centrifugatlon devlces whlch were used to obtain the data set forth ln Tables XIII-l through XIII-6 of Example XIII.
In each lnstance, the stated amount of speclfic microorganlsm as lllustrated ln Table XIV-l below was added to 7.5 ml. of blood ln the first serles of tubes and to 8 ml. of blood ln the second serles of tubes.
The blood was then deposlted ln the respectlve lysls-centrlfugatlon tubes and the tubes were then held at 21-C for the tlme perlod set forth ln Table XIV-l below. Each tube was next sub~ected to centrlfugatlon and the concentrated contents plated on growth medla as descrlbed ln the speclficatlon.
As can be seen from the data set forth ln Table XIv-l, certain specles of bacteria propagate or dle ln the origlnal tube when held for a perlod of 24 hours.
Surprlsingly, these same specles did not substantlally grow or die in the second series of new tubes contalning the specimen transport system. While it is not recommended that the centrlfugation tubes be held for lengthy periods of tlme, lt has been found ln the hospital envlronment that such tubes are held for tlme periods before processlng. Whlle the data shows no substantlal propagation of most specles wlthin the new tube at 24 hours, lt ls belleved that the tubes should be processed as qulckly as possible and certalnly before a hold tlme of 12 hours has been completed.
Furthermore, to assure agalnst growth of some specles of bacterla such as Enterobacter cloacae, sodlum chlorlde can be added, such as ln the urlne examples as set forth ln Example XVII below. Sodlum chlorlde can be present ln an amount from about 0.1% to about 10% by welght of the final process treatlng solutlon and blood sample, and preferably in the range of from about 1% to about 5%
and most preferably at about 3%.

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, Lr) o Lr) o lOg 2029353 EXAMPLE XV
USE OF AN ENZYME COMPONENT
A series of lysls-centrlfugatlon devlces were assembled the same as the second serles of devlces contalnlng the specimen transport system as ln Example XIV. To each tube was added 8 ml. of blood contalning 842 CFU of E. coll and 20 ug/ml. of the antlblotlc cefotaxlme as well as the stated amount of beta-lactamase enzyme lllustrated ln Table XV-l below.
The beta-lactamase enzyme used was beta-lactamase (~aclllus cereus), lot No. 203435, order No. 426205, Calblochem-Behrlng Corporatlon, La Jolla, Callfornla, NOTE: beta-lactamase I - 13 unlts of actlvlty to beta-lactamase II - 1 unlt of actlvlty. The blood was then deposlted lnto the respectlve lysls-centrlfugatlon tubes and each tube was sub~ected to centrlfugatlon as described in the speciflcatlon. Llke quantltles of mlcroblal pathogen-antlblotlc-enzyme comblnatlon were plated on small petrl plates as descrlbed ln Example XI.
The results are set forth ln Table XV-l below.

Table XV-l E. coll - Cefotaxime 20 ug/ml.
Unlts Of Percent Enzyme Recovery S-Factor 0 58 .02 . 01 100 . 011 0.1 100 .052 1.0 99 33 2.0 99 1.17 99 .82 5.0 99.8 .95 As can be seen from Table XV-l the beta-lactamase as an lntegral part of the specimen transport system wlll effectlvely functlon to block the actlvity of the antlbiotic and prevent kllllng of the mlcroblal pathogen whlle contalned wlthin the lysls-centrlfugation tube.
As a comparlson, a second serles of tubes were assembled as descrlbed ln Example 1 and to each tube was added 765 CFU of E. coll, 20 ug/ml. of cefotaxlme, the unlts of beta-lactamase enzyme as lllustrated ln Table XV-2 and 7.5 ml. of blood. The tubes were then centrlfuged and samples were cultured as descrlbed above, and the results are set forth ln Table XV-2 below.

Table XV-2 Unlts Of Percent Enzyme Recovery S-Factor 0 93 .08 1 90 .04 .155 The results of Table XV-2 when compared wlth Table XV-l lndlcate that the addltlon of the enzyme does not satlsfactorlly lmprove the S values when used ln a lysls-centrlfugatlon tube whlch does not contaln the speclmen transport system.
Further tests were made comparlng a flrst serles of lysls-centrlfugatlon tubes ldentlcal to those prepared ln con~unctlon wlth Table Xv-2 above and contalnlng no speclmen transport system; a second serles of lysls-centrlfugatlon tubes ldentlcal to those used ln con~unctlon with Table XV-l above but contalnlng no enzyme; and a thlrd serles of lysls-centrlfugatlon tubes 111 202~3S~
whlch were the same as the second series of tubes but whlch contained the indlcated amounts of beta-lactamase enzyme as set forth in Tables Xv-3 through Xv-8 below.
The blood containing between 200 and 1000 CFU of the lndlcated bacteria was added to each tube and the tubes were processed as described above in this example and the results are set forth in Tables XV-3 through XV-8 below:

Table XV-3 Escherichia coli 25922 Second First Series Third Series Series of Tubes of Tubes Antibiotic ug/ml of Tubes No EnzymeWith Enzyme Units .01 .1 1. 2. 2.5 3. 4. 5. 10.

Cephalothin 20 .04 .05 .40 .90 1.30 Cefamandole 20 .35 .35 1.0 .63 0 Cefoxitin 25 .27 .76 .91 1.76 Cefotaxime 20 .08 .02.01 .05 .33 1.2 .82 .95 Moxalactam 100 .02 .01 .03 .05 .004 .04 .002 Moxalactam 50 .03 .04 .05 Moxalactam 40 .01 .10 .22 .12 .16 Moxalactam 20 .33 .20 .16 .65 Moxalactam 10 .84 1.01 .92 Cefo~ .O01 .003 .84 l.Ol o C~
~r,~

~i ;i Table XV-4 Staphylococcus aureus 25923 Second First Series Third Series Series of Tubes of Tubes Antibiotic ug/ml of Tubes No EnzymeWith Enzyme Units .01 .1 1. 2.2.5 3. 4. 5. 10.

Cephalothln 20 .08 .04 .03 .002 .50 .85 1.65 0 Cefamandole 20 .006 .03 .81 .62 .81 Cefotaxime 20 .28 .52 .96 1.02 Cefob 50 .009 .025 .72 .80 STS = Specimen Transport System o '~2 Table XV-5 Rlebsiella pneumoniae 632-2 Second First Series Third Series Series of Tubes of Tubes Antibiotic ug/ml of Tubes No Enzyme With Enzyme Units .01 .1 1. 2. 2.5 3. 4. 5. 10.
Cefotaxime 20 0 0 .03 .58 Moxalactam 20 - - .23 .22 0 Table XV-6 Enterobacter cloacae 1344-2 Second First Series Third Series Series of Tubes of Tubes Antibiotic ug/ml of Tubes No Enzyme With Enzyme Units .01 .1 1. 2. 2.5 3. 4. 5. 10.
Cefotaxime 20 .12 .013 .79 .52 Moxalactam 20 - - .12 .26 STS = Specimen Transport System 2 CV
CJ~

Table XV-7 Haemophilos influenza 19418 Second First SeriesThird Series Series of Tubes of Tubes Antibiotic uq/ml of Tubes No Enzyme With Enzyme Units .01 .1 1. 2. 2.5 3. 4. 5. 10.
Cefotaxime 20 .003 .001 .153 .46 Moxalactam 40 - - .005 .004 0 Table XV-8 Streptococcus pneumoniae 6301 Second First Series Third Series Series of Tubes of Tubes Antibiotic ug/ml of Tubes No Enzyme With Enzyme Units .01 .1 1. 2. 2.5 3. 4. 5. 10.
Cefoxitin 25 .39 .28 .33 .18 Cefotaxime 20 .001 0 .23 .62 o STS = Specimen Transport System c~

Cl~

116 20293~3 The data shown ln Tables xv-3 - xv-8 show that the addition of the enzyme to the specimen transport factor system of the sub~ect lnventlon effectlvely enhances the neutralization properties thereof for the above indicated class of antibiotics.

EXAMPLE XVI
STERILIZATION OF ENZYME-CONTAINING SPECIMEN
TRANSPORT SYSTEM IN A SPECIALIZED APPARATUS
Lysis-centrifugation tubes containing the antibiotic deactivation system utilized in the second series as set forth in Tables.XV-3 through XV-8 above were made up. To a first serles of these tubes was added the amount of beta-lactamase enzyme set forth in Table XVI-l below. The tubes were then sub~ected to cobalt sterilization and thereafter 8 ml. of blood containing the microbial pathogen and the antibiotic as set forth in Table XVI-l were added thereto and processed as set forth in Example XV. A second series of the tubes were steam sterilized and thereafter the indicated amount of beta-lactamase enzyme was added thereto and thereafter the 8 ml. of blood with the lndicated amount of microbial pathogen and antibiotic was added thereto and the tubes were centrifuged and processed as set forth ln Example XV. The results of these tests were set forth ln Tables XVI-l and XVI-3 below.

Table XVI-l COBALT STERILIZATION
E. coll 25922 Staph. aureus 25923 cefotaxlme 20 ug/ml cephalothln 20 ug/ml Unlts of Percent Percent Enzyme Recovery S-Factor Recovery S-Factor 0.1 67 .01 100 .04 1.0 83 .06 97 .17 5.0 81 .41 97 .33 Table XVI-2 STEAM AUTOCLAVE STERILIZATION
E. coll 25922 Staph. aureus 25923 cefotaxlme 20 ug/ml cephalothln 20 ug/ml Unlts of Percent Percent Enzyme Recovery S-Factor Recovery S-Factor 0.1 100 .05 17 .002 1.0 99 .33 26 .50 5.0 99.8 .95 34 1.65 As can be seen by a comparlson of the data ln Table XvI-l wlth Table XVI-2, the loss of enzyme actlvlty due to cobalt sterlllzatlon ranges from 20~ to 80~, dependlng on the concentratlon of the enzyme.
However, Table XVI-l clearly lllustrates that cobalt sterlllzatlon can be effectlvely utlllzed, and when used, lncreased amounts of the enzyme should be added to the tube prlor to the sterlllzatlon. It should be noted that other chemlcals are antlclpated for use wlthln the specimen transport system dependlng somewhat on the type of antlmlcroblal factors whlch are antlclpated to be present ln the sample. For example, other water-soluble compounds whlch are antagonlstlc to other classes of antlmlcroblal substances such as sodlum ll8 202~35~
hypochlorlte, heavy metals and the like include substances like sodium bisulfite and sulfhydryls, for example. As indicated, the specimen transport system of the subject invention finds speclal utility in the lysis-centrifugation tube such as set forth in U.S.
4,131,512 and U.S. 4,212,948. In addltion, the specimen transport system finds utility in the lysis-centrifugation tube as sbt forth in U.S. 4,164,449. In addltion, the speclmen transport system of the sub~ect invention can be utillzed in a blood treating tube for neonates which simply will lnclude a standard slngle stopper vacuum tube designed to draw between 1 and 2 milliliters of blood. The tube would contain no substance other than the specimen transport system of 1~ the sub~ect lnvention and saponln if deslred. The blood can be treated upon in~ection in the tube and then directly plated upon growth medla.
The above examples illustrate the beneflclal effect of the speclmen transport system of the sub~ect invention when used in a lysls-centrlfugatlon tube for analyzlng mlcroblal pathogens within blood samples.
However, the specimen transport system of the present lnventlon flnds utillty ln protectlng mlcroorganlsms ln sample fluids other than blood which are collected and 2~ later analyzed for the presence of microbial pathogens.
For example, the specimen transport system of the sub~ect invention can protect mlcroorganisms present in swabs, urlne, sputum, splnal fluid and other body flulds during translt. It ls well known that these flulds also contain both humoral and chemlcal antimlcrobials (if the patient is belng treated with antlblotlcs). Wlth urlne samples, the concentratlon of antibiotlcs may actually exceed that present ln serum. An example of a modlfled speclmen transport system for neutrallzing antibiotlcs in urine is present ln Example XVII below.

EXAMPLE XVII
MAINTAINING THE MICROBIAL INTEGRITY
OF A URINE SPECIMEN
The following example was performed to test the ability of the specimen transport system urine cocktail to block conventlonal therapeutic antiblotics and hold the microblal population, present ln the urine, stable for up to 24 hours.
The followlng dry mlxture was placed ln each of a serles of tubes:
0.03 grams of sodium polyanethosulfonate 0.005 grams of thloglycolate-lS 0.1 grams of ICN free-base cystelne 0.1 grams of sodlum blcarbonate.
The various antlbiotlcs, llsted ln Tables XVII-l -XVII-6 below, were added, at the concentratlons also speclfled thereln, to the tubes contalning the above-described specimen transport system urlne cocktail and to an equal number of tubes without the urlne cocktall. Flve mllllllters of sterlle urine was then added to all tubes after whlch the tubes were vlgorously mlxed. Control tubes contalned elther urine alone or urlne plus the above-descrlbed speclmen transport system urlne cocktall. No antlblotlcs were added to control tubes. The mlcroorganlsms llsted ln Tables Xv~
XVII-6 below were ad~usted to a McFarlln of 0.5 and then dlluted 1:100 wlth sterlle culture medla. A 0.1 mllllllter allquot of a slngle mlcroorganlsm was added to each urlne contalnlng tube and the resultant mlxture vlgorously agltated. A ten microliter allquot from each tube contalnlng the resultant mlxture was immedlately 20293~3 inoculated on tryptic soy agar plates and spread with a sterile spreader. The inoculated plates were incubated overnight in an envlronment and temperature approprlate for the microorganism employed. The tubes were then allowed to stand at room temperature for 24 hours.
Additional ten microliter aliquots were plated as before herein described at the time intervals indicated in Tables XVII-l - XVII-6 below. All plating was done in quadrupllcate and the S-Factor recorded as an average of the quadruplicate plating in Tables XVII-l - XVII- 6 below.

Table XVII-l Escherichia coli 25922 Hour Tlme Polnts Antlblotlc** 2 4 6 24 *No Drug -- .92 1.05 .84 No Drug -- 2.01 TNTC TNTC
*Amlkacln (210) -- .73 .62 .33 20Amlkacln (210) -- 0 0 0 *Amplcillln (210) -- .92 .75 .70 Amplclllln (210) -- .02 .001 0 *Carbenlclllin (200) 1.12.90 -- .31 Carbeniclllln (200) .83.37 -- .003 25*Cefamandole (200) .88.45 -- .31 Cefamandole (200) .37.33 -- .005 *Cefobid (500) --1.16 1.63 1.68 Cefobld (500) -- .03 0 0 ~Cefotaxlme (200) --1.03 1.55 1.65 30Cefotaxime (200) --.009 0 0 *Cefoxitin (250) .72.33 -- .17 Cefoxltln (250) .23.05 -- .002 20~9353 ~Cephalothln (200) -- .47 .86 .53 Cephalothln (200) -- .01 .01 .04 *Chloramphenlcol (180) -- 1.03 1.03 .89 Chloramphenlcol (180) -- 0 0 o 5*Erythromycin (80) -- .70 .75 .59 Erythromycln (80) -- 1.36 1.49 .42 *Gantrisin (1000) .70.93 -- .56 Gantrlsin (1000) 0 0 -- 0 *Gentamlcin (60) -- .81 .38 .28 10Gentamicin (60) -- 0 0 0 *Piperacillin (600) .76.67 -- .33 Plperaclllin (600) .48.SS -- .01 *Tetracycline (9o) 1.121.16 -- .56 Tetracycline (90) .20.02 -- 0 15*Tobramycin (40) -- 1.05 1.06 .90 Tobramycin (40) -- 0 0 0 -- Time point not lncluded ln reconstructlon.
! * Speclmen transport system urlne cocktall ls present.
** Number ln parenthesls represents flnal con-! centratlon (ugJml) of antlblotlc ln urlne TNTC - too numerous to count Table XVII-2 Klebslella pneumonlae Hour Tlme Polnts Antlblotlc** 4 6 24 *No Drug 1.10 1.31 1.08 No Drug 1.00 3.7- TNTC
TNTC
30*Amlkacln (210) .86 .64 .27 Amikacin (2101 0 0 ~Amplclllln (210) 1.13 1.18 .67 Amplcillin (210) 1.30 TNTC TNTC

*Carbenlclllln (710) 1.06 .83 .33 Carbenlcillln (710) .15 .01 .03 *Cefamandole ~200) 1.15 1.07 .49 Cefamandole (200) .16 .11 .02 5*Cefobid (500) .67 .90 .90 Cefobid (500) .007 .007 0 *Cefotaxlme (200) 1.00 1.333.83 Cefotaxlme (200) .02 .008.015 *Cefoxltln (250) .64 .80 .49 10Cefoxitin (250) .04 .05 .14 *Cephalothin (200) .70 .81 .18 Cephalothin (200) .10 .02 .03 *Chloramphenicol (180) 1.22 1.07 .59 Chloramphenicol (180) .84 .87 .39 15*Erythromycln (80) 1.27 1.001.08 Erythromycln (80) 1.63 2.25TNTC
*Gantrlsln (1000) .82 .671.00 Gantrisin (1000) 2.29 .60- 3.8-TNTC TNTC
20*Gentamicin (60) .94 .73 .22 Gentamicin (60) 0 0 0 *Moxalactam (1000) 3.48 3.522.30 Moxalactam (1000) .11 0 0 *Piperacillin (600) .52 .84 .46 25Piperacillin (600) 1.07 .15 .34 *Tetracycline (90) .75 3.02 .23 Tetracycllne (90) .90 .97 .23 *Tobramycin (qO) .85 .95 .53 Tobramycln (40) 0 0 0 -- Tlme polnt not included ln reconstructlon.
* Speclmen transport system urlne cocktall is present.
** Number in parenthesis represents flnal con-centration (ug/ml) of antibiotic in urine TNTC c too numerous to count Table XVII-3 Pseudomonas aeruqinosa Hour Tlme Polnts Antlbiotic** 4 6 24 *No Drug .92 .77 .58 No Drug 1.48 2.49 TNTC
*Amikacln (210) 1.33 1.41 .47 Amikacin (210) .58 .15 .01 *Carbenicillin (710) 1.16 1.36 .80 10Carbenicillin (710) .88 .38 .08 *Moxalactam (1000) 1.10 .86 .41 Moxalactam (1000) .52 .18 .06 *Piperacillin (600) 1.54 1.05 .91 Piperaclllin (600) .32 .98 .23 15*Tobramycin (40) .90 .42 0.5 Tobramycln (40) .22 .05 0 -- Time point not included ln reconstruction.
* Specimen transport system urine cocktail is present.
** Number in parenthesis represents final con-centration (ug/ml) of antiblotlc ln urlne TNTC - too numerous to count Table XVII-4 Proteus vulqarls Hour Tlme Polnts Antlblotlc** 4 6 24 *No Drug .57 1.07 3.11 No Drug .88 1.04 TNTC
*Amlkacln (210) .72 .92 1.67-swarm 30Amlkacin ( 210 ) .15 0 0 *Cefamandole (200) .50 .50 .34 Cefamandole (200) .20 .21 .007 2029~53 *Piperacillin (600)2.17 1.80 3.7-swarm Piperacillin (600)0 0 0 *Tobramycin (40).67 .92 swarm Tobramycin (40~.18 .03 0 -- Time point not included in reconstruction.
Specimen transport system urine cocktail is present.
** Number in parenthesis represents flnal con-centration (ug/ml) of antlbiotic ln urlne TNTC ~ too numerous to count Table XVII-5 Enterobacter cloacae Hour Time Points Antlblotlc** 4 6 24 *No Drug 1.0 .77 2.47 No Drug 2.21 7.91 TNTC
~Amikacin (210) 1.12 .62 .11 Amikacin (210) 0 0 0 *Ampicillin (210) .99 1.68 3.60 20Ampicillin (210) .18 .07 .02 *Carbenicillln ~710) .76 .76 .1~
Carbenicillln (710) .25 .23 .20' *Cefamandole (200) .89 1.44 1.11 Cefamandole (200) 1.13 .48 .44 25*Cefobid (500) .07 .13 .04 Cefobid (500) .03 .003 .0005 *Cefotaxlme (200) .92 1.04 .59 Cefotaxlme (200) .12 .02 .05 *Cefoxltin (250) 1.01 1.14 2.1 30Cefoxltln (250) .25 .52 4.34 *Cephalothln (200) .88 1.03 .49 Cephalothin (200) 2.41 4.37 TNTC

*Chloramphenicol (180) .97 .94 .94 Chloramphenicol (180) 1.06 .97 .76 *Erythromycin ~80) .95 1.04 1.06 Erythromycin (80) 1.23 1.27 TNTC
5*Gantrisin (1000) .78 1.01 .99 Gantrisin (1000) 1.74 4.021.04-TNTC
*Gentamicln (60) .68 .55 .13 Gentamicin (60) 0 0 0 *Moxalactam (1000) 5.28 5.68 5.80 10Moxalactam (1000) .10 0 0 *Piperacillin (600) .91 .88 1.84 Plperaclllln (600) .65 .24 .09 *Tetracycline (9o) .85 1.73 1.23 Tetracycllne (9o) 1.03 2.43 .58 15*Tobramycin (40) .92 .81 2.12 Tobramycin (40) 0 0 0 -- Tlme polnt not lncluded ln reconstruction.
* Specimen transport system urine cocktail ls present.
*~ Number ln parenthesis represents final con-centration (ug/ml) of antibiotlc ln urlne TNTC - too numerous to count 2~29~3 Table XVII-6 Staphylococcus aureus *SPECIMEN TRANSPORT SYSTEM URINE
REGULAR URINE
Hour Tlme Polnts Antlblotlc** 0 2 4 6 24 *No Drug 1.00 -- .91 1.05 1.20 No Drug 1.00 -- .98 1.06 TN
10*Amlkacln (210) 1.00 -- .73 .37 .33 Amlkacln ~210) 1.00 -- .071 .008 .001 *Amplclllin (210) 1.00 .68 .70 __ .63 Ampiclllln (210) 1.00 1.57 1.52 -- .86 *Carbenlclllln (710) 1.00 .58 .57 -- .43 15Carbenlclllln ~710) 1.00 .70 .80 -- .37 *Cefamandole ~200) 1.00 .78 .68 -- .57 Cefamandole ~200) 1.00 .70 .63 -- .14 *Cefobld (500) 1.00 -- 1.151.15 1.17 Cefobid (500) 1.00 -- .58.42 .08 20*Cefotaxlme (200) 1.00 -- .83.82 1.00 Cefotaxlme (200) 1.00 -- 1.18 .82 .26 *Cefoxltln (250) 1.00.79 . 9 -- .76 Cefoxltln (250) 1.00.79 .57 -- .21 *Cephalothln (200) 1.00 -- .87 1.2 2.03 25Cephalothln (200) 1.00 -- .74 1.03 .39 *Chloramphenicol ~180) 1.00 -- .73 .80 .63 Chloramphenicol (180) 1.00 -- .41 ~.28 .086 *Erythromycln (80) 1.00 -- .81 .85 .78 Erythromycln (80) 1.00 -- .23.090 .012 30*Gantrlsln (1000) 1.00 -- .79.84 .66 Gantrlsln (1000) 1.00 -- .89.21 2.22 *Gentamlcln (60) 1.00 -- .71.81 .36 Gentamicin (60) 1.00 -- . 00 0 *Moxalactam (1000) 1.00 -- . 8.76 .62 35Moxalactam (1000) 1.00 -- .86 .65 .26 20293~3 *Piperacillin (600) 1.00 .45 .58 -- .38 Piperacillln (600) 1.00 .72 .47 -- .19 *Tetracycline (9o) 1.00 .64 .73 -- .73 Tetracycline (9o) 1.00.067 .012 -- o 5*Tobramycin (40) 1.00-- 1.02 .99.93 Tobramycin (40) 1.00-- .093 .013 o -- Time point not included ln reconstructlon.
* Specimen transport system urlne cocktail is present.
** Number in parenthesis represents flnal con-centration (ug/ml) of antibiotlc in urine TNTC - too numerous to count Tables XVII-l - XVII-6 clearly demonstrate the ablllty of the speclmen transport system urlne cocktall 1~ to block conventlonal therapeutlc antlmlcroblals ln the urine and to hold the mlcroblal count relatlvely constant ln the absence of antimlcroblals.
It should be noted that wlth normal urlne mlnus antibiotlcs the common pathogenic organisms will grow (E. coll, ~. pneumonaie, P. aeruqlnosa, P. vulqarls, and E. cloacae) over a 24 hour perlod at room temperature. Hence, lf the urine speclmen is not analyzed promptly, lt can lead to a false posltlve result. In the presence of average urlne concentrat,lons of antlbiotlcs (lOx that of blood serum) sensltlve pathogenlc organlsms rapldly dle. Thls could lead the laboratory to the concluslon that the speclmen does not contaln a slgnlficant number of pathogenlc organlsms (105) when ln reallty the speclmen dld contaln the pathogens at thls level at the tlme of collectlon. In other words, lf two or more hours have elapsed between collectlon and laboratory processlng, the count obtalned may be as low as 103, l.e., considered not slgnlflcant.

The urlne speclmen transport system achleves two ma;or lmprovements, namely:
1. It ls capable of effectlvely blocking the cidal effects of antlbiotlcs for at least 6 hours, and ln the most of cases, for up to 24 hours.
2. The number of organlsms present at time zero in the presence or absence of antlblotics remains constant for up to at least 6 hours.
In conclusion, the unlque features of this urine speclmen transport system allows the urlne specimen to be held for up to 24 hours prior to processlng with no deleterious effect on the microbial integrity of the sample. Refrigeration is not required, and the system lS is effective ln the absence or presence of antimicrobials.
As can be seen from the above examples, the speclmen transport system whlch falls withln the scope of the sub~ect lnventlon has many uses. The ablllty of the speclmen transport system to hold the mlcroblal count constant may allow for detectlon of signlficant microbial specles whlch would otherwise be masked by the overgrowth of more rapldly dividing organlsms.

EXAMPLE XVIII
INCREASING HYPERTONICITY TO CREATE
HEIGHTENED BAC~TRIOSTATIS EFFECT
The following dry mlxture was placed in each of a series of tubes:
0.03 grams of sodlum polyanethosulfonate 0.005 grams of thioglycolate o.l grams of ICN free-base cystelne o.l grams of sodium bicarbonate Various percentages, by weight thereof, of sodium chloride, lndicated ln Table XVIII-l below, were then 129 20293~3 added to individual tubes containing the above-described specimen transport system urine cocktail. A
flve milliliter aliquot of sterilized urine was then added to all tubes contalning the above-descrlbed specimen transport system urlne cocktail and to an equal number of tubes without the urlne cocktail. Culture media containlng Enterobacter cloacae ATCC ~1344-2, was ad~usted to a McFarlin of 0.5, representing approximately 5 x 108 microorganisms per mlllillter of culture media, and then dlluted 1:100 in sterile culture media. A 0.1 milliliter aliquot of diluted microorganlsms was added to all urine containing tubes and the resultlng mixture vigorously agltated. Ten microllter allquots of the mlxture were plated on agar plates such as described ln Example VII. The remalnlng mlxture was allowed to stand at room temperature for 24 hours after which time, a second ten microliter aliquot was plated as described in Example VII above. All plating was done in duplicate and the survival index (S-Factor) was calculated for each as described in the examples above. The average S-Factor for each time point was determined and is recorded in Table XVIII-l below.

20293S~

Table XvIII-Enterobacter cloacae (ATCC ~ 1344-2) S-Factor Sample STS* NaCl (%)*~ 0 Hour 24 Hours 1 -- -- 1.00TNTC***
2 ~ -- 1.001.72 3 + 1 1.001.27 4 ~ 2 1.00 .80 + 4 1.001.03 6 + 8 1.000.69 * STS Specimen transport system urine cocktall lS ** percentage percentage sodlum chlorlde by welght *** TNTC too numerous to count In the absence of the speclmen transport system urlne cocktall, see Sample 1 ln Table XVIII-l above, the mlcroorganlsms present ln the urine will qulckly multlply and thus prevent the cllnlclan from obtalnlng an accurate count of the number of mlcroorganlsms per mllllllter of urlne.
The results, dlsplayed ln Table XVIII-l above, lndlcate that whlle the speclmen transport system urine cocktall can decrease the rate of mlcroblal repllcatlon, the addltlon of such salts as sodlum chlorlde lncrease the effectlveness of the urlne cocktail in holding the bacterlal count, ln urlne, stable over a 24-hour period.
The preferred range of salt, as determlned from the results dlsplayed in Table XVIII-l above, is from about 2.5 percent to about 4.0 percent, by weight thereof.
From the results of the salt tltratlon experlment above, lt was concluded that addltlon of about three (3) percent, by welght, sodlum chlorlde to the speclmen 131 20293~3 transport system urlne cocktall should prevent the overgrowth of Enterobacter cloacae in urlne over a 24-hour period. To verify thls conclusion, the specimen transport system urlne cocktall prepared as descrlbed above, was added to a series of tubes. A second serles of tubes was prepared by addlng the identlcal cocktall plus 0.15 grams of sodlum chlorlde, the equlvalent of three (3) percent, by welght, sodlum chlorlde. A thlrd serles of tubes were set aslde wlthout cocktall. A
flve-mllliliter allquot of sterlle urlne was then added to each tube, lncludlng those tubes wlthout cocktall.
Four stralns of Enterobacter cloacae, ldentlfled ln Table 40 below, were grown separately and ad~usted to a McFarlln of O.S. Each straln was then diluted 1:100 in sterile culture medla and a 0.1 mllllllter allquot of each added to lndlvldual urlne tubes as ldentlfled ln Table XvIII-2 below. After vlgorous mixing, a ten-mlcrollter allquot from each tube was plated on agar plates as descrlbed ln Example XVII. Thereafter, the mlxture was allowed to stand at room temperature and at the time lntervals lndicated ln Table XVIII-2 below, another ten (10) mlcrollter allquot was plated as descrlbed ln Example VII. The results of each tlme polnt glven ln Table XVIII-2 below represents an average 2~ survlval lndex ~S-Factor) for quadrupllcate platlng.
The results, glven ln Table XVIII-2 below, conflrm an lncreased stablllzatlon of colony formatlon for all four strains of Enterobacter cloacae afforded by additlon of about three percent, by weight, sodlum chlorlde to the speclmen transport system cocktall.
Hypertonlcity may also be lncreased by utllizlng other salts, carbohydrates or sugars. It ls expected that appropriate concentratlons to approach the effect of sodlum chlorlde in thls example may be calculated wlth the knowledge dlsclosed hereln.

Table XVIII-2 Enterobacter cloacae S-Factor (ATCC:various strains) HOUR TIME POINTS~

4 +ADS+ 6 +ADS+ 24 +ADS+ 48 +ADS+
Strain - +ADS NaCl - +ADS NaCl - +ADS NaCl - +ADS NaCl 1344-2 3.21.28 1.97 4.75-TN 1.23 .91 TN 3.88-TN 2.05 TN TN 4.64-TN
3118 3.00.87 .98 3.00-TN .95 1.05 TN 4.25-TN .98 TN TN 4.14 0 2294 2.5-TN1.00 .76 5.53-TN .89 .89 TN 2.38-TN .82 TN TN 2.25 0879 2.5.95 1.03 4.02-TN .86 1.06 TN TN 1.07 TN TN 1.29-TN
* "-" indicates urine without specimen transport system cocktail; "+ADS" indicates urine plus specimen transport cocktail; "+ADS+NaCl" indicates specimen w transport system cocktail containing 3 percent, by weight thereof, sodium chloride.
TN = too numerous to count.

o CD
C~

20293~3 EXAMPLE XIX
EE`FECT OF URIN-E SPECIMEN TRANSPORT SYSTEM ON
MICROBIAL INTEGRITY IN T~IE ~/R~ l!;M;~!; OF ANTIBIOTICS
Tables XIX-l - XIX-3 below lllustrate the effect of the specimen transport system on quantltatlon ln the presence and absence of the antlblotlcs over 24 hours.
The followlng dry mlxture was placed ln a serles of sterlle tubes:
0.03 grams sodlum polyanetholsulfonate 0 . 005 grams thloglycolate 0.1 gram of ICN free-base cystelne 0.1 gram of sodlum blcarbonate 0.15 grams of sodlum chlorlde The varlous antlblotlcs, llsted ln Tables XIX-l -XIX-3 below, were added, at the concentratlons also speclfled, to the tubes contalnlng the above-descrlbed speclmen tLar.s~oL~ system urlne cocktall and to an equal number of tubes without the urlne cocktall. Flve mllllllters of sterlle urlne was then added to all tubes after whlch the tubes were vlgorously mlxed. Control tubes contalned elther urlne alone or urlne plus the above-descrlbed speclmen tr2nsport system urlne cocktall. No antlblotlcs were added to control tubes.
The mlcroorganlsms llsted ln Tables XIX-l - XIX-3 below were ad~usted to a McFarlln of 0 . 5 nnd then dlluted 1:100 wlth sterlle culture medla. A 0.1 mllllllter allquot of a slngle mlcroorganlsm was added to each urlne contalnlng tube and the resultant mlxture vlgorously agltated. A ten-mlcrollter allquot from each tube contalnlng the resultant mlxture was lmmedlately plated as descrlbed ln Example XVII above. The tubes were then allowed to stand at room temperature for 24 hours. Addltlonal ten mlcrollter allquots were plated as descrlbed ln Example XVII above at the tlme polnts 35 lndlcated ln Tables XIX-l - XIX-3 below. All platlng 134 202~353 - was done ln quadrupllcate, and the S-Factors recorded ln Tables XIX-l - XIX-3 below represent an average of the quadrupllcate platlng.
The results set forth ln Tables XIX-l - XIX-3 lndlcate that the salt contalnlng speclmen t~an~o~
system urlne cocktall was able to hold the colony count of Proteus vulqarls, StrePtococcus, pneumoniae, and Streptococcus Pvoqenes relatlvely stable ln both the presence and absence of most antlblotlcs.
Table XIX-l I. Proteus vulqarls S-Factor (ATCC ~23315 ) ~OUR TIME POINTS*
Antlblotlc** 4 6 24 lS + -- + -- +
No Drug 1.06 1.07 1.38 3.15 1.12 TNTC
Amplclllln ( 210 ) . 77 .19 1.19 . 04 . 87 . 004 Cefoxltln (2s0) .73 .002 .79 .0008 .25 0 20 Chlo. ,'-q1col (180) 1.01 .53 .56 .32 .46 .06 Erythromycln ( 80 ) . 79 1.13 1. 50 1. 78 . 94 1. 07-TNTC
Gantrlsln (1000) 1.47 .56 1.28 1.32 1.19 TNTC
Mezloclllln (500) 2.14 .31 1.51 .50 1.31 ` 0 -- Tlme polnt not lncluded ln reconstructlon.
* Speclmen transport system urlne cocktall ls present.
** Number ln parenthesls Lep-esen~s flnal con-centratlon of antlblotlc ln ml..oy~ per mllllllter of urlne TNTC - too - _ uus to count 202~35~

Table XIX-2 I. Streptococcus ~n. --1 ae S-Factor (ATCC ~23315) HOUR TIME POINTS*
SAntlblotlc** 4 6 24 + _ + _ +
No Drug 1.19 .82 .95 .73 1.27 4.80 Amplclllln ( 210 ) 1. 60 . 54 1. 34 . 43 1. 08 . 07 10 Cefamandole ( 200 ) . 42 1. 04 . 75 1.12 1. 36 . 28 Cefoxltln (250) 1.08 .98 1.11 .94 1.01 .20 Cephalothln ( 20 0 ) 1. 0 4 . 5 3 1. 4 2 . 5 3 2 . 5 4 . 21 ChloL ,' 1Col (180) .57 .77 .80 .31 1.03 .30 Erythromycln (80) .99 .73 .83 .73 .78 .32 15 Gantrlsln ( 1000 ) 1. 00 . 96 1. 00 . 96 1.10 14 . 54 Mezloclllln ( 500 ) 1.18 . 71 1. 05 . 29 1. 03 . 01 -- Tlme polnt not 1nrl~lcled ln reco~.sLLu.,Llon.
* Speclmen transport system urlne cocktall ls present.
20 ** Number ln parenthesls Lepres~.. 1 s flnal con-centratlon of antlblotlc ln mlcLoyL per mllllllter of urlne 20293~3 Table XIX-3 I. Streptococcus pyoqenes S-F~ctor (ATCC 123315) HOUR TIME POINTS*
5Antiblotlc** 4 6 24 + _ + _ +
No Drug 1.02 1.95 .93 3.10 .84 TNTC
Amplclllln ( 210 ) . 90 1. 50 1. 00 1. 50 . 79 1. 00 10 Cefamandole ( 200 ) . 52 . 41 . 88 . 09 1. 28 . 26 Cefoxltln ( 250 ) . 60 . 30 . 94 . 24 . 96 .10 Cephalothln (200) 1.35 .63 1.50 .58 1.62 .08 Chloramphenlcol ( 180 ) . 84 1.15 1. 05 1. 06 . 75 . 22 Erythromycln ( 80 ) . 5q . 97 . 33 . 81 . 54 . 76 15Gantrlsln ( 1000 ) . 84 2 . 01 . 94 2 . 34 . 78 1. 56-TNTC
Mezloclllln (500) .95 .74 1.33 .51 .62 .10 -- Tlme polnt not lncluded ln reconstructlon.
* Speclmen transport system urlne 20cocktail is present.
** Number in parenthesis represents final con-centration of antibiotlc in micrograms per mllllllter of urlne TNTC - too numerous to count 202g3~3 EXAMPLE XX
EFFECTIVE CO~c~~ ATION OF SODIUM
POLYANETHOLSULFONATE ( SPS ~ FOR A SPECIMEN
TRANSPORT SYSTEM TO ~1~1~;5~'KVls MICROBIAL INTEGRITY
AN ANTIBIOTIC-CONTAINING URINE SPECIMEN
The followlng dry mlxture was placed ln each of a series of sterile tubes:
0.005 grams thloglycolate O . l gram ICN free-base cystelne 01. gram sodlum blcarbonate 0.15 grams sodlum chlorlde Dlfferent amounts of SPS, deslgnated by welght percent thereof ln Table XX-l below, were added to the tubes contalnlng the above-descrlbed speclmen transport system urlne cocktall and to the control tubes. The varlous antlblotlcs listed in Ta~le XX-l below were then added to one half of the tubes contalnlng the above-descrlbed speclmen transport system urlne cocktall.
Flve mllllllter allquots of sterlle urlne were next added to all tubes. Control tubes contalnlng urlne but no antlblotlc were est~hl 1 ~hed, half of whlch contalned the above-descrlbed speclmen transport system urlne cocktall plus the varlous amounts of SPS deslgnated ln Table XX-l below. StaphYlococcus aureus, ATCC t25923, were grown, prepared and allquoted lnto all tubes as descrlbed ln Example XVII above. All tubes were plated as descrlbed ln Example ll above at the tlme polnts lndlcated ln Table XX-l below.

Table XX-1 Staphylococcus aureus S-Factor (ATCC #25923) liOUR TI~OE POINTS*
SPS9 **~ Antibiotie** 2 4 6 24 + _ + _ + _ +
-- No Drug ND ND ND 1. 39 ND 1. 50 ND 2 . 3-TNTC
0 0 . 6 No Drug . 7 0 ND 6 . 0 ND ND ND . 4 4 ND
1. 0 No Drug ND ND 1. 4 ND . 83 ND . 66 ND
2 . 0 No Drug ND ND . 85 ND . 83 ND . 91 ND
6.0No Drug .87 ND .76 ND ND ND .63 ND
5 0.6Gentamlcin (60 ug) .63 .02 .51 0 ND ND .51 0 1.0 Gentamicin (60 ug) ND ND 1.12 0 1.03 .0q 1.22 0 2.0 Gentamicin (60 ug) ND ND .75 0 .75 0 .47 0 6.0 Gentamlcin (60 ug) .87 .12 .76 .01 ND ND .53 0 0.6 Tetracycline(90 ug) .64 .07 .73 .01 ND ND .6 0 20 1.0 Tetracycline(90 ug) ND ND 1.0 0.1 1.3 0 .67 0 2 . 0 Tetracycline (90 ug) ND ND . 68 0 . 74 . 007 . 5 . 002 6.0 Tetracycline(90 ug) .91 1.09 .87 .34 ND ND ND ND
-- Time point not included in reconstruction.
* Specimen transport system urine cocktail is present.
** Number in parenthesis represents final coneentration of antibiotic in mierograms per milliliter of urine.
*** ~inal sodium polyetholsulfonate eoneentration, by weight thereof, per tube.
TNTC = too numerous to eount + Contains Speeimen transport system.
- Does not eontain Speeimen transport system.
ND = Not Done 2Q293~3 The results set forth ln Table XX-l above reveal an optimum range for SPS ln the specimen transport system urlne cocktall to be between about O . 696 and about 2 . 096, by welght thereof.
Whlle thls lnventlon has been descrlbed ln relatlon to lts preferred embodlments, lt ls to be understood that varlous modlflcatlons thereof wlll now be apparent to one skllled ln the art upon readlng the speclflcatlon and lt ls lntended to cover such modlflcatlons as fall wlthln the scope of the appended clalms.

Claims (42)

1. An apparatus for the collection and treatment of microorganisms in a specimen comprising:
(a) a container of generally elongated shape having a first end and a second end;
(b) a piston slidably mounted in said container;
(c) a shaft attached to said piston and extending through said first end, said shaft being detachably connected to said piston by complementary thread means;
(d) a detachable cap removably attached to said second end;
said cap, said container and said piston forming a sealed chamber, in which the pressure in the chamber may be decreased by moving said piston toward said first end;
said cap being puncturable for insertion of said specimen, and;
(e) a water-soluble specimen transport system deposited in said container, said specimen transport system comprising a water-soluble additive at a concentration effective to prevent replication of microorganisms present in said specimen, when said specimen is mixed with said water additive therein to form a solution, and to reduce the cidal activity toward said microorganisms of antimicrobial factors present in said specimen so that at least some microorganisms will be capable of replication upon dilution of said solution on medium capable of supporting replication of said microorganisms.

2. The apparatus of claim 1, wherein said apparatus further comprises a means to lock said piston to prevent movement of said piston within said container.

3. The apparatus of claim 1, further comprising:
(f) a substance effective for preserving the viability of microorganisms of interest in said specimen in said container.

4. The apparatus of claim 3, wherein said substance for preserving the viability of said microorganisms of interest comprises a growth base effective for supporting the general nutritional needs of the microorganisms of interest.

5. The apparatus according to claim 3, wherein said substance for preserving the viability of the microorganisms of interest in the specimen comprises starch.

6. The apparatus according to claim 3, wherein said substance for preserving the viability of the microorganisms of interest in the specimen comprises agar.

7. The apparatus according to claim 3, wherein said substance for preserving the viability of the microorganisms of interest ln the specimen comprises hemoglobin.

8. An apparatus for the collection and treatment of microorganisms in a specimen comprising:
(a) a container of generally elongated shape having a first end and a second end;
(b) a piston slidably mounted in said container;
(c) a shaft attached to said piston and extending through said first end, said shaft includes a narrow portion causing said shaft to break at said narrow portion when said shaft is bent;
(d) a detachable cap removably attached to said second end;
said cap, said container and said piston forming a sealed chamber, in which the pressure in the chamber may be decreased by moving said piston toward said first end;

said cap being puncturable for insertion of said specimen, and;
(e) a water-soluble specimen transport system deposited in said container, said specimen transport system comprising a water-soluble additive at a concentration effective to prevent replication of microorganisms present in said specimen, when said specimen is mixed with said water additive therein to form a solution, and to reduce the cidal activity toward said microorganisms of antimicrobial factors present in said specimen so that at least some microorganisms will be capable of replication upon dilution of said solution on medium capable of supporting replication of said microorganisms.

9. The apparatus of claim 8, wherein said apparatus further comprises a means to lock said piston to prevent movement of said piston within said container.

10. The apparatus of claim 8, further comprising:
(f) a substance effective for preserving the viability of microorganisms of interest in said specimen in said container.

11. The apparatus of claim 10, wherein said substance for preserving the viability of said microorganisms of interest comprises a growth base effective for supporting the general nutritional needs of the microorganisms of interest.

12. The apparatus according to claim 10, wherein said substance for preserving the viability of the microorganisms of interest in the specimen comprises starch.

13. The apparatus according to claim 10, wherein said substance for preserving the viability of the microorganisms of interest in the specimen comprises agar.

14. The apparatus according to claim 10, wherein said substance for preserving the viability of the microorganisms of interest in the specimen comprises hemoglobin.

15. A method of collecting and transporting a fluid specimen suspected to contain microorganisms comprising the steps of:
depositing the specimen in a container in which there is residing a water-soluble specimen transport system effective for preserving the microbial integrity of the specimen;
said container being of generally elongated shape having a first end and a second end, said container further comprising:
a piston slidably mounted in said container;
a shaft attached to said piston and extending through said first end, said shaft includes a narrow portion causing said shaft to break at said narrow portion when said shaft is bent;
a detachable cap removably attached to said second end;
said cap, said container and said piston forming a sealed chamber, in which the pressure in the chamber may be decreased by moving said piston toward said first end;
said cap being puncturable for insertion of said specimen, and;
wherein said specimen transport system comprises a water-soluble additive at a concentration effective to prevent replication of microorganisms present in said specimen, when said specimen is mixed with said water additive therein to form a solution, and to reduce the cidal activity toward said microorganisms of antimicrobial factors present in said specimen so that at least some microorganisms will be capable of replication upon dilution of said solution on medium capable of supporting replication of said microorganisms.

16. The method according to claim 15, wherein said specimen transport system comprises an effective amount of an agent selected from the group consisting of sodium polyanetholsulfonate and sodium amylosulfate.

17. The method according to claim 16, wherein said specimen transport system further comprises an effective amount of a sulfhydryl-containing substance non-toxic to said microorganisms.

18. The method according to claim 16, wherein said specimen transport system further comprises a hypertonicity-increaser effective for providing an increased bacteriostatic effect.

19. The method according to claim 16, wherein said specimen transport system further comprises a substance effective for inhibiting the replication of gram positive microorganisms.

20. The method according to claim 16, wherein said specimen transport system further comprises a substance effective for buffering the pH of said specimen in a range from about 6.5 pH units to about 7.5 pH units.

21. The method according to claim 15, wherein said specimen transport system further comprises a substance effective for preserving the viability of microorganisms of interest in said specimen in said container.

22. The method according to claim 21, wherein said substance for preserving the viability of said microorganisms of interest comprises a growth base effective for supporting general nutritional needs of microorganisms.

23. The method according to claim 21, wherein said substance for preserving the viability of said microorganisms of interest in the specimen comprises starch.

24. The method according to claim 21, wherein said substance for preserving the viability of said microorganisms of interest in the specimen comprises agar.

25. The method according to claim 21, wherein said substance for preserving the viability of said microorganisms of interest in the specimen comprises hemoglobin.

26. A method according to claim 21, wherein said specimen transport system comprises an inhibitor of sulfa drugs.

27. The method according to claim 21, wherein said specimen transport system further comprises an antibiotic-specific enzyme.

28. The method according to claim 15, wherein said apparatus further comprises a means to lock said piston to prevent movement of said piston within said container.

29. A method of collecting and transporting a fluid specimen suspected to contain microorganisms comprising the steps of:
depositing the specimen in a container in which there is residing a water-soluble specimen transport system effective for preserving the microbial integrity of the specimen;
said container being of generally elongated shape having a first end and a second end, said container further comprising:
a piston slidably mounted in said container;

a shaft attached to said piston and extending through said first end, said shaft being detachably connected to said piston by complementary thread means;
a detachable cap removably attached to said second end;
said cap, said container and said piston forming a sealed chamber, in which the pressure in the chamber may be decreased by moving said piston toward said first end;
said cap being puncturable for insertion of said specimen, and;
wherein said specimen transport system comprises a water-soluble additive at a concentration effective to prevent replication of microorganisms present in said specimen, when said specimen is mixed with said water additive therein to form a solution, and to reduce the cidal activity toward said microorganisms of antimicrobial factors present in said specimen so that at least some microorganisms will be capable of replication upon dilution of said solution on medium capable of supporting replication of said microorganisms.

30. The method according to claim 29, wherein said specimen transport system comprises an effective amount of an agent selected from the group consisting of sodium polyanetholsulfonate and sodium amylosulfate.

31. The method according to claim 30, wherein said specimen transport system further comprises an effective amount of a sulfhydryl-containing substance non-toxic to said microorganisms.

32. The method according to claim 30, wherein said specimen transport system further comprises a hypertonicity-increaser effective for providing an increased bacteriostatic effect.

33. The method according to claim 30, wherein said specimen transport system further comprises a substance effective for inhibiting the replication of gram positive microorganisms.

34. The method according to claim 30, wherein said specimen transport system further comprises a substance effective for buffering the pH of said specimen in a range from about 6.5 pH units to about 7.5 pH units.

35. The method according to claim 29, wherein said specimen transport system further comprises a substance effective for preserving the viability of microorganisms of interest in said specimen in said container.

36. The method according to claim 35, wherein said substance for preserving the viability of said microorganisms of interest comprises a growth base effective for supporting general nutritional needs of microorganisms.

37. The method according to claim 35, wherein said substance for preserving the viability of said microorganisms of interest in the specimen comprises starch.

38. The method according to claim 35, wherein said substance for preserving the viability of said microorganisms of interest in the specimen comprises agar.

39. The method according to claim 35, wherein said substance for preserving the viability of said microorganisms of interest in the specimen comprises hemoglobin.

40. A method according to claim 35, wherein said specimen transport system comprises an inhibitor of sulfa drugs.

41. The method according to claim 35, wherein said specimen transport system further comprises an antibiotic-specific enzyme.

42. The method according to claim 29, wherein said apparatus further comprises a means to lock said piston to prevent movement of said piston within said container.