JPH04197169A - Stabilization of sample for microbioassay - Google Patents

Abstract

PURPOSE: To provide an apparatus for stabilizing a microbial level in a sample by pressing a dissolvent and a specific sample transport system in a syringe- shaped centrifugal separating container.

CONSTITUTION: A sample transport system 260 containing a dissolvent in a composition effective in preserving a biological just likeness is placed in a syringe-shaped apparatus having a piston 230 and a shaft 220 in a centrifugal separating container 210. A mouth 270 of the container 210 is made hermetically sealable with a closing member 250 and a tube 285 is attached to the mouth 270 when collecting a sample. The piston 230 is pulled by the shaft 220 to suck the sample into a cylinder. The sample transport system 260 is dissolved in the sample in the cylinder. The tube 285 is removed to cover the mouth 270 with a cap 290 and the container 210 is closed.

COPYRIGHT: (C)1992,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION TECHNICAL FIELD This invention relates to the analysis of microorganisms in a sample. In particular, the invention provides a method for determining the nature or microbial integrity of a sample.
:] from the time of its collection until the time laboratory analysis is initiated.

BACKGROUND ART Accurate laboratory analysis of samples suspected of containing microorganisms is of critical importance in the fields of pharmaceutical and food technology and safety, among other things. Techniques are being developed to improve the speed and sensitivity of microbiological identification, medicines are being developed to combat infections in patients, and sanitary requirements for food processing wastes are being mandated by law. However, it is clear that problems remain.

For example, sepsis, the presence of pathogenic microorganisms in the blood, is one of the most dangerous types of infections encountered.

There is unanimous agreement in the medical community that sepsis is second only to meningitis in terms of dangerous infections. Although modern medicine provides an arsenal of antibiotics and antifungals, the mortality rate for sepsis is approximately 25%. Furthermore, when shock is accompanied by sepsis, the mortality rate increases to over 60%. Debilitating illness, major surgery, administration of immunosuppressive drugs or anti-cancer medications predispose patients particularly to sepsis. Early diagnosis of the causative agent along with the use of appropriate antibiotic therapy is important in the fight against sepsis. It is therefore urgent that physicians know as quickly as possible not only that a patient has sepsis, but also the identity and/or antibiotic susceptibility of the microorganisms involved. Proper and timely diagnosis of sepsis therefore depends on the rapid consequential analysis of microorganisms in the patient's blood. Furthermore, during microbial analysis in patient blood, it is necessary that the blood sample be free from contamination with microorganisms from the hospital environment.

Other examples of disorders caused by microorganisms are very common in young children, pregnant women, patients with persistent disorders, who have had devices inserted into their urinary tract (e.g. catheters) or who have urological conditions that affect urination. A common occurrence is the presence of pathogenic microorganisms in the urine. This disorder can result in a localized infection within the bladder or kidneys. When confined to the bladder, the infection is usually well controlled with antimicrobial therapy. However, once the kidney becomes infected, the lesions continue to progress to chronic pyelonephritis and sepsis despite treatment.

In the food technology field, contamination is sometimes a problem that endangers human health. For example, contamination of milk can occur even where processing steps that sterilize harmful microorganisms are used, as equipment malfunctions, human error and sometimes unexplained circumstances contribute to rendering the process ineffective. Are known.

In such cases, rapid and accurate analysis of samples from food processing equipment and the food itself is critical to establishing the source of contamination, as is correcting processing. A variety of techniques are used for microbial analysis. Simple quantitative analysis involves determining the number of microorganisms in a given sample, independent of microbial identification. Quantification may involve introducing a known amount of the sample (perhaps diluted to a known amount in broth) onto nutrient agar and allowing colonies to form. Identification and/or determination of antibiotic susceptibility of detected microorganisms may be desirable. Analyzes for identifying microorganisms and/or establishing antibiotic susceptibility usually involve placing individual colonies on differential media.

In these instances, accurate quantification and identification of specific microorganisms is much more important than a simple determination of whether a specific microorganism is present. Therefore, it may not be sufficient to determine whether a sample is “positive” or “negative” for microorganisms; rather, if the sample is positive, it may be necessary to know how many of a particular species of microorganism are present in the sample. It may be necessary.

For example, it is normal for humans to always have microorganisms in their mouths and throats. These normal microorganisms, hereinafter referred to as normal flora, generally do not cause disease in their normally present numbers. However, microorganisms that may be part of the normal flora can multiply to the extent that they become disease-causing microorganisms (pathogens). Thus, for example, the difference between a normal human throat and a diseased human throat lies not in the identification of specific microorganisms that are alive at the time of analysis, but in the number of those microorganisms present in the patient's throat. I can recognize that it might be possible. The bloodstream is generally sterile. However, for example, temporary screen syndrome, which is not normally a cause for alarm, can occur when a small number of microorganisms enter the bloodstream through a cut or ulcer.

Quantification of microorganisms in a blood sample is very important to distinguish temporary bacteremia from sepsis and possibly contamination of the sample. While quantification is extremely important in the analysis of blood samples, determination of the identity of the microbial pathogens present is still important.

Although taxonomic identification of microorganisms may not be necessary for patient treatment, determining the microorganism's susceptibility to antibiotics may be important in selecting continuous drug therapy. This may be done by identifying the microorganism by genus and species, since drug manufacturers often predetermine the efficacy of a drug for a particular biological taxonomic group. Separately, a drug effect (antibiotic susceptibility) test may be conducted.

In some liquids, the concentration of microorganisms in the sample is so small that the presence of microorganisms may not be apparent in the test area using conventional methods. Improvements useful in detecting low concentrations of microorganisms have recently been published that have greatly improved the detection of sepsis in the blood before the microorganisms have multiplied to the extent that the patient is in critical condition.

A recently developed method and apparatus for concentrating and detecting microorganisms in sample liquids is described in the ``Method ofDe
tecting Microbial Paro
gens Employing aCushion
U.S. Pat.
ForDetecting Microbial P
attrogens Employing aCush
U.S. Patent No. 4,2 titled
Published in No. 12゜948. The technology disclosed in the above-mentioned patents includes (in the case of blood sample analysis) microorganisms,
It involves pre-lysis of blood cell compounds followed by centrifugation to concentrate them from other components, including antimicrobial factors, present in the blood. The concentrated microorganisms are then placed on a nutrient medium such that the substances inhibiting microbial growth present in the sample are diluted to a minimum of 60 microorganisms. This technique has previously been demonstrated to provide clearer cultures than traditional liquid broth cultures, pour plate cultures or filtration methods using solid matrix f-liquids. Gordon Dorn, Geoffr
ey A, Land O and George E, 'W
ilson, ”Improved Blood C
u1tuveTechnique Ba5ed on
Centrifugation: Cl1nical
Evaluation”, 9 J, C11nical
Microbiology 391-396 (19
79).

Despite the increasing sophistication in techniques for detecting and characterizing microorganisms in samples, microbial analysis is limited by the accuracy of the techniques and the microbial nature of the sample being analyzed. Problems remain in the field.

"Microbiological resemblance" means that a sample taken at one time (1Q) and analyzed at another time (t, It is meant to provide an accurate representation of the microbial population.

There are a minimum of three major factors that contribute to the lack of microbial resemblance of samples in tl.

The first is that samples often contain antimicrobial factors that may previously kill the microorganisms of interest. The second factor is tl, even in the absence of antimicrobial factors.
This means that the microorganism of interest may not survive in the sample.

Third, microorganisms comprising the sample may reproduce more rapidly than, for example, in the patient from whom the sample was taken. Rapidly growing but relatively harmless or unrelated microorganisms become overwhelmingly present in the sample to the extent that the more harmful species of interest are not detected in the laboratory being analyzed.

Even if the laboratory correctly identifies the microorganism being grown, failure to detect important microorganisms can lead to misinterpretation of the contamination problem. In either case, the sample analyzed at t will not give an accurate picture of the microbial problem in the patient or other source. Resolving the issue of physical resemblance may be important to a patient's recovery, since drug therapy prescribed by a physician depends on laboratory determination of the type of infecting organism and the extent of infection. good. False negatives regarding food processing equipment or the food itself are harmful to public health. Furthermore, incorrect identification of contamination in food-related areas may prevent discovery of the source of contamination or cause unnecessary disposal of the product. Discovery of the source is often necessary to prevent future incidents of contamination.

Several problems arise when antimicrobial agents, such as antibiotics, are present in the sample. For example, a patient who has been given an antibiotic by a doctor may have levels of that drug in his or her blood or urine. I can't get enough of it.

If a urine sample is collected at the time (by way of example), the urine may contain live microorganisms and antibiotics. The antibiotic may continue to kill microorganisms in the sample to the extent that no living microorganisms remain at t. The laboratory may analyze the urine sample and conclude that the patient no longer has a microbial problem. However, this may be inaccurate. Unlike the specimen, the patient's system may remain inoculated with microorganisms from the source of infection. Although the antibiotic level in the sample was sufficient to kill the microorganisms therein, this does not necessarily reflect the infection status in the patient. Furthermore, living microorganisms require microbial identification and antibiotic susceptibility testing. If there are no live microorganisms in the sample that arrives at the laboratory, the laboratory usually cannot accurately determine the identity or antibiotic susceptibility of the microorganisms. If a drug that may be effective in eliminating a particular microorganism is not sufficiently effective in the patient's system to destroy the infecting microorganism, a less effective drug may be taken by the patient. If the microbial integrity of the sample taken from the patient is sufficient to destroy it, it is not recommended, but it is not compulsory.

Natural bactericidal substances found in samples such as blood can also alter the microbial purity of the sample before it is analyzed, causing inaccurate results.

Even if antimicrobial factors are not present in the sample, the problem of microbial resemblance remains. If the sample contains live microorganisms in maru but not in tl, the microorganisms are unlikely to be detected by the laboratory because the detection technique is primarily based on microbial reproduction. . Such conditions would result in negative reports and potentially harmful consequences if microbial infection or contamination were left untreated.

If too much is regenerated in the sample, laboratory analysis will falsely indicate that the patient, food, or food processing equipment is highly contaminated. Many patients are reluctant to be given incorrect drug therapy, which is unnecessary and potentially harmful in itself. Also, rapidly regenerating microorganisms can cause other harmful microorganisms in the sample to remain dead in the sample even though they are rapidly regenerating in the patient. . Appropriate drug therapy depends on the identity of the microorganism in question, so patients with more aggressive
Due to the removal of undetected microorganisms, appropriate treatment is not taken and harm is caused.

In the case of food analysis, the source of the malignant microorganism may not be discovered, resulting in false identification of the source of contamination.

The problem of lack of microbial similarity in samples is compounded by hospital inefficiencies in transporting samples to the laboratory and backlogs that occur in the laboratory of samples to be analyzed. Many textbooks and textbooks on microbiological techniques recommend sample retention times of less than 2 hours, which is impractical to operate at this standard of efficiency. This problem is further exacerbated when the sample must be transported from a remote location, such as a doctor's office, food processing plant, or sewage treatment plant, to a central laboratory. The longer it takes to transport the sample to the laboratory, the less accurate the analysis will be because the microbial susceptibility of the sample is compromised.

Ideally, therefore, when testing a liquid for different levels of microorganisms, the level of microorganisms present in the sample should not change between the time the sample is taken and the time it is tested. However, the environment of the test sample typically differs from the environment of the sample being tested due to variations in temperature, light, nutrient availability, and the like.

This can lead to large differences in the number of microorganisms present at the time of testing. For example, the amount of urine produced by a doctor's body over a period of several hours may be reduced due to the effects of antibiotics taken by the doctor.

Similarly, microorganisms present in the water can quickly deplete all available nutrients in the sample container and die before the test can be performed. In any case, the longer the time between sample collection and testing, the greater the probability of inaccurate test results.

Although the problem of sample properties has been addressed with techniques, none of the known methods have been completely effective and some have introduced further problems.

The simplest method published by the prior art is rapid transport from the sample collection point to the analysis point. For microorganisms that are particularly sensitive to transport, immediately onto nutrient plates,
Practically speaking, it was proposed to culture the wires at the patient's bedside. As already pointed out, it is often difficult to ensure that the sample was transported within the selected time frame. Even if confirmed, up to 50% of the microorganisms of interest may be killed within 15-20 minutes if the sample contains antibiotics.

Transport to the laboratory within 2 hours or less may therefore be insufficient. Streak culture at the bedside involves a loss of sterile technique, and there remains the problem of transporting the plates to the laboratory. The presence of antibiotics may still pose a problem.

In the past, sample transportation was often performed in sterile containers, beginning in an attempt to improve sample quality.

However, even if the sample is collected in a sterile container, the microbial identity of the sample may be compromised during transportation of the sample because the initial sterility of the container does not prevent the microorganisms in the sample from dying or overgrowing. Maybe. Furthermore, the sterility of the container may be lost if a sample, such as urine, is collected as soon as the closure is removed for urination.

U.S. Patent Nos. 4,145,304 ('304) and 4,
No. 174,277 ('277), methods and structures for the removal of antimicrobial agents were published. The mixed resin bed adsorbs antibiotics and prevents them from killing microorganisms of interest. Resin bed systems require multiple physical intrusions into the sample in which the sample must be collected from the patient, transferred to a resin bed for antibiotic adsorption, and removed from the resin bed.

The more physical penetration a sample undergoes, the greater the risk of microbial contamination from the skin of the handler or the environment. This resin bed is insoluble and therefore requires physical handling before the sample is analyzed.

Loss of microorganisms may result from non-selective adsorption. Additionally, mixed resin systems fail to maintain microbial cells in non-replicating, viable conditions.

Several systems have been taught for use with urine samples that deal with the problem of uncontrolled growth of the particular species of interest that skews the analysis. However, many of these systems require that the sample be prepared at a common chemical level, e.g. glucose,
Since it is tested for things like bilirubin, the focus is on killing any bacteria that may be present. In the system taught for microbial resemblance preservation,
Antibiotic inhibition is generally not covered. Therefore, methods of preserving the actual number of microorganisms in the presence or absence of disinfectants are not addressed by known urine sample processing agents.

Maintaining samples at about 4° C. from the time of collection to the time of analysis is another known method for attempting to maintain sample quality.

Low temperatures reduce microbial growth, so antibiotics that only affect microbial replication may become ineffective. However, this method is impractical in this field; low temperatures, while ineffective controls on the growth of other microorganisms, may be detrimental to the viability of the underlying microorganisms. Furthermore, the action of antibiotics may not necessarily be inhibited by cryogenic methods. An example of a microorganism that may be killed by cold is 5trep, which the late physician was interested in detecting as the cause of lobar pneumonia.
tococus pHeumoniae. Therefore, it is preferred that the sample be maintained at room temperature between 21 and 25°C.

Other ways to improve sample quality include Amie
S [C, Am1es Oyobi F, Path, 58C
anadian j.

Public Health 296 (1967)
) as well as 5tuarts (RoStuart et al.
e+The problem of transport
tof Specimes For Cuxture
of Gonococci”, 45Canadi
an J, Public Health 73 (1g
54)] includes. Although these methods may support some improvement in sample quality for the microorganisms of interest, accurate microbial quantification is affected by the possible presence of antibiotics in the sample and the different nutritional requirements of different microorganisms. Failure to address the effects of sample retention time.

Another problem that remains unaddressed by previous methods for microbial detection is the possibility of introducing additional microorganisms into the sample from external sources. This "contamination" of the sample will lead to inaccurate results because, for example, the patient may be assumed to have microorganisms that are not actually present in the blood. The more frequently it is transferred from container to container, the more it undergoes physical handling. For example, system A (Becton-Dickenso
n) requires containers containing preservatives to be used due to poor urine collection. Thus, it reduces the handling required, provides a way to immediately preserve the microbial integrity of the sample, and, in preferred embodiments, provides a collection container that can be used for other processing steps in the analysis of the microorganism of interest. It is desirable to provide

Therefore, once a sample has been removed from the processing container (not yet) in a collection dish and placed on top of the growth medium, any microorganisms of interest present in the sample, including any nuisance microorganisms of interest, will be removed. minimizes the risk of contamination, reduces or eliminates the requirement for sterility of collection containers for specimens, and provides the necessary inactivation of antimicrobial agents during transport of specimens to ensure growth and identification. steps have been taken to maintain the viability of at least some of the microorganisms of interest, and preferably to maintain the microbial integrity of the sample from the time of sample collection (to) to the time of sample analysis (11). What is needed is a method and means for receiving a liquid sample suspected of containing microbial pathogens and antimicrobial agents.

Now t. Preserves the microbial integrity of patient samples and other samples such that analysis up to 72 hours later results in a much more accurate representation of the microbial population in the sample than previously possible. I found out that it is good to be done. This was accomplished by providing a mixture of individual chemicals that dissolved in the aqueous sample to form a unique mixture that acted synergistically as a preservative of the sample's microbial identity.

1 preservative is a proprietary mixture that prevents the replication of the microorganism of interest, improves the survival of the microorganism, and protects against antimicrobial factors that may be present in the sample until laboratory analysis begins. It means to prevent the action.
"Microorganism of interest" means a microorganism that is tested in a laboratory procedure.

e.g. all that may be present in a given sample,
Preserving potential microbial viability may not be necessary or desirable. For example, in the food industry, harmless or even beneficial microorganisms may be present in the food, which are of no interest for identification in the laboratory. However, laboratories are interested in testing for microorganisms potentially harmful to human health. Therefore, the latter 6 preservation of microorganisms of interest is addressed by the present invention. In addition, the growth of microorganisms of no interest is prevented from masking harmful microorganisms during analytical procedures, and grow,
We must continue to ensure that harmless microorganisms are prevented from depleting nutrients and causing the death of other microorganisms. The present invention is effective in inhibiting the replication of such potentially interfering microorganisms. Therefore, the present invention allows for a longer elapsed time between sample collection and sample analysis than was previously possible without sacrificing accuracy. It also prevents the action of antimicrobial factors that might destroy the microorganisms of interest, even if the samples are analyzed within a short period of time, even within the two-hour processing time recommended by the prior art. , allowing for more accurate analysis.

Furthermore, there is no reason why the disclosed sample transport systems would not be advantageous for improving the accuracy of analysis of samples for periods exceeding 72 hours. If we maintain the viability of some small number of microorganisms of interest, we will improve the microbial identity of the analyzed sample beyond what is possible according to the prior art, and as a result Improve laboratory analysis.

Novel methods, articles and compositions for microbial pathogen detection are disclosed. In another aspect, the present invention relates to novel techniques and means for selectively separating microorganisms from sample liquids containing antimicrobial agents. In yet another aspect, the invention relates to methods and means for use in detecting microbial pathogens that provide improved recovery of microorganisms. In aspect-specific aspects, this invention, when quantified later,
Methods and means for accurate quantification of the number of microorganisms present in a sample liquid at a given time.

A sample receiving reservoir article is disclosed that includes a means for preserving the microbial integrity of the sample.

SUMMARY OF THE INVENTION In accordance with the present invention, a device for stabilizing microbial levels in a sample is provided which comprises a container and a composition effective to preserve the microbial integrity of the sample. The composition is placed in a container configured such that the sample and composition are mixed when the sample is introduced into the container.

Also in accordance with the invention there is provided a method for the collection and transportation of a liquid sample, which comprises mixing with the sample in a container a composition effective to preserve the biological integrity of the sample. Ru. The structure of the container is such that the sample and composition mix when the sample enters the container.

Also disclosed are compositions and methods for inactivating antimicrobial agents and maintaining microbial identity in a sample after sample collection and before microorganisms of interest are analyzed in accordance with the present invention. .

According to a preferred embodiment of the invention, the mixing of the composition and the sample is carried out by means of a screw on the cap (thereby allowing the examiner or sample to remove and replace the cap, as would occur if a rubber stopper is used). A sliding piston in the container (reducing the chance of contamination) and a removable piston rod that may be removed from the piston (leaving no part of the rod outside the container, allowing the container to be placed on a test tube stand or propped up on a table). The process is carried out in a container consisting of a container or a centrifugal separator.

In addition, according to a preferred embodiment of the present invention, it is effective in preserving the microbial integrity of a sample (hereinafter referred to as a sample transport system), and the antimicrobial factor in a sample containing an antimicrobial factor and a microorganism is A special set of aqueous solution soluble compositions and methods of their use are provided that are effective for inactivating:

(1) Immediate inhibition of the bactericidal action of penicillins, cephalosporins, aminoglycosides, and antibiotics that require the growth of microorganisms for efficacy (microorganisms with troublesome nutritional conditions susceptible to the lethal effects of dioxygen) (3) Complete neutralization of the normal bactericidal effect of human blood and the original bactericidal components in other breeding cows (4) Keep the number of viable microorganisms stable during the period (5) Providing optimal nutritional requirements for the microorganism of interest.

This procedure is applicable to all types of body fluids such as blood, bone marrow, vertebral and pleural fluids, body secretions, urine, etc. as well as non-liquid samples from patients in which microorganisms may be extracted into aqueous solutions. I can do it. The microbial identity of water samples, food samples, surface contaminated samples of food preparation or processing equipment, and other samples are also preserved by the present invention. Generally, when employed with a blood sample, a cytolytic agent will be used. Mucolytic agents may advantageously be used in conjunction with saliva. Effective cytolytic or mucolytic agents are D
U.S. Patent No. 4,053°3 (
This is a detoxified saponin published in No. 52). The novel power composition of the present invention is used in a sample collection or transport container, and the microbial pathogen therein is analyzed after the sample has been collected, e.g., by a method in which the microbial pathogen is placed on top of a culture medium for the microbial pathogen. It can be mixed with the sample beforehand. The novel composition of the present invention can be in the form of an aqueous solution contained in the sample collection transport container described above. However, the novel composition of the sample transport reservoir is preferably placed in said container in the form of solid particles which are soluble in the sample liquid or optionally in the aqueous extract of the sample.

The present invention is a Method For Detecting
Microbial Pathogens Employ
No. 4, 1, issued December 16, 1978, entitled
31.

The basic method published in No. 512 is adopted.

” Apparatus For Detecting
Micxobial PattrogensEmpl
oying A Cushioning Agent”
No. 4,2, issued July 15, 1980, entitled
No. 12,948. Also in accordance with one aspect of the present invention, a novel method for assembling and sterilizing a lysis-centrifuge device is provided, which includes:

(a) placing a liquid relaxation agent and a sample transport system in the form of solid particles in a lysis-centrifuge tube, such as that disclosed in the 1948 patent; Φ) evacuating said tube; to sterilize the tube at the evaporation temperature of the liquid softener, e.g.
'C for a sufficient period of time, eg, about 30 minutes, and then cool the tube to room temperature.

Further, the system of the present invention is described in "5surface 5eparation Te" published August 14, 1979.
chnique For The Detection
of Microbial Patrogens”
It can be used to implement the lysis-centrifugation technique disclosed in U.S. Pat. No. 4,164,449. for example,
While the lysis-centrifuge tube is retained in the process, the sample may be retained in a container, such as the tube.

Surprisingly, the novel system of the present invention suppresses the replication of microorganisms contained in a sample for up to about 72 hours after the sample is collected. It is believed that replication may be inhibited in some microorganisms even for longer periods of time than the present invention is utilized.

The sample delivery system of the present invention contains very high concentrations of specific chemical compounds that serve to neutralize antibiotics and/or normal human serum factors. This increased concentration is
Conventional gravy systems currently used in many microbial testing laboratories are It cannot be easily incorporated. However, if the sample of interest has a high concentration of microorganisms, e.g. urine, the invention provides that the transport container contains the sample and the composition of the invention, and that when the analysis is started this composition is present in the gravy system. It may be possible to use it in conjunction with conventional gravy systems such as dilutions. The sample transport system of the present invention will effectively inactivate many antibiotics and other antimicrobial factors mixed in the sample liquid and will stabilize the viability of the microorganisms of interest.

In order to reduce the concentration of the inactivating chemical agent to a concentration that is not inhibitory to the microorganism of interest, the resulting mixture of sample and disclosed composition can first be diluted on a growth medium during analysis. Normally necessary. However, the present invention is particularly useful and advantageously employed in methods that require dilution prior to microbial analysis. For example, cotton swabs, saliva, urine, processed blood and the aforementioned U.S. Pat. No. 4,164,4.
49.4,131.512 and 4,221,948 generally require high dilution factors and are therefore well preserved by the present invention.

The lysis-centrifugation system described above is a suitable example for the detailed description of the invention. If the sample transport system is placed in a centrifuge tube for blood sample processing prior to centrifugation and addition of the microorganisms onto the medium, the microorganisms of interest will be exposed to antimicrobial agents present in the liquid sample, such as antibiotics and In fact, it will be protected against attack by bactericidal serum factors.

Conversely, in the absence of the present invention, microbial pathogens may be disrupted in centrifuge tubes prior to processing resulting in undesirable false negative assay results or inaccurate quantification of culture. The basic benefits of using the present invention can be explained in a sea layer diagram according to the following theory. Diagnostic signs of shock and microorganisms in the bloodstream, such as infectious vasocoagulation (blood clots) and elevated temperature (fever) and low blood pressure, sepsis does not imply infection of the bloodstream itself. In this theoretical model, there is a primary infection elsewhere, such as in the kidneys, lungs, or elsewhere, and the microorganism continues to be inoculated into the bloodstream at a sustained rate. The immune system and/or antibiotics continue to eliminate the microorganism at a constant rate. Only if the rate of inoculation is less than the rate of elimination will the patient survive the crisis of sepsis. Therefore, based on this theoretical model, once the sample is withdrawn, microbial inoculation from the primary source ceases on the sample and antimicrobial factors in the patient's blood are still active. , conventional blood culture systems produce a significant number of false-negative cultures. Therefore, during transport to the laboratory for processing, these agents may kill any live microorganisms present at the time of withdrawal, rendering the test negative. This concept is particularly important for immunologically competent patients or those on a broad spectrum of antibiotics. Therefore, in conjunction with a lysis-centrifugation system, the improvements of the present invention can be used to remove immune system and bactericidal antibiotics prior to dilution on agar plates, which is a unique feature of the lysis-centrifugation method. , by immediately blocking the known harmful effects of those factors, the microbial status of the blood sample is precisely preserved.

Employing this sample transport system for urine analysis means that it is present in the urine container from which a diagnostically relevant aliquot of urine will be withdrawn for direct microbial analysis.

Therefore, the practice of the present invention provides an accurate assessment of the microbial status of a urine sample by immediately inhibiting the known adverse effects of bactericidal antibiotics and by acting as a bacteriostatic agent even in the absence of antimicrobial agents. save.

Employing the present invention with throat culture swabs, vaginal swabs, tissue, bone marrow, and other samples also advantageously preserves the microbial integrity of the samples.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS At the outset, it should be noted that, as used herein, the unit designation 1 μg'' refers to micrograms.

By way of illustration and not mere use with respect to the present invention, embodiments of the present invention may include collection of urine or blood samples, typically
It is described as being used for storage and transportation.

Referring to FIG. 17, one aspect of the present invention is that the container 21
The device may be described as having the same shape as a typical Fisherman's hospital syringe, having a piston 230 and a shaft 220.

The closure member 250 is attached to the screws (17th and 18th) on the cap.
, 19 and 21) or a pierced stopper (Fig. 23)
(Fig.). The advantage of twisting the cap with a stopper is that the twist or screw-on cap must not come off. This reduces the chance of contaminating test personnel or samples or creating aerosols and other potential hazards in the air. When the closure member 250 takes the form of a cap, the device has a sealable mouth 270.

Composition 260 is a composition effective to preserve the microbial integrity of a sample and may be designed to prevent an increase or decrease in the number of microorganisms present in the sample liquid. Composition 260 may be in any form, such as a tablet, powder or liquid, and a preferred composition, herein referred to as a sample transport system, is described in further detail below.

Referring to FIG. 21, when collecting a urine sample, tube 285 is placed over mouth 270 and placed into sample tip 282 containing urine for sample collection. Attach the piston 230 and pull up the shaft 22o to draw urine into the cylinder.
Raise it up. Composition 260 dissolves in the urine in the cylinder. Once sufficient sample has been withdrawn, tube 285 is removed and dropped into the sample cup, thereby reducing the chance that urine from tube 285 will come into contact with anyone. Next, attach the cap 290 to the opening 270.
and close the container 290. (Figure 18) 1 No. 17
, 18 and 21, the method of removing the shaft 220 is shown in the form of a thin section 240 of the shaft 220, and as shown in FIG. ) 220 can be folded. The removal means allow the entire device to be placed upright on a table or in a rack. 20th
22, the shaft removal scheme includes a threaded connection 24 between piston 230 and shaft 220.
It takes the form of 1. Removal of the shaft 220 allows the device to be used in devices that typically require test tubes or other containers that may be placed on a rack or used in a centrifuge device.

Reference is made to Figures 19 and 22, which illustrate aspects of blood sample collection. A perforated membrane 100 may be provided to seal the port 270. Pull the piston 2θ0 to create a low pressure area within the cylinder. A stop 215 may be provided to secure the piston 230 and preserve its low pressure area. When the piston 230 is stopped at that location,
The shirt 220 may be removed and discarded. Stop 215 may also take the form of stop members 252 and 254 as shown in FIGS. 24 and 25.

Referring again to FIG. 22, needle 277 is connected to a source of liquid to be sampled. Examples of such sources include a venous connection to a patient's vein or artery, a tube extending into a conventional syringe from which the sample is drawn, or a sample tip containing a liquid. The needle 277 pierces the membrane 275 and liquid is drawn into the cylinder by low pressure. The needle 277 is then removed and the membrane 275 is resealed. A cap 290 is then placed over the mouth 270 to protect the membrane. Composition 260 may be dissolved in a liquid and samples transported to a laboratory for testing. In the laboratory, samples can be centrifuged in the device by means of the concave surface 280.

Referring to FIG. 23, an embodiment of sample collection through needle 277 is shown in which closure member 250 is in the form of a flexible stopper, composition 260 is in powder form, and container 21
0 is made of glass. The pressure within container 210 is lower than the external pressure. Needle 277 punctures flexible stopper 250 and liquid enters container 210 and mixes with composition 260. The needle 277 is then withdrawn and the flexible plug 250 seals the hole. The device may then be sent to the laboratory for testing.

The choice of container material is not critical. For example, glass or plastic may be used. Glass holds vacuum better than plastic, but plastic does not break easily. Other containers that may be used are common syringes and 5ars
U.S. Patent No. 4,459,99, issued to tedt.
No. 7, a special purpose syringe and a glass vacuum test tube suitable for puncturing with a needle through a flexible stopper.

In embodiments using powdered or liquid compositions, a one-way valve may be used to prevent the composition (or liquid with which it is contacted) from flowing out of the mouth. In the case of a powder, a screen may be used to keep the powder in the cylinder while the liquid passes into the container.

DETAILED DESCRIPTION OF THE SAMPLE TRANSPORT SYSTEM In a preferred embodiment of the invention, the composition effective to preserve the microbial integrity of the sample comprises antimicrobial agents such as antibiotics and sterilizers in the sample, e.g. normal human blood. It will inactivate the sex drug among other things. The novel sample transport system of the present invention includes relatively high concentrations of specialized chemical agents. The sample suspected of containing the microorganism of interest may be a liquid, such as blood or urine, or a semi-solid or solid in which the microorganism is collected and suspended in an aqueous solution. This may be done, for example, by swabbing a sterile cotton swab over the solid surface of interest, holding the swab, and dipping the swab into a suitable solution that is effective in supporting the viability of the microorganism of interest. good. As another example, muscle tissue can be prepared by taking a portion of the tissue and placing it in an aqueous receiving solution that can permeate and diffuse into the tissue to preserve any microorganisms in the tissue. The microorganism of interest may then be transported to a laboratory for later analysis.

Nutrients effective to support the viability of the microorganism of interest are present in the transport medium. The "effective nutrient" added to the sample can be anything from sterile distilled, deionized water to fully commercially prepared meat broth for microbial culture, depending on the nature of the sample and the characteristics of the microorganism of interest. . The criteria for being "effective" is that the sample of the invention should be prepared so that at least some of the microorganisms of interest that were alive in the sample at the time of sample collection (1,) are replicated at the beginning of sample analysis (tl). It is the ability to support the growth of a microorganism of interest up to 11 in the presence of a bacteriostatic agent added as part of the transport system. In most instances, using the present invention, the power t,'! Microbial survival is at least 50% and often more than 80%. However, according to the present invention, t
Even if the survival rate is not high, the present invention provides an advantage over the technology because the survival of microbial species in the microorganism is improved, resulting in better identification and antibiotic susceptibility testing than was previously possible. provided.

In such cases, the effective amount of nutrients will be pure water, for example if the sample is not aqueous in nature. Inclusion of an effective amount of added nutrients depends not only on the nature of the sample but also on the identity of the microorganism of interest. Furthermore,
An appropriate balance must be achieved between providing nutrients that are effective for microbial replication and using bacteriostatic agents to prevent microbial replication during sample transport. Different microorganisms have different nutritional requirements. The nutrients supplemented in the context of the present invention are used when the sample is diluted on the culture medium (e.g. agar plates) to such an extent that the agents of the present invention are no longer effective in inhibiting the replication of the microorganism of interest. , the microorganism of interest should be allowed to survive up to ζ so that the viable microorganism of interest can reproduce in order to be tested and identified.

For example, since both blood and urine naturally contain sufficient nutrients required during the period of transport of the microorganisms of interest, there is generally no need for the addition of nutrients to achieve the aforementioned results. However, if the microorganism of interest is collected with a microbial attachment device, such as a cotton swab, effective nutritional components must be provided together with the disinfectant. For example, cotton swabs are commonly used to collect samples from a patient's throat. Additionally, it may be desirable to add nutrients to the microorganisms of interest, even to samples such as blood and urine, to prolong their viability. The following specific examples demonstrate the use of effective nutrients within the sample transport system of the present invention.

Addition of a growth substrate that does not inhibit the microorganism of interest and is effective in supplementing its nutritional requirements, if the sample itself does not naturally contain nutrients with this effect. It is desirable to do so.

One effective growth substrate is Mueller-Hinto
n Gravy (B B L Microbiology
Systems, Cockeysville.

Md 21030. (available from ). This contains bovine extract (3g/l) and casein acid melt (7.5g).
/l) and starch (1.5g/l). Another effective growth substrate is Tryptic Soy Broth (BBL
Micro-biology, Cockeyvi
lle, MD 21030. It is available from

The composition of the chosen growing substrate will, if the growing substrate contains some effective nutrient that would otherwise be added separately, account for the total concentration of that particular nutrient. You should be careful to adjust the amounts as you know. For example, it may be desirable to add starch to the nutrient medium, especially if Haemophilis is the microorganism of interest. Mueller-Hlnton The gravy contains starch, so the amount added is Mueller-Hlnton.
The contribution from inton becomes tam consideration.

In order to preserve the microbial integrity of the sample, the sample transport system of the present invention combines effective nutrients and replication inhibitors that provide nutrients for the microorganisms of interest but inhibit the replication of all microorganisms in the sample. combination has been achieved. In combination with suitable replication inhibitors, nutrient additions, if necessary, have been found to be effective at approximately 0 to 10% (w/v of growth substrate per combined volume of sample and transport system). There is. The preferred range is 0.1 to 5.0'4. More preferably, it is about 1 to 3%.

If Haemophilis is the microorganism of interest, it is preferred to use starch for the sample, but starch is not believed to be necessary for all samples or microorganisms of interest. If starch is desired, about o,oos to 2. θ
% (w/ of growth substrate per volume of sample and transport system)
v) is known to be effective. More preferably about 0
．． The range is from 0.01 to 1.5 inches. A range of about 0.1 to 1.01 is highly preferred.

Agar is also a desirable but not essential nutrient.

It provides a surface for growth and keeps the microorganisms dispersed in the liquid medium. The range of agar that may be used is about 0 to 5% (by weight per total volume of sample and transport system), preferably 0.5 to about 2 inches, and more preferably 0.1 to 1.0 inches.

Effective nutrients for samples suspected of containing Haemophilis include hemoglobin. Hemoglobin is also 5treptococcus
pHeumoniae is also improved, which is desirable if this is the microorganism of interest. Surprisingly, when hemoglobin is used in the transport system of the present invention, Hae
NADP to maintain mop h i l i s
() lyphosphopyridine nucleotide) sources need not be added. Haem.

philis strains contain the so-called “X” factor and the so-called “■” factor (N
ADP). Although hemoglobin supplies the 'X' factor, the need for the addition of an exogenous source of NAD P is not apparent when using the mixture of the invention.

Inactivation of antimicrobial factors is also part of the functionality of the invention.

For example, according to one embodiment of the invention, inhibitors for aminoglucoside antibiotics and polymyxin B are included in the sample transport system.

Typical aminoglucoside antibiotics include gentamicin,
Includes tobramycin and amikacin. Aminoglucoside and polymyxin B all have a net positive charge. When this charge is blocked, these compounds lose their potency.

Accordingly, in accordance with one aspect of the invention, this positively charged blocking agent is included in the sample transport system. A preferred compound is sodium polyanetholesulfonate.

Sodium polyanetholesulfonate inhibits the effects of aminoglucoside and 4-remixin B in direct proportion to its concentration. Surprisingly, the concentration required to completely inhibit these antibiotics is at least about 0.06% w/v of sodium lepolianetholesulfonate based on the sample volume, which is not toxic according to prior techniques. It was found to be at the concentration that was taught to be present. Another such inhibitor compound is sodium amylosulfate.

The sample transport system of the present invention contains sufficient sodium polyanetholesulfonate, which ranges from about 0.06 to
6.0%, preferably about 0.1-2.0% (weight of SPS relative to total weight of sample liquid and sample transport system composition). Motsutsu and preferably about 0.3 to 1.0 qb of 5PS (sodium polyanethole sulfonate) (828 qb based on the total weight of sample liquid and sample transport system composition).
weight).

In the present invention, the harmful effects of sodium polyanetholesulfonate on microorganisms comprising: ) was removed by dilution method.

The SPS concentrations used in the present invention include aminoglucosides, streptomycin, and polymyxin B as previously discussed.
is sufficient to block the effects of High concentration of S
PS is also effective in controlling the replication of microorganisms ranging from - to t, thereby reducing the effectiveness of antibiotics that require microbial replication for activity. Since the prior art teaches that SPS is toxic to microorganisms at concentrations above 0.03%, it is surprising that SPS can be used in methods involving detection and identification of microorganisms. Low concentrations of SPS, such as those taught in the prior art to be non-toxic, are ineffective in the present invention to achieve the desired results.

The sample transport system of the present invention preferably includes a water-soluble component effective to inhibit the action of penicillins and cephalosporins, and in combination with other components of the sample transport system,
It exerts a bacteriostatic effect on the replication of microorganisms in a sample without imparting a bactericidal effect to the microorganisms of interest. Sulfhydryl group-containing compounds such as L-cysteine, N-acetylcysteine, thioglycolate, glutathione and mercaptoethanol are suitable antibiotic inhibitors of the penicillin and cephalosporin classes. Surprisingly, however, concentrations used in the past have been suboptimal in achieving the desired goal of antibiotic inhibition, and higher concentrations, which are taught in the prior art to be toxic to microorganisms, are It has the advantage of blocking antibiotic inhibitors and acting as a bacteriostatic agent in combination with sample transport system components.

It has been discovered that microbial resemblance of samples may be used as a method of preservation. Other effective antibiotic inhibitors that may be used in the sample delivery system of the present invention are enzymes specific to the antibiotic. If used, the combination of enzymes and other sample transport system components has been found to have effects not possible using enzymes alone; It will be done.

The ingredient effective in blocking the action of penicillins and cephalosporins may be used in dry form, such as salt or lyophilized form, so that it may be used in a dry mixture. preferable. However, a liquid blocking component such as mercaptoethanol may be used, if desired, as part of a liquid sample diluent provided in the liquid mode of the sample transport system or in combination with a dry mixture.

Combinations of one or more sulfhydryl group-containing compounds may be used, with particular combinations being preferred.

Approximately 8.2μ in the sample liquid and sample transport system
L-cysteine in an amount of M~8.25mM is a preferred inhibitor of penicillin and cephalosporin present in the sample transport system. The most preferred amount varies depending on the sample. For urine samples, it is approximately 0.82-41
．． 3mM0 range, very preferably 4.1-24.8m
Preferably, M is used.

For throat cultures and other samples, the preferred range is about 0.82-24.8 mM, most preferably about 0
．． 82-B, 3mM. In particularly preferred embodiments, the sample delivery system of the present invention comprises about 8.2 μM to 82 μM.
5mMc7) amount of nocysteine and approximately 0 to 42.5mM (
molar equivalents of thioglycolate based on the molecular weight of thioglycolate as the active ingredient. Contains a synergistic mixture of thioglycolate and cysteine. This combination inactivates penicillins, cephalosporins and aminoglycosides very effectively and also reduces the viscosity of the system thus formed and increases the shelf life of the dry mixture. Thioglycolate and related compounds by themselves cause an undesirable increase in the viscosity of the transported sample. However, it has been found that the combination of cysteine and thioglycolate described above, for example, greatly reduces the viscosity of blood after dissolution. Furthermore, the combination allows for a thioglycollate proportion that is less toxic to microorganisms with troublesome nutritional requirements, etc. Another advantage is that cysteine is easily oxidized and the presence of thioglycolate preserves the microbial integrity of the sample, for example during the preparation of lysis-centrifuge tubes or in sample transport system mixtures. The purpose is to help maintain cysteine in a reduced state for shelf-life stability.

Cysti/
An example of a combination of cystine and thioglycolate is an initial concentration of 1.2 wL% cystine and 0.1 wt% thioglycolate in the sample liquid and sample transport system, as disclosed in said patent. Contains a final concentration of about 0.018 wt% cysteine and about 0.002 wt% thioglycolate as final diluted on the growth medium.

Due to cysteine's tendency to oxidize, it is desirable to add cysteine during the last step in tube manufacture prior to placing the centrifuge tube in vacuum and autoclaving. Purity of cysteine is important. Due to the high concentration of cysteine required in the sample transport system, this compound should have a purity of 9 parts or higher. If cystine contaminated with cystine is used, cystine will precipitate during blood processing.

This is an undesirable property because cystine precipitates resemble small colonies of microorganisms on agar plates. The inclusion of a combination of thioglycollate and cysteine is said to protect anaerobic microorganisms (e.g. cross-) from being harmed by the oxygen present in the blood sample during transport of the sample to the laboratory. It has a second effect. This is due to the fact that thioglycolates and other sulfhydryl compounds are excellent oxygen scavengers. Cysteine is much more effective than other sulfhydryl compounds, on a dulam or molar basis, in inhibiting the bactericidal action of penicillins, cephalosporins, and the aminoglycosides that comprise them, and as mentioned above, the additional benefit of the presence of cysteine is Its purpose is to reduce the viscosity of the microorganisms, which improves the sedimentation of microorganisms in centrifuge tubes. Preferably, cysteine in its free base form is utilized to avoid the need for the addition of high concentrations of pH adjusting agents, such as the requirement for cysteine-Hct2. However, the latter may also be used.

If other sulfhydryl compounds other than cysteine are to be used in conjunction with cysteine, appropriate concentrations may be used to achieve an effect as close as possible to that of cysteine.

Thioglycolate is approximately 4.4 to 43.8 m in blood samples.
M, preferably in the range of about 8.8 to 35.1 mM, very preferably about 17.5 to 30.7 mM.

Glutathione is effectively present in the blood at approximately 1. (52)～
16.3mM, preferably about 3.25mM to 13.5mM
M, very preferably about 6.5-11.41 nM is used.

For samples other than blood, it is preferred to use higher amounts of thioglycolate or glutathione.

Thioglycolate is effectively about 4.4-52.6mM
, preferably from 8.76 to 43.8, most preferably from about 17.5 to 35. Use OmM. Glutathione is preferably about 1.6 to 19.5 mM, more preferably about 3
．． 25-16.3mM, most preferably about 6.3mM.
51-13.0mM is used.

- According to another aspect of the invention, a sulfa compound inactivator is present in the sample transport system. Sulfa compounds are believed to exert their antimicrobial effects by interfering with the bacterial folate pathway. That pathway is essential for the synthesis of nucleic acids, which are the primary compounds of microbial DNA. Therefore, preferably p-aminobenzoic acid (PABA) may be added to the sample transport system as a competitive inhibitor of sulfa compounds. The preferred concentration of PABA is between 5 and 500 microns per sample and sample transport system combined.
g/ml is most preferably in the range of about 10-100 μm.

However, the suppression of replication provided by a combination of other sample delivery system components may be limited to the presence of very high concentrations of sulfa compounds or the sample retention time at which the sulfa drug begins to exert a bactericidal effect on the microorganism of interest. Addition of PABA is only necessary in environments where it is desired to extend the range to a certain extent.

The sample delivery system of the present invention can also contain enzymes such as β-lactamases and vericilinases that react with and inactivate the antibiotics. Typically about 1 to 20 active units of such enzymes are effective in the system to provide an inhibitory effect in combination with other components of the sample transport system. Example W demonstrates the synergy achieved using enzymes with other sample transport system components.

The sample transport system of the present invention may include other compounds depending on the use of the system, for example, when processing blood, a lysing agent, e.g.
“Detoxification of Sa”
U.S. Patent No. 3,883,42 entitled "Pon ins"
It may contain purified saponins as disclosed in No. 5. The composition may also contain anticoagulants such as citrate or ethylenediaminetetraacetic acid (EDTA).

The antibiotic inhibitors of this invention serve in combination as bacteriostatic agents.

Additionally, it may be desirable to add additional bacteriostatic agents to prevent the replication of any microorganisms in the sample. The bacteriostatic agent selected should not be bactericidal against the microorganism of interest, as defined above. The choice of bacteriostatic agent depends on the type of sample and the identity of the microorganism of interest. It is also unlikely or unlikely that the microorganisms can be present in a particular sample to the extent that it is not necessary to use special bacteriostatic agents that are intended to control the growth of the microorganisms in the special system. isn't it. Thus, carbohydrates, sugars or salts, such as sulfur chloride or their equivalents, can be used in urine samples to control the replication of more rapidly growing microorganisms, such as K2±2 Emi Hongxi and Proteus. For swab collected samples and other samples, it is desirable to increase the hypertonicity of the aqueous sample or sample receiving liquid. Suitable salts include sodium chloride, potassium chloride, and ammonium salt (NH4)2S0.
4 and NI (4N03 and other nitrates, sulfates, acetates, and mixtures thereof. Suitable sugars such as sorbitol, mannitol, glucose, etc. are included. Preferably sodium chloride or potassium chloride is
~171.1 mM, preferably about 8.5-136.
9 mM, very preferably about 17.1-85.5
Used in the mixture of sample and sample delivery system in the mM range.

It may be desirable to add substances that are not bactericidal to the microorganism of interest and are effective in inhibiting the replication of Gram-positive microorganisms. For example, 5treptococ
us faecalis and 5tpptococcus
Since A. agala-□ ctiae are rapidly growing, they would not be able to be killed or isolated together with the gram-positive microorganisms, so the use of substances that are bactericidal against the gram-positive group. is not desirable. Effective growth inhibition of microorganisms without killing them can be achieved by combining sample transport system components with dyes such as brilliant green or malachite green. Also effective in combination with other system ingredients is ox bile extract (fresh bile that has been dehydrated). Brilliant green is 0-4.1 pNJ
Used within the range of It is 1006H ~ 3.3 IJ
Preferably, M is added. Very preferred is about 20
0 nM to 2.1. If malachite green is used, the concentration in the final sample solution should be about 0 to 5.5, preferably 100 nM to 4.4 IIM, and most preferably 2.7 to 27.4 A. be. Other dyes may be used at concentrations that are non-bactericidal and suppressive of Durham positives, and the suppression is reversible upon appropriate dilution.

The ox bile extract is used at about O-0.002 w/v%, preferably 0.00005-0.0016 w/v%, most preferably about 0.0001-0.001 w/v s. Since ox bile extract is completely dehydrated fresh bile from the ox gallbladder, there cannot be any molecular weight or product-to-product correspondence.

Therefore, the amount given is the product purchased from Dirco,
Estimate based on catalog #0128-02.

It may be desirable to add additional bacteriostatic agents in the sample. Calcium propionate, methylparaben, potassium sorbate, sodium nitrate and sodium benzoate are mainly used in E, Co
11. Klebsiella Oyobi Enterob
It has been discovered that it acts as a bacteriostatic agent against S. acteviaceae. These drugs generally contain approximately 0.1
~10 w/vs+preferably 0.01 to about 8. Ow/
v%.

Most preferably 0.1 to about 5% w/v is effective.

Calcium propionate is highly preferred. Based on the molar equivalent of propionic acid as the active ingredient, it is approximately Q
~42.1 rrM, preferably about 424-33.7
mM, very preferably 421.4 tt M to about 21.2 mM is used.

It is desirable to maintain the pH of the system between about 6.5 and 7.5. It is therefore appropriate to adjust highly acidic or basic samples and then buffer them with an effective pH buffer. Urine pH is one indicator of the body's natural defense mechanisms. Extreme pfl (acidity) may therefore kill microorganisms of interest in the sample prior to analysis. Extreme pH is t
may exhibit rapid replication of microorganisms masking microbial resemblance in L. However, the pH buffer must be compatible with the system. preferred pH
The buffer is sodium bicarbonate. For urine it is about 1.2-238. Om M + preferably about 2.4~
59.5 mM may be present. The concentration may be varied to achieve the desired buffering result. For other samples except blood, which generally do not require buffering, the concentration may range from about 0 to 60 mM, preferably from about 0.6 to 24.0 mM, depending on the pH adjustment requirements. .

The sample transport system chemical component is preferably a dry mixture that is used in a sample collection container for an aqueous sample and dissolves into the aqueous sample when it is introduced into the collection container. This collection container is used for sample transport and other processing steps.
It is highly preferred if it is used to reduce sample handling and reduce the risk of contamination. Examples of collection/processing vessels can be found in the Examples section. An example of the use of a dry mixture in conjunction with urine may be found in Example II. It is more convenient to use dry, aqueous mixtures for most of the sample transport system components. It is highly desirable to use L-cysteine or any sulfhydryl group-containing material used in dry mixtures to increase the shelf life of the sample transport system mixture. When using a liquid sulfhydryl compound such as mercazotoethanol, it is desirable to provide a sealed container with an inert atmosphere, such as N2 gas, to prevent oxidation.

For samples that are not aqueous in nature or are collected using absorption devices such as cotton swabs, it is necessary to use an aqueous liquid as part of the sample transport system. This aqueous liquid includes a diluent that is effective in combination with the dry mixture components to preserve the microbial integrity of the sample. With the exception of agar, optional nutrients and substances that are liquid in nature, such as mercaptoethanol, which may be desirable for certain microorganisms of interest,
All of the sample transport system components may be included in the dry mixture. Agar must be pre-dissolved in an aqueous solution by appropriate heating. One embodiment of the swab sample collection and delivery system utilizes a nutrient consisting of growth-sustaining broth and agar such that the appropriate volume of aqueous solution that receives the swab contains an effective amount of the swab and agar. In this embodiment, one compartment in the swab receiving device contains an aqueous receiving liquid, and the compartment contains a dry mixture of sample transport system components near the time the swab/sample is collected and placed into the sample transport device. It can be broken up with a cotton swab to release the liquid so that it mixes and dissolves with the aqueous gravy-agar solution. It may be practical to add the compounds that make up the sample transport system to an aqueous receiving solution rather than to a dry mixture because of the requirement that the compounds be at low concentrations. An aliquot of a concentrated stock solution of the component may be added to the aqueous receiving solution rather than mixing a small amount of the dry component with the dry mixture.

Therefore, in one embodiment of a sample transport system for samples collected by swab, an aqueous receiving solution is prepared according to the following method.

Preparation of diluent Preparation of stock solution (2) Preparation of diluent without Brilliant Green Mue
ler-Hinton gravy (MF(B) (BBL;
Cockey V i l leMd,)
4.4 agar
0.22 Starch 0.8
Preparation of Brilliant Green-containing diluent Mueller-Hi
nton gravy 4.42 agar tempura
0.21 starch 0.82 water 75- Boil the mixture and then add 20 μy/mt
Add IJ Ant Green (2N7100 dH20).

1007! Autoclave an aliquot at 121°C for 15 minutes. When it has cooled to room temperature, the color should be lime green.

50 - 25 dt in a sterile plastic conical tube and stored in a cold room at 4°C (C) 1: Preparing a 100 hemoglobin solution (2) Adding 100 - deionized water (3) Adding a stirring bar to a beaker (4) Adding the solution Stir for a minimum of 30 minutes without heating (5) to dissolve any bubbles or any floating powder on the back of the glass and completely dissolve.

Continue doing this until all the powder is dissolved.

(6) 2 horn f' paper (Whatman 934 A
The solution was pre-filtered using H-glass fiber) and filtered for 10
Clean the filtration equipment after 0-300 ml filtration. 30 at a time
Do not filter above 0-.

(7) Autoclave a 100- aliquot at 121°C for 15 minutes (8) 50-50- in a sterile plastic conical tube.
(d) Stock solution is diluent 1 mixed with 1 part hemoglobin solution.
Department.

Final concentration of raw aqueous receiving solution: 1:200 hemoglobin 0.07875 MH
B undiluted concentration 2.2Chi starch 0.55% (0.15% is the undiluted concentration of MH
From B) Agar 0.1% Brilliant Green 0.00025chi (2.5μ
2 shoulders) *GIBCODri-Form he-r-globin. Catalog 8M0023CLL-cysteine, SPS,
Make a dry mixture of thioglycolate, sodium chloride and calcium propionate giving the following concentrations in the transport system aqueous receiving solution.

L-cystiy O, 25% (2,06
m M) S P S O 6% Thioglycolate 0.01% (108,6m M
) Sodium chloride A 2.0% (34,3
3rrM) 7' o Hi, t 7-acid calcium A 3.
Ol (20,52 rrM) This dry mixture is preferably added to the appropriate volume of aqueous receiving solution at the time of sample collection.

Other aspects will be apparent from the above disclosure. It is envisioned that a completely dry mixture would be more suitable for aqueous samples such as urine or blood. A dry mixture and a separate aqueous receiving solution may be preferred for samples absorbed on cotton swabs. Yet another embodiment is a fully liquid system in which the components, normally in a dry mixture, are pre-dissolved in an aqueous receiving solution and stored in a non-oxidizing environment.

As an example of an aqueous sample collection device, a urine collection/transport device is disclosed that combines the dry mixture disclosed above with an additional pH buffering material so that the patient may urinate directly into the collection/transport device. and the dry mixture is directly mixed and dissolved with the urine sample.

The device is then sealed and transported to the laboratory. The sample volume is standardized by the instrument so that the concentration of the aqueous dry mixture dissolved is adequate to preserve the microbial integrity of the sample.

Example I Preservation of Microbial Similarity of Reconstituted Samples in the Absence of Antibiotics The reconstituted samples are Pseudomonas aerugin.
Suspension of osa (IXIO' pieces/d) (Ar
nerican 5ociety of Mi-cro
isolated from clinical specimens and fixed according to known procedures approved by
It was prepared by inoculating with a sterile cotton swab. The cotton swab was mixed with 2.26% Muel 1er-Hinton broth (MHB) (BBL Microbiology
Systems, Cockeysville, M.
d, 21030), 0.55% starch (0, 15% HMHB), 0.10% agar and 0.079%
Hemoglobin (7) Mueller-Hinton Broth Mixture (hereinafter referred to as MHBM) or any of the sample transport system compositions listed in the table below is placed in 5-. The results show that the sample transport system is effective in maintaining the microbial integrity of the samples. Viability, treated (sample transport system) or untreated (MHBM only)
It was determined by applying 0.01- of a sample of 0.01 mm to three chocolate agar plates at various time points. The number of colonies grown on each plate was counted, and the average of the three plates was taken. A survival rate of 1.00 indicates 100 chances of survival, and 1
．． Values greater than OO indicate growth, and values less than 1.00 indicate death.

The sample transport system used in the above examples was
It is clear that the microbial integrity of the samples was preserved to allow quantification of the number of microorganisms of interest 72 hours after sample collection. Without the use of the sample transport system of the present invention, uncontrolled growth of microorganisms will occur. For example, at 24 hours, samples without the present invention showed a 58-fold increase from sample collection to the time of analysis. A laboratory that analyzes a sample without the addition of a sample transport system can expect to obtain false positive results for microbial populations that were present in the sample at the time of sample collection. Even a time as low as 4 hours below the collection time will skew the results.

With transportation system composition 1. ool, 020.791
．． 081.020.66 Transport system without composition 1,
00.2.91 Q6go 58.1358,135
8.13*2 qb NaC7; 3% Calcium Propionate: 25 Thicysteine; 2,5xlO% Friliant Green: 0.6 Chi8PS; 0,01% Thioglycolate; 2.2% Mueller-Hinto
n Meat juice; 0.55% starch (0.15% Mueller
-) From 1inton Broth) ; o, t %
Agar: 0.07875% hemoglobin (all wt% of total volume).

Example ■ Preservation of microbial susceptibility of reconstituted samples in the presence of antibiotics Reconstituted samples are prepared as described in Example ■.

Test the same sample transport system. Antibiotics are added at the expected average highest serum level concentration. Without the present invention, the microbiological integrity of the sample begins to clearly deteriorate even 4 hours after sample collection. In the table below,
It can be appreciated that false negative cultures are quite possible. Quantification without using the compositions of the invention would be highly inaccurate.

Example 1 Synergistic Effects of Combined Sample Transport System Components on Preservation of Microbial Similarity of Reconstituted Samples Reconstituted samples were
Aeru 1nO8a was prepared as described in Example I using the microorganisms listed in the table below.

The survival results show that the combined components of the sample transport system have a synergistic effect compared to the individual components. For example, in Tables 1-4, the sample transport system maintains survival at a relatively constant level over time. Growth is occurring for other individual treatments, and in some cases overgrowth of the microorganism dominates the plate (
TNTC value). This overgrowth may be particularly unsatisfactory if multiple microorganisms are present, as is the case in the actual sample. In Table 1-1, it can be seen that SPS, NaCA' or MHB when used alone do not allow quantitative survival at 24 hours.

The following are tested alone or in combination with other ingredients:

Mueller-Hinton gravy (■ field) beef oil extract
[1,3 ticazeic acid hydrolyzate 1.75 Ti starch 0.15% Mueller-Hinton gravy mixture (MHBM)
Mueller-Hinton gravy 2.0ch starch 0.
55% (MBB7b40.15%) agar
0.1o Hemoglobin 0.07875 Tibrilliant glycine Mueller-Hinton gravy mixture Mu
Eller-Hinton gravy 2.2 starch
D, 55 ts (from MHB., 15%) Agar o, 1 ots Hemoglobin D -07875% Brilliant Green 0.00025 ts Transport System Brilliant Green Mueller-HintOn
Meat juice mixture + 0.25% cysteine + 0.64r sp
s +2% NaC1! +0.11 f og! J All numbers following the Korat microorganism body are America.
nType Culture Co11ection
Culture numbers from Rockville, Maryland are shown. sps = sodium polyanetholesulfonate.

Table 1-1 Individual components [146-8240,5 Ticysteine 1.00 1.02 0.
98 2.050.6 sps 1.
00 1.10 1-07 0-2% NaC11-
DO1-230-210-006Mueller-Ho
-0O6 gravy 1. Q:l O,870,84
0.04 Mueller-Hinton gravy mixture 1
．． 00 1.64 3.60 7.85 Brilliant Green Mueller-1,001,321-411-
(12) JzntOn gravy mixture combined ingredient delivery system 1.00 1.01 0.
97 0.76 Table 1-2 0.5 Ticysteine 1.00
0.65 0.19 0.030.6%sps
1.001.11 1.022.722%Na
C11,000,941-210,72Mue 11e
r-Hinton gravy 1-00 1.09
1.62 x Mueller-Hinton gravy mixture 1,00 1.3 (52). 33 8.10 Brilliant Green Mueller-1,001-040
,980,69H1ntOn Meat Juice Mixture Combined Component Transport System 1-00 0-90 [1,850-6
6TNTC=Individual components in Table 1-3 that are too large to count 0 4 6-8 240
．． 5cysteine 1.00 1. [
]8 1.21 3. (510.6 sps
1.00 1.05 1.54 2. LgJ2
alb NaC11,l]:11-11 1-12 1
-82Mueller-Hinton gravy 1.
00 1.34 1. (51 6.90Mueller
-HintOn Gravy Mixture 1,00 1.56 1.
95 4.32 Brilliant Green Mueller-
1,001,5! i 1,86 1.54I(in
ton gravy mixture combined ingredient transport system 1.000.94 0.9
1 0.68 Table 1-4 Individual components 0 4 6-8 240
．． 5cysteine 1.00 1.3
0 1.46 4.080.6%sps・
1.00 1.65 3.73 Public 2% NaC
”l 1.00 1.44 2-3
2'IWmMueller-Hinton gravy
1.00 2. al 4.57 TNrcM
Ueller-Hinton Meat Juice 1.01 1
．． 9814.11 48. '43 Brilliant Green Mueller-1-On 0-94 0-93
0-35 Hinton Gravy Mixture Transport System 1.00 0.94 0.
860・Gloss TNTC=Too many cylinders to count 1-5 Table 0.5 Ti Cysteine 1.00 1.
40 2.00 Real Q, 6% SPS
1.00 2.31 3.98 'I[2
%NaC11,001,C03,08 resistance τMuelle
r-Hinton gravy 1.00 2.99
7.18 rMueller-Hinton gravy mixture 1.00 11.14 24.8360. SiBrilliant Green Mueller -1,001,59
2,012,83 HintOn Gravy Mixture Combined Ingredient Delivery System 1. [Il 0.92
0.85 2.49 TNTC = too many cylinders to count Individual components in table N-6 0 4 6-8 240
．． 5% Vsfin 1.00 3.04 6. (52)
'IWI'CD, 6% SPS 1.
00 4.0218.20 'INI'j:2% 5
alt i, oo 2-33 6-
29 TNrCMueler-Hinton gravy
1.00 3.66 15.40 Actually Muel
ler-Hinton gravy mixture 1.00 4.40
28.4459.87 Brilliant Green Muel
ler-1-On 2-62 6-16 72.46
HintOn Gravy Mixture Transport System 1.00 0.98 1. Q
3 0.67 TNTC = too many cylinders to count 1-7 Table 0.5 Ticysteine 1.00 2.
71 5.68 Actual [1.6% SPS
1.004.87 Vcffi296 NaC11
,003,417,20TINI'j:Mueller
-Hinton gravy 1.00 4.90
Chu■yτMue 1ler-Hinton gravy mixture
1.00 5.33 53.0996.41 Brilliant Green Mueller-1-Do' 6.87
31-9859.17HxntOn Gravy Mixture Transport System 1. oo 1.08 1.
03 0.70 TNTC = Example too many to count ■ Preservation of the microbial fragility of a throat swab sample from a normal supplier spiked with pathogens together with the normal flora found in the throat The effectiveness of the sample transport system of the present invention in preserving the microbial integrity of throat swab samples containing known amounts of pathogenic microorganisms is shown in the following table. Overgrowth of normal flora has been shown to mask pathogenic microorganisms in the sample for analysis.

Normal throat flora was collected from 20 healthy donors (3 swabs per donor). The swab was then inoculated with macropathogens 104-106.

The microorganisms present on each swab are then extracted at time 0 by vigorous agitation in the selected transport system 5d. The swab was discarded and the liquid portion was collected for subsequent quantitative analysis at 0, 4.6 and 24 hours in an attempt to determine the relative survival of the pathogen relative to the normal 70-l excess that was present on the swab. kept at ℃. Next microorganism 'E -' C011+ P -a e r
ug l nos a * S -a ga la
Ctla e+ H-1nfluenZae+ S-m
, E-cloacae + K.

pHeuwcOniae+ s, aureus, S
, faecBlis, was tested.

Regarding the transport system of 5tuart, the overgrowth of normal flora within 6 hours made elucidation difficult.

The low growth (0.39) observed at 24 hours could be due to either death of the pathogen or screening of the microorganism by excess normal 70-L. Similar results were observed with the Am1es transport system. The amount of overgrowth will vary depending on the pathogen being analyzed. Microorganisms with difficult nutritional conditions (e.g. Hae
mophllis xllfluenza) is more sensitive to overgrowth. Excessive growth of normal throat flora is effectively inhibited by the sample delivery system of the present invention, which prevents overgrowth of pathogens by normal flora over a period of 24 hours without the presence of antibiotics. .

Addition to agalac t 1ae Sample transport'/Sfm 11. (D 1.00
0.87 0.83 normal flora + 5 treptococcus
ccus 1. [ ] [1i, Q9 -3 -”a
The sample transport system used in this example was 1.5% Na
Cl, 2.0% cysteine, 0.6% sps and 0.01% thioglycolate.

2Stuart et al., “The Problem of
Transport ofSpecxmens for
r Culture of Gonococci”,
45Canadian J, or Public H
S published in earth, 73 (1954)
Tuar t system.

3. Overgrowth of normal flora made accurate calculation difficult.

Example V Preservation of Microbial Toughness of Reconstituted Samples from To-0 to T1 = 4 Hours in the Presence of Antibiotics In many manuals, samples should be analyzed within 2 hours after collection. I am recommending that. However, this assumed safe time may not be appropriate in all situations. The present invention represents a significant improvement over prior art delivery systems that do not prevent significant deterioration of microbial susceptibility even within a short time of 15-20 minutes.

Am1es transportation system, C, Am1es etc., 58C
anadian J, Public Health,
296 (1957) (Curtin Mathes
The formulation (11 parts distilled water) is as follows.

-Sodium chloride 3. DI-Potassium chloride 0.2I-Calcium chloride
0.1g-Magnesium chloride 0.
1 Ii Meerin dihydrogen potassium 0.2 1
! -Dipotassium hydrogen phosphate 1.15 g-
Sodium thioglycolate 1. OI-agar
7.37 IiS tuart transport media, 45 Canadian J, Public
Heae'th 73.75 (1'956) is 6i BactO agar in distilled water 1900 ml as follows:
2 ml brought to pJ (7,2) using IN NaOH
Thioglycolic acid. In addition, 20% of 100 Jll/(in water"/r % > sodium glycerophosphate and 201+
17 of CaCl2 (1”/% in water) ■ Add CaCl2. 1 W/v% of 20+ILl
CaCl2 was added and the solution was diluted to 1NH (pH 7,4 in J).
Make it. Then add 41 0.1% methylene blue.

The sample transport system of the present invention, described in the diagram below, has the following formulation.

2 t NaCl 0025 t L-cysteine (free base) 3 t PropioΔcalcium 2.5 X 10-' % Brilliant
Green 0.64 5PS 0.01% Thioglycolate 2.2
% Mueller-Hinton gravy 0.55 g Starch 0.1 % Agar 0.7875 g Hemoglobin Antibiotics were added at average peak serum levels as determined by published reports. These values are shown in Example 1, Table Vl-2.

Microbial count/d levels were tested at each time shown on the graph (Figures 11-16).

In Figure 11, En
The microbial integrity of reconstituted samples containing T. cloacae is known to deteriorate within -20-30 minutes in the presence of the antibiotic tobramycin 40 pg/1111. The control sample transport system keeps the number constant over time.

In FIG. 12, it can be seen that the sample transport system shows excellence after 4 hours, exceeding the 2 hours recommended for sample analysis with conventional techniques.

In Figure 13, Escherichia Co1
One reconstituted sample was tested. The sample transport system showed excellence in maintaining the microbial integrity of the sample in the presence of 21 μg/w4 amikacin.

Figure 14 shows 5 treps using the present invention compared to the conventional system in the presence of 21 μg/at ampicillin.
describes the preservation of the microbial acuity of Tococcus pHeumoniae.

Figure 15 describes the effect of 100 μg/ml of moxalactam in reconstituted E, coei samples. The sample transport system can preserve microbiological integrity beyond the two hour transport time.

Figure 16 shows Klebslella in the presence of cephalothin.
The influence of the sample transport system on pHeumonia is described. Am ze s and S tuar
The T system accepted a slightly higher amount of inoculum than the sample transport system, but the former two had a lower number of microorganisms/ml after 3 hours.
shows a dramatic decline in

Example ■ Relative average microbial accuracy (swab) sample transport is Am
erican Type Culture Co11e
ctzon (ATCC), Rockville,
Md, to obtain a microbial pathogen, and one pathogen l
Test by inoculating x10' onto multiple sterile swabs. Each inoculated swab was injected with 0-25% (2-06mM) L-cyx
Nine (free base), 0.6% SPS, 0-019
b (108-6mM) thioglycolate, 2-096
(34-22mM), 3.0% (20,52mM) calcium propionate, 2.2% MBuller-
Hinton broth, −0.55% starch, 0.1% agar, 0.7875% (1.2 μM) hemoglobin and 2
．． 5 X 10-'% Brilliant Green (0,5
μM), or 45 Canad
ian J, of Public Health 73
(1954), the transport medium or Am
i e s transport medium (without charcoal) (58Canad
ian J, ofPublic Health 29
6 (1967)).

A specific concentration of the selected antibiotic or no antibiotic is added to each individual aqueous preparation. Antibiotic concentrations are selected according to published values of the average highest serum values that would be found in a patient. This level is shown for each antibiotic in Table Vl-2. (10 times the antibiotic average peak serum level is used for urine samples not tested in this example). The number of bacteria in each sample was determined by transferring 0.0111 to each of the 6 chocolate plate media at 4 times for each of the 3 transport solution preparations, culturing at 67°C for 24 hours, and counting the number of colonies. Calculate and determine the number of viable microorganisms.

In the chart below, a value of 1.00 is 100 chances to survive. At time 0, all test samples show a value of 1.00. Values greater than 1.00 indicate microbial replication that occurred during the transport period multiplied by a factor of 1.00. A value less than 1 indicates that the number of microorganisms has decreased during transport (mortality that has occurred).

A value of 0.5 therefore indicates that half of the original number of microorganisms has been lost. The values in the chart are averaged for the Durham-negative organisms tested (see chart below) and the Gram-positive organisms tested for a given antibiotic group.

] 1fill@J-N'')! 9
%-/'-' Referring to Figure 1, both C
The above-mentioned rice m! is incorporated by reference in the pending application of
No. 4,131,512 and its division, FF No. 4,212,948, disclose a centrifugal article 20. The combined patents are directed to methods and apparatus that provide improved rapid quantification of blood samples for the presence of microbial pathogens. The blood sample is lysed, deposited onto a dense, immiscible, hydrophobic, non-toxic liquid softener, and centrifuged. The microbial pathogens contained in the lysed blood sample collect in a layer close to the interface between the relaxation agent and the blood sample remnants, and in this concentrated form can be cultured and quantitatively enumerated.
It can be easily separated from the rest of the blood sample.

As shown, the article 20 includes an elongated tubular centrifuge vessel 22', which has a conventional injectable closure member 24 hermetically closing its upper end and sealingly closing its lower end. There is an injectable closure member 26 and a metal holder. Article 20 contains an effective amount of emollient 28. If an elongated tubular centrifuge vessel 22 is used, the sample transport system is deposited as a layer 30 of particulate solids on the relaxation agent 28. Although the sample transport system is contained in an aqueous solution, e.g. solid granular powder 30
is insoluble in the liquid relaxation agent 28 and has a higher self-stability than the liquid solution formed by its components. In addition, the particulate solid sample transportation system includes article 20, described below.
Enables new sterilization techniques to be carried out internally. In a preferred embodiment of the invention, the sample transport system comprises a sample transport system which, when the sample liquid, e.g. Cysteine is approximately 0.5
~2.5 wt%, approximately 0.1-1 thioglycolate
．． Sufficient is present in the aqueous solution or layer 30 to contain about 6 Wt % and about 5 to 500 μg of p-aminobenzoic acid. Furthermore, since this particular embodiment is used for processing blood samples, the total volume obtained contains about 0.02-i wt% purified saponin and about 0.01-Q EDTA;
5 wt Chi? Also includes. When the sample transport system is in the form of a water bath, the centrifuge container 22 collects blood at a rate of about 7.5 cm.
ml! Bring it in. Preferably, the sample transport system accounts for at least 3 voj%, preferably about 5 to 3 Qvoj%, of the total liquid in the centrifuge vessel 22, which includes the sample transport system, sample liquid, and buffer. .

When the sample transport system is in the form of a granular bed 30, the elongated tubular centrifuge vessel 22 draws approximately 8 rnl of blood.

In the most preferred embodiment of the invention, layer 30 includes 0.0967 g cysteine and 11.008 g thioglycolate.
1 Sodium polyanethole sulfonate 0.048y1
Purified saponin 0.018 g and EDTA O,00
Contains 8y. It should be noted that EDTA is not necessary to prevent clot formation as long as adequate amounts of polyanetholesulfonic acid are present. For example, another satisfactory blood treatment system (layer 30) includes 0.048 g of sodium polyanetholesulfonate, 8 g of cysteine, 0.009 g of thioglyflate, and 0.0 g of purified saponin.
19 f! Contains.

The combination of sample transport system and urine preferably has a phantom of 0.6 to 2. about 0.5 to 2.5 wt % of the free radical cysteine and about 0.5 wt % of the free radical cysteine and about 2 wt % of the thioglycolate. Sodium carbonate and about 2.5 to 4. OWe contains % sodium chloride.

Bicarbonate (IJ) is added to the urine sample transport system to regulate the normal acidity of the urine and maintain neutrality.

Addition of salt, for example in the form of sodium chloride, increases the bacteriostatic efficacy of the system in the absence of antibiotics in the urine. The free base OL-cysteine, for example IC'N cysteine, is bicarbonate in the L-cysteine-sodium bicarbonate mixture, as previously found).When combined with the IJum buffer it does not produce a gaseous reaction. Therefore, it is preferably replaced with the previously employed L-cystine-HCl.

Centrifuge vessel 22 can be made of siliconized glass or hard plastic such as polycarbonate or polypropylene. Injectable closure member 24
and 26 can include a rubber housing sealing plug. Both injectable closure members 24 and 26 each have a notch to increase the ease of injection with conventional needle types.

The evacuated space 32 has an injectable closure member 2 therein.
4 and 26 from the opening of the centrifuge vessel 22 without creating an overpressure that would dislodge them from the opening of the centrifuge vessel 22. , maintained below a predetermined atmospheric pressure.

With particular reference to the injectable closure member 26 at the lower end of the centrifuge vessel 22, it is noted that the inner surface 34 of the injectable closure member 26 is oriented at an angle to the wall of the centrifuge vessel 22. .

Note that article 20 is specifically designed for use in angled rotor centrifuges, and that angled interior surface 34 is a complement to the angle of the rotor. However, it should be noted that the apparatus of the present invention can be used in conventional rocking packet drop-down centrifuges. In the latter instance, it should be perpendicular to the bottom of the article 20 and is otherwise used in the same general manner as described below with respect to the article 20 described in FIG. Surface 34 should be smooth and substantially free of porous spaces and crevices in which microbial pathogens can become trapped. Furthermore, the circular sealing area around the circumference of the surface 34 where the injectable closure member 26 mates with the wall of the centrifuge vessel 22 is such that the interface forms a large circle in which microbial pathogens can enter and temporarily remain. It should be tightly sealed to avoid creating circumferential cracks.

The angle of inclination of the smooth surface 34 with respect to the wall of the centrifuge vessel 22 is determined according to the centrifuge in which the article 20 is centrifuged.

As mentioned above, when using a swinging packet drop centrifuge, the surface 34 will be perpendicular to the bottom of the article 20. However, when using an angled rotor centrifuge, surface 34 has the complement of the rotor corner. Therefore,
Generally for a rotor angle range of about 60 to 10 degrees, the surface 34
The angle of inclination in the centrifuge vessel accordingly ranges from 30° to 60°. The angle of inclination shown by arc 36 is therefore generally a complement to the angle at which device 20 is placed in the centrifuge during centrifugation. For example, the tilt angle 36 described in FIG. 1 is approximately 34 degrees. Therefore,
For example, if article 20 is placed in an angled rotor centrifuge where centrifugation occurs at approximately 56 degrees, the liquid contained in article 20 will be forced onto surfaces 34 that are at a substantially right angle.

The amount of softener 28 used should be sufficient to completely cover surface 34 during centrifugation.

The amount of emollient used can vary depending on the parameters of the particular system chosen, such as the stopper design, the volume of residual blood, and the volatility of the emollient used. A preferred amount of emollient can include about 3.3-4 Q vol% of emollient and volume of residual blood sample removed from article 20 and tested for the presence of microbial pathogens.

Generally, the emollients of the present invention may include dense hydrophobic, water-immiscible liquids. As previously noted, high density 1, as used herein, refers to bodies that are unsupported by a mixture of blood and blood processing fluids or any other sample fluid suspected of containing microbial pathogens in the presence of centrifugal force. say. Additionally, the mitigating agent should be nontoxic to microbial pathogens and relatively inert to butyl rubber, silicone rubber, and other types of elastomers used in the manufacture of injectable closures. The density of the relaxant is 1c! rL
Approximately 1.2-2. You can benefit from the OS. Generally, fluorinated hydrocarbons having the properties described above and having molecular weights between about 300 and 850 are preferred. Moreover, it has the above-mentioned properties and 771.
Vapor pressure at 1 atm 0.06psi (0,3II
Hg) to approximately 0.58 psi (30iIIHg),
Preferably it has a vapor pressure approximately equal to that of water.

Therefore, it has the above-mentioned properties and has a boiling point of about 200-420'''F (
93-216°C), preferably about 225-280″F
'(106-138°C) can be used.

The emollient preferably has a temperature of at least 0.2 at 77°F and 1 atm.
We are looking for a specific heat equal to or greater than g -caz/fi 0C, most preferably equal to or greater than water. The emollient also provides a vapor pressure such that it does not shatter the injectable closure from the tube during the manufacturing step, e.g. > J-) creping. 3
M Company of Minneapolis
.

Minneso brand name: FLUORINERT
It has been found that fluorinated hydrocarbons sold under Especially FLUORINER
T series types FC-75, FC-48 and FC
'-43 is particularly useful.

Although the exact function of such mitigating agents is not completely known, it is believed that they improve the collection of microbial pathogens that are transferred from suspension into blood samples centrifuged by at least one method (25). . First, the emollient acts to seal the interstitial spaces, crevices and crevices of both the smooth surface 34 of the centrifuge container 22 and the interface between the centrifuge container 22 and the injectable closure member 26. Thus, microbial pathogens that would otherwise be trapped in such interstitial spaces and thus not recovered are recovered by the mitigating agent 2B when removed from the article 20. Second, the mitigating agent is believed to act to cushion the impact of microbial pathogens that are shed from the turbidity in the blood sample during centrifugation. The moderating effect of O reduces the risk of damage to microbial pathogens that would otherwise occur during impact. Furthermore, while some of the microbial pathogens do pass into the mitigating agent, virtually none pass through it completely, with the majority forming a surface at the interface between the mitigating agent 28 and the blood sample. , gather in one layer.

After the relaxation agent 28 is placed into the centrifuge article 20, the blood reservoir sample transport system 30 may also be placed therein.

Once the sample transport system 30 is placed into the centrifugation article 20, the injectable closure member 24 is placed in place;
The space 32 has a desired pressure lower than atmospheric pressure, for example 25 columns of mercury.
Can be evacuated to ~3 in. According to one embodiment, centrifuge vessel 20 is then sterilized using the novel technique. If the centrifuge container contains FLUORINE
The interior of the tube, and the solid particle sample transport system 30, is heated to the vaporization point of the RT material, e.g., at least 120° G, and held for a light minute time, e.g., about 30 minutes, and the solid particle sample transport system 30 is thermally
It has been discovered that O can be sterilized with RT steam. After one degree of cooling, the centrifuge container 20 is simply cooled to room temperature and packaged, eg, for sale.

Referring to Figures 2-9, the analysis sequence is diagrammatically set forth, illustrating a preferred embodiment of the invention. For example, for the detection of microbial pathogens in blood samples, procedures performed according to embodiments of the present invention can be conveniently carried out using the following equipment:

Said centrifuge article 20 containing a relaxation agent 2B and a sample transport system 30, said container having a volume of 12-1411 LI
! I can get it.

Sterile glass syringe and one 1”/2 in 21?
-Disposable hypodermic needle.

One sterile glass syringe and one 1 in 18 DESO disposable hypodermic needle.

One 5/s in 25 gauge hypodermic needle with cotton tip (used as extraction port) 25) Blood agar plate 25) Chocolate agar plate.

It is noted that centrifugation article 20 or an equivalent article can be used and well-known laboratory equipment and culture media can be used for carrying out the novel method of the present invention. In particular, it is noted that the above-mentioned culture medium is only exemplary and it is generally preferred to use one that is not capable of detecting the most commonly known microbial pathogens. The blood agar plate medium mentioned above is basically a mixture of sheep blood and basic nutrients such as brain and heart infusion (inf
A blood plate is used, which is maintained on an IJ dish with an agar solidifying agent.

Chocolate agar plates are designed for the growth of difficult pathogens such as Hemoph 1lus in some nutritional conditions.

Thus, although a variety of equipment may be used in the method of the present invention, the methods and materials listed above may be conveniently used within the scope of the present invention in the following manner.

To use the centrifuge article 20 described in FIG. Reason 34
Wait for the injectable closure member 26 to fit over the lower end of the article 20. In practice, due to handling such that the sharp layer of 25) is not always present, mixing of the relaxation agent 28 with the solid particles of the sample transport system 30 may occur.

The unstable mixing of the relaxation agent 28 and the sample transport system 30 results in rapid dissolution of the solid system 30 into the aqueous sample (blood) and rapid separation of the resulting liquid phase in step 25 by centrifugation. Therefore, the method described here does not have any disadvantageous effects.

Next, a predetermined amount of blood sample taken from the patient, for example 8 LA.
' is injected using a conventional type syringe 4o into the evacuated space or centrifuge article 20 shown in FIG. Or another Bect. n Dickinson Trademark'V
Using standard post-needle equipment with a conventional vacuum blood collection device, such as those sold under 'AcuShiner',
It is also possible to draw the sample directly into the article 20. Then, the article 2o containing the blood sample 38, the sample transport system 30, and the emollient 28 is assembled, ensuring that the anticoagulant, the red blood cell lysing agent, and the sample transport system 30 are thoroughly mixed with the blood sample 38. Add to mixture. This stage of mixing is illustrated diagrammatically in FIG. This mixing step ensures that the sample transport system 30 containing the lysing agent is thoroughly mixed and lysed by the blood sample. This lytic action ensures contact between the antimicrobial agents and the chemical components of the sample transport system 30, thus ensuring that any pathogens contained in the blood sample 38 are protected from antimicrobial activity.

After the blood sample 38 is processed in this manner, the microbial pathogens in the processed blood sample 42 pass out of suspension and the dense relaxation agent 2B
and centrifuge to collect at the interface with the remainder of the sample liquid. The microbial pathogen actually deposits on the sidewall of centrifuge vessel 22 near the top of smooth surface 34 at point 22a. This centrifugation step is represented schematically in FIG. The speed and time of centrifugation can vary widely depending on the material of construction of centrifuge article 20 and the type of centrifuge device.

The centrifugation is performed at approximately 1500-6000 G for centrifugation article 20 containing treated blood sample 42 and relaxation agent 28;
This is conveniently carried out preferably by applying about 1500 to 3000 G. As shown in FIG. 5, an angled rotor centrifuge is used which places the centrifuged article 20 at an angle of, for example, 56 degrees (indicated by arc 43) during centrifugation. Therefore, if the smooth sloped surface 34 is 34 degrees to the inner wall of the centrifuge, the processed blood sample 42 and relaxation agent 28 will be forced onto the sloped surface 34 at a relatively vertical angle during centrifugation. . When using a rocking packet centrifuge, the centrifuged article 20 is centrifuged at substantially 0° relative to a horizontal plane. Therefore, in such a case, the angle of surface 34 would be approximately 90° and injectable closure member 26 could be replaced by an injectable rubber closure member with a flat internal surface.

Once the centrifugation step is complete, the centrifuge article 20 can be removed from the centrifuge and the majority of the processed blood sample 420 from which the microbial pathogens have been separated can be removed. As used here, the term acoustic residual treated blood.

Alternatively, "residual blood 1" refers to a blood sample that has been centrifuged in such a way that any microbial pathogens that were present are collected at the bottom of the sample, thereby leaving a residual portion of the sample substantially free of microbial pathogens. This step 6. For ease of removal, an injectable closure member 2 is provided with a pull-out needle 44, for example in the form of an ordinary hypodermic needle with cotton wool attached to the shank end.
Pass through 4. A second hypodermic needle with syringe barrel 45 is then passed through the injectable closure member 26 to remove the majority of the residual processed blood sample 42 from which the microbial pathogens have been separated. For example, if the centrifuge container has a volume of 12
~If you have 14 go, about 1.3~1.71n! 1'/ to remove all processed blood samples 42 except
A 2 in 18-dage needle can be used. As shown, the majority of the residual blood sample drawn from the interior of the centrifuge vessel 22 is located above and within the liquid interface of 25) and near the top of said sloped smooth surface 34.
To avoid disturbing the layer of microbial pathogens that forms on the sidewalls of centrifuge vessel 22, it is preferred to draw at a point opposite the sidewall near the top of the sloped smooth surface 34.

Most of the residual blood is removed at this stage. However, a small amount of residual blood should remain in the centrifuge container 22 so that the relaxation agent is included in the remaining liquid by approximately 3.3 to 40.0 vot%. Large amounts of such mitigating agents, such as approximately 20 vol.
It is preferred that the emollient is no more than %.

Once the majority of the residual blood sample to be processed has been removed, both needles may be withdrawn from the injectable closure members 24 and 26, and the centrifuge article 20 is removed, as shown diagrammatically in FIG. Subjected to some second mixing. However, if desired, the withdrawal needle 44 can be left in place on the injectable closure member 24 to assist in removing the fluid-containing pathogen at a later stage. The second mixture is separated from the bulk of the remaining processed blood sample 42 and serves to resuspend the microbial pathogens forming the layer. Resuspension of the collected microbial pathogens in the remaining small portion of the residual processed blood sample 42 ensures more or less uniform recovery.

Once the mixing stage is resuspended, a small portion of the remaining processed blood sample 4
The microbial pathogens in 2, the mixture of microbial pathogens in the residual processed blood sample and the dense softening agent can be removed from the centrifugation article 20. This stage is illustrated in FIG. As noted above, if desired, an extraction subcutaneous injection needle 44 may be inserted through the injectable closure member 24 to facilitate removal of remaining constructs. A syringe barrel fitted with a hypodermic needle can then be passed through the syringable closure member 26 to withdraw the mixture of palliative agent 2B, a small portion of the remaining residual blood sample 42, and the microbial pathogen.

Note that better retrieval is achieved when the hypodermic needle used for construct removal is injected into the lower end of the angled smooth surface 34. It is believed that the angle of surface 34 acts, in part, as a funnel through which residual liquid containing microbial pathogens flows. This mixture 48 of dense liquid softener 28 and the remaining small portion of residual processed blood sample 42 with recovered microbial pathogens should be approximately 11/2 rnl of liquid. This liquid is then distributed onto a suitable growth medium. And this step is illustrated diagrammatically in Figure 9.
ing. Using the apparatus described above, the material can be distributed as follows.

Blood agar plates receive 0.4 - of the aqueous solution and are incubated at 36°C in an anaerobic environment. The chocolate agar plates of 25) receive 0.41 g of the aqueous solution and are incubated at 36° C. in candle jars. The growth medium should be inspected daily for the presence of colonies. Microbiological analysis techniques may be used. blood l v
The number of microbial pathogens in a tl can be determined by multiplying the number of colonies by a factor. This correction factor takes into account the recovery of a given microorganism, the volume of blood, the amount of dense relaxation agent employed and the amount of final mixture applied to the medium. In the general example given above, this correction factor is 0.5.

The foregoing operations result in a dilution of at least about 1:60 on the growth medium of the remaining small portion of the residual processed blood sample 42. This ensures that any residual amount of chemical in the sample transport system is diluted to such an extent that it does not inhibit the growth of microbial pathogens therein. The sample transport system of the present invention neutralizes or suppresses sterilizing agents. For example, sodium polyanetholesulfonate generally neutralizes and cysteine generally inhibits. Furthermore, the effect of oxygen on cysteine after removing the sample from centrifuge vessel 22 destroys its inhibitory effect on microorganisms. The dilution operation described above is performed on the drug and/or sample transport system.
It may be necessary to dilute the ingredients to a level that is neither bactericidal nor inhibitory to microbial growth.

Furthermore, for antibiotics that exert only an inhibitory effect on microorganisms that may be present in a blood sample, but not a bactericidal effect, a dilution of 1:60 is generally adequate to reverse the inhibitory effect on microorganisms. be. An example of this group of compounds is cancer? This is IJ Shin. Therefore, a 1:60 dilution generally prevents inhibition of the growth of many microorganism/antibiotic combinations. Nevertheless, there are certain microorganisms that are particularly sensitive to the bactericidal or inhibitory action of certain groups of antibiotics. For example, if the highly sensitive strain S. aureus (minimal suppression?
(concentration 0.24/ml) and the blood sample was treated with sodium polyanetholesulfonate, p
-Antibiotics that cannot be blocked by aminobenzoic acid or cysteine-thioglycolate containing 20 μg/rnl normally give a blood sample of 0.4 μg/rnl.
If the above method of adding ml of antibiotic is followed, the microorganism will not grow on conventional agar plates (medium 2C]tl). This combination gives a final dilution of approximately 1:60. Therefore, in this example, 0.3% of the antibiotic applied to the plate would inhibit the subsequent growth of the S. aureus strain.
Give a final concentration of 6 μg/rnl. Furthermore, the dilution of the antibiotic is not instantaneous and initially high levels of the antibiotic at the surface of the agar plate have a lethal effect. In order to avoid this problem and still preserve the known advantages of improved lysis-centrifugation techniques using the novel sample transport system of the present invention, one additional modification is needed: larger Petri dishes. required. The clinical laboratory is one for testing antibiotics.
A 50III x 20 plate medium was used in all cases. This plate medium has 60-80ml of medium! It contains il- and has a surface area 2.25 times that of the conventional 1001+(1)X201+III Petri plate medium. When a 0.4-sized blood sample is uniformly streaked onto the surface of a large O2 plate, there is a 2.25-fold increase in the diffusion rate and approximately (1:200) to (270)
) is obtained. In the above example, this would be an antibiotic degree of 0.1-0.075 μg/rILl.

Using this plate, the final concentration of antibiotic should be below the minimum degree of inhibition and the microorganisms should grow in a normal manner. Therefore, it is not necessary to use large plates in all cases, but should be used when complicated microorganism-antibiotic combinations with nutritional conditions are suspected, such as the S. aureus-cephalothin combination.

Referring to FIG. 10, another embodiment of the invention is described that includes an apparatus for collecting and transporting a sample of bodily secretions.

The device 100 is closed at one end 104 and the opposite R
a 106 contains a long flexible tube 102 which is open. A cap 108 closes the open end. Within the tube near the closed end 104 is a rupturable umbour 110 containing a liquid growth medium suitable for microbial pathogens. An absorbent material 112, which can be a suitable absorbent, such as cotton wool, is placed near the ambour 11-0. Absorbent material 112 contains sample transport system solids 114 dispersed therein. Within the open end 116 of the tube is placed a swab member 116 that includes a handle 118 and an absorbent end 120 for collecting body secretions from, for example, a lesion.

The sample transport system solids 114 can be the same materials disclosed for use in the lysis-centrifuge vessels described above, and the amount of solids 114 and growth medium and body shunts collected at the absorption end 120 is In operation, cap 106 is removed and swab 116 is exposed from within tube 102. The swab is brought into contact with bodily secretions, for example from an open lesion, and reinserted into the tube 102 and the open end 106 is capped. after that,
a portion of the tube 102 near the closed end 104;
The ambule 110 is broken, Fj is completely dissolved therefrom, the non-growth plot is fully saturated, the absorbent material 112 is sufficiently soaked, and the solid material 1127 of the sample transport system is dissolved. The resulting liquid containing a dissolved sample transport system is absorbed into the swab end 120 and a medium for maintaining the microbial pathogens present at the tip and body secretions absorbed into the tip 120 of the swab 116. and a sample transport system that inactivates antimicrobial factors that may be present. The swab 116 is later removed from the container 102 and microbiologically analyzed in the manner described above.

The following examples are given to provide a better understanding of the invention and are not intended to limit its scope.

In Examples 1-XVI, CFU = 1 rnl of blood initially inoculated into the tube.Connie forming unit of the current organism. Treat the blood in charge 7.5-.

% Recovery - Percentage of recovered mimics in the gradient spectra of all the organisms found after treatment.

S coefficient = survival index = number of CFU recovered from all contents in the tube/number of CFU introduced by several people: S-1 means no sterilization, s = o, i means survival of 10 .

EXAMPLE ■ The effect of sodium polyanetholesulfonate (5 ps) on dentamycin was tested including the original centrifuge article described in US Pat. No. 4,212,948. In the original description, the container contained FLUORINERT FC480,3r as a relaxation agent.
nl and 0.005 mr PPG as blood processing fluid.
and [ ] 0.5 d of distilled water containing 0.89 g of EDTA and 0.0048 g of sps, together with 0.018 g of purified saponin as a solubilizing agent. The tube is sterilized with a -7.4 aqueous solution and drawn with sufficient vacuum to draw approximately 7.51 nl of human blood. A second tube is prepared except that sodium polyanetholesulfonate is added to the aqueous solution in an amount equal to a final concentration of 0.6 wt% of processing liquid and blood sample.

Next, a series of the original tubes and the original tubes with added sodium polyanetholesulfonate were added to a blood sample containing 76 μg of sulfuric acid with a known amount of OS siaph.
ylococcus aureus and test.

The blood is lysed and the tubes are left at room temperature (approximately 72'F) for 2 hours before centrifugation, mimicking clinical conditions.

The material is then centrifuged as described above, and the tangled material is spread onto a growth medium and tested for recovery. The results are listed below.

Table ■-1 Dentamycin (6μFiAl) Original system 133 100. 06
Original system +〇, 6chi sps 203 80.
The 9 sps system supported a recovery rate of 72% (an 11,0-fold improvement) while the original tube had an overall recovery rate of 6%.

Example ■ Inactivation of Amvicillin with Thioglycolate This example was carried out in the same manner as Example V1, except that the stated amount of thioglycolate was added to the second and sixth series of tubes. Ta.

Part ■-1 Omotehara System 196 33.
002 original system + 1% thioglycolate 8490 8
9. 040 original system + 6% thioglycolate 4
66 89. Thioglycolate amount for 13 tatami treated liquid and blood sample 6Thioglycolate system had an overall recovery rate of 12.5% (179 times improvement), while the original tube had a recovery rate of 0.07.

EXAMPLE ■ Inactivation of ampicillin and dentamycin by a novel cysteine-thioglycolate combination The preliminary tests listed below demonstrate the properties of antibiotics added to liquid two-component processing materials and the thioglycolates described. Except for the amount of cysteine,
Same as example of punishment■, it was carried out in the evening.

Table rx-i Ampicillin (21 μg/m7) Hara'/3 Qi & 196 33
．． 002 original system + 0.5% thioglycolate
245 980.80 + 0.2% cysteine (original system + 0.1% thioglycofu) 696 9
91.1 + 1.2% cysteine 203 1
00. 2 Hara System + 5% Thioglycolate”
287 86. 8+, 2% cysteine” Lid for total volume of treatment liquid and blood sample
The comparison in the -i and lX-2 tables clearly demonstrates the use of the thioglycolate-cysteine combination.

EXAMPLE , 1 saponin 2
．． Treat with an aqueous solution containing 5 wL% of thioglycolate and cysteine (based on the total volume of treated fluid and blood) as indicated in the table, if present. The viscosity of each sample is measured at a temperature of 23.5-24.5°C. The results are listed below.

Ten mu
Go to IV- (A) Dimensions, ■
to 0O-Lj”) (a) ■
N size he
-9 (A) (A)
℃ Ma (I) 〇? -To 0 CI Fv-story +++++++++
, b b + b b % %
,, SEKAIKIb llh Ik k Th I
lh%.

Straw Example M Effect of Sample Transport System on Blood Sample Microbial Similarity Modification The data in the table below illustrates the following important aspects of the invention. 1. In the presence of an inoculated antibiotic at an average serum level, au
For r8us, 13 out of 19 antibiotics kill 50% or more within 2 hours. Escherchiac
Regarding oli, antibiotic 9 has an excessive bactericidal effect. (See Tables Xl-1 and Xl-2).

2. For the new system, the maximum sterilization rate is 70%, and a reduction of 50% or less of the inoculum is S, aur
This occurred with regard to antibiotics for E. coli (25) and E. coli (25). Adding large plates to the new method virtually eliminates the bactericidal effect seen in these four cases (S=0.8 vs. 0.6: S=0.
9 vs. 0.5; S = 0.8 vs. 0.5 and S =
0.5 vs. 0.6: where S = 1.00 = 100% survival).

In summary, a novel system combined with effective dilution (i.e., the use of large Petri plate sites) can reduce blood and therapeutic antibiotics against bacteria present in blood samples during transportation and processing time. can effectively prevent the bactericidal action of

The data in Tables Xl-3 to Xl-10 below confirm the general effectiveness of the present invention against other pathogens commonly isolated from the blood of patients with sepsis.

The tests described below were performed on each pathogen using various antibiotics. The concentrated residue from the tube was applied to small and large plates.

A series of original lysis-centrifuge devices are assembled as described in Example 2. A second series of dissolution-centrifuge devices was constructed as in Example 2, except that the aqueous phase was changed as follows.

0.5 flu/flu of distilled water containing 0.005 m of polypropylene glycol is placed in a brass tube.

A total of 0.17 g of the powder mixture is then added to the container. The mixture contains the following ingredients:

1.8 g purified saponin, 4.8 g sodium polyanetholesulfonate, 0.8 g thioglycolate and 9.6 g cysteine.

The tube was placed in an autoclave and killed, and the final temperature was 6.6~
It is 6.8.

7.5 mt of blood is drawn into the tube with a light vacuum.

On each side, add the specified amounts of specific microorganisms and antibiotics listed on the thorns to the blood 7.51. Then dissolve the blood-
Place in a centrifuge tube and hold the tube at room temperature for 2 hours to mimic clinical conditions, then centrifuge as described herein. Then, the concentrated residue of the container containing suitable growth gravel is 1001! Equal aliquots are plated on five agar plates of size llX20101 and the raw stomachs are observed.

1 d of supernatant remaining after centrifugation is applied to 5 plates.

[, 5 taphylococcus aureus (A
TCC#25923) No thick drug 7591 (6)-'r'ntamicin 133 1
00(4)-Dopramycin 155 9
9(21)-Amikacin 7699(20
) - Penicillin 546 68 (21)
-Amugi Shirin 403 84 (20)-
Sef7ofn214 100(25)-Sefokishiti 7
158 98(18)-chloramphenicol 476 99(9)-tetracycline
218 100(8)-Erythromycin
512 100 (100)-gantricin
642 95(5)-Clindamaishi 7 1
80 99°(9)-Methicillin 58
8 99° (8) - Vancomaishi 7 286
98(60)-Piperaziline 350
82 (100) - Moxalactam 676
100(71)-carbenicillin 483
100(20)-cefotaxime 472
99C15D)-ticarcillin 606
501-1 Table type flat plate, 9±, 2 745 99 1.1 ±, 2
．． 1±, 04 852 99 1.0±, 1.
4±, 2'259 95. 8±, 21
．． 0±, 5 152 99 1.1±, 2.
01±, 01 305 96. 9±, 2.
003±, 003 319 94 1.0
±, 1.1±, 03 1177 76. 3±, 3
．． 5±, 1 991 99. 6±, 2.6
±, 2 495 99.8 1.1 ±, 3.5
±, 4 584 100 1.2 soil, 1.1±, 0
5 623 72. 8±, 21.0±, 2
441 99.7 1.0±, 17 1.2±,
9 123 78 1.3±, 48. 9±
, 1 408 92 1.1 ±, 3.6 ±
, 1 1190 52. 8±, 2.005±
, 007 396 99 1.2±, 1.2±
, 05 2345 49 1.2±, 4.03
±, 02 438 99.8 1.1±, 3.3±
, 1 595 98. 5, 2.01±,
01 55398 ・9t・1A9 only- No drug 214 98(6)-Dentamicin 468 100(4)-) Pramycin 129 100(21)-Amikacin 255 100(20)-Penicillin 526 99(21)-Ambicillin 490 10 Mouth (20) - Cephalothin
413 100(25)-Cefoxitin
466 99(18)-chloramphenicol 323 99(9)-tetracycline 3
68 99(8)-Erythromycin 30
5 99(100)-gantricin 140
99(i50)-ticarcillin 574
99.8(20)-Carbenicillin 169
4 99.6(60)-piperaziline 3
64 100 (2Q) - 77 Amandole 16
7 99.5(14)-Kanamycin 2
03 10 mouths (9) - Methicillin 198
99.8 All tubes were placed at room temperature for 2 hours prior to processing, and the small plates were placed in a culture pot containing 20 rnl of agar medium.The numbers in parentheses are 1 for blood - 1.2±, 38 for the microorganisms in question.
86 95 1.1±, 2-02±, 01
167 97 1.1 ± 1.0
．． 6±, 6 399 99
．． 9 ± , 2.05 ± , 03 420
98. 8±, 11.4±, 3
206 95 1.3 s , 3
．． 2±, 1 144 98
．． 9 ± , 2.04 ± , 04 353
99. 5±, 1.3±, 86 148
89. 8±, 3.8±, 1 39
5 99.6. 7±, 3.5±, 04
320 98 1.1 ±,
21.0±, 4 212 97 1.1, 3
1.5±1.0 282 97
．． 8±, 3.5±, 4 651 99 1
．． 3±, 7.2±, 1 417'
98 1.0 master, 2.7±, 4 1084
96. 9±, 2.4±, 2 1
53 100. 7f, 4.1 ±
, 03 176 95. 3±,
11.1±, 2 146 99 1.1±, 3
(20°C).

do.

represents the final concentration of antibiotic used.

The experiment described above was carried out in such a way that the reduced residue from the container was placed in a 1[ID x 2[U] Petri plate containing medium, containing 60 to 80 grams of medium, and was repeated, except that the plate was applied to a 150 ml x 2D Petri IJ plate with a surface species approximately 2.25 times that of the lQQimx2[]jn Petri plate used to generate the data.

The results are shown below in Section Xl-2.

1 [, Escherichia coli (ATCC#
25922) No drug 237 91(6)-dentamycin + +(4)-tobramycin + +(21)-amikacin
+ +(20)-penicillin
+ Ya(21)-Ampicillin +
+(20)-cephalothin 102
100(25)-Cefoxitin 454
100(18)-chloramphenicol 21
7 99.5(9)-tetracycline +
+(8)-bithromycin 305
100 (100) - Gantricin 140
99.8(150)-ticarcillin +
+(20)-carbenicillin +
+(60)-Piperaziline +
+(2D)-cefamandole 167 9
9(14)-Kanamycin 330.
All 100 tubes were prepared at room temperature (20 large plates containing 8 Qrnl of agar medium).The numbers in parentheses indicate the amount of microbial remains of blood imzib. 1.3±, 3 172
941.5± , 6 + + + + + + + + + + + + + + + + 10 + + + , 1 length, 09 288 92
．． 8±, 1.5i-, 140699, 9±, 6 1.0-f-, 34209 (51,1±, 4++++, 9±, 2 212 99 1
．． 0±, 21.4±, 6 231
991.0± , 1++++ ++++ ++++ , 5± , 2 209 98
．． 8 Sat, 1.05 Shi, 03 3
52 99.6.5±, 210C) for a period of time.

"do.

represents the final concentration of antibiotic used.

) Not tested.

n Hehe, he (I)-PLl") FC'l w
-1 Table Xl-10 No drugs 9793.6 length, 1. 6±, 2
Carbeninrin 9899.3±, 6. 8±
, 3 Cefotaxime 9798.5±, 2. 5
±, 2chloramphenicol 9697.7±64.
6±,4 Clindamycin 99.7100.
4 length, 2. 3±, 2 erythromine 969
9.5±, 2. 8±, 2 is also 51 degrees, protecting new cocktail microorganisms from the bactericidal effects of antibiotics. As would be expected for an antibiotic that does not exert a bactericidal effect, we obtain the same actual recovery of the original tube and the modified tube co-microorganisms containing the sample transport system. When dealing with a few specific microorganism-antibiotic combinations, such as S. aureus and cephalothin, a large volume C1:267> is clearly required. The data of On et al. indicate that large dilutions are necessary only for a few antibiotics such as cephalothin, tetracycline, erythromycin and for microorganisms such as +cocci. Aminocricosides, penicillins, 7-mubicillins and chloramphenicol are completely neutralized with this cocktail, but cephalothin is only partially neutralized.

EXAMPLE ■ A series of original tubes as described in Example M and tubes containing a sample transport system as described in Example M yield positive blood cultures from blood from a patient suspected of having sepsis. It is used for processing.

In each case, blood from the patient is placed into the original tube as well as a tube that has been modified and contains the sample transport system.

Centrifuge the tube and inoculate the shrunken residue into small Petri plates as described in Working Example M.

The results of the test are set out in Table X11-1 below.

Table ■-' Number of cultured microorganisms/M1 original system New 1, Ac1 netobacber
sp, NO41,42
・FiXterobacteragglomerans
[], 7Q, 73, Enterobac and ercloa
caeQ, 1NO4, NG [1, i 5, Escherichia coli
G16 2.96,
1.1
．． 77, 1
3.0 92.88,
#7.5 12.
59, # #
2.1 10.21Q,
＃＃NG
Q, 111, 1
N() 0.112,
Flavobacterium N
G O, (513, His shiopla
sma capsulabum iO,57,
(44・
1・62・(515, #
# 13.5 14.216,
Klebsiella 0XyLOC
a s, s
20-(517,
2.1 3.918, K
lebsiella poeumoniae Q,
j Q, 119,
11.9 19.32
0, 130.8
78.121,
1 (52). 0 182.022,
# Q, 5
NG23,
0.3 1.024,
l 7.3
8.425, Lisberiamo
nocytogenes O, 49,526, Ps
eudomonas aeruginO5a 13
1.3 83.027, Pseud
omonas fluorenscens Q,i
N01 No table 0 None Dopramycin and cefazolin - cefoquintin + 383 Penicillin and tobramycin + (514
None +71 Ticarcillin and rfutamycin +38
5 None Metoxin Penicillin Toterramycin - 50 Amphotericin B +62 N+05 +134 None +86 Dentamycin and Ticarcillin Dentamine +62 To-7'' Ramycin and Carbenicillin - 67 None +12 Tobramycin and Carbenicillin +2
27 Dentamine and ticarunrin +15 +227 Cefuoquintin-58 No tobramycin and carbenicillin Conclusion 1. In 68% of positive samples, new tubes give higher microbial counts/ml f of blood. The lowest is 5, and the highest is 514% (the number of pages increases by 514%).

2. The original system missed 5 positives and the new system missed 6 positives. In the -6,4 case, the original system gave a much higher number. However, this level is within expected experimental variation.

4. 36% of patients were not on antibiotics at the time of blood collection. The new system gave a higher number in 50% of such cases. The lowest figure was 134 cm, and the highest figure was 514%.

5, 17 cultures became positive simultaneously at the same time.

25) cultures (one for Li5teria and one for Esc
hrichia coli) became positive one day earlier in the new system.

As shown in the table, in 68% of the samples, the modified device containing the sample transport system gave higher counts than the original device (5 to 514% increase); in 5 cases , negative on the original instrument and positive on the new instrument. Most samples were positive at the same time, but if the new instrument detected a positive culture one day earlier (E, co
li and Listeriaaf)25).

Interestingly, even when patients do not have antibiotics (6 patients), the new device seems to give a larger number. C
This indicates that the new device containing this sample delivery system inhibits the patient's immune system more effectively than the liquid blood processing solution of the original device.

Example ■ A first series of original lysis-centrifuge devices is assembled as described in Example ■. The 20th lysis-centrifuge device is assembled in the same manner as the second series of devices in Example V.

A third series of lysis-centrifuge devices is assembled as follows.

FLUORINERT FC480 as a emollient in the article disclosed in U.S. Pat. No. 4,212,948.
, 3 together with the following compounds in the form of dry granular powder: Thioglycolate 0.008 g HolJ Sodium Anethole Sulfonate 0.048
g Add 0.018 g of purified saponin. The third series of tubes is evacuated to a light level to draw 8 ml of blood. This series of tubes is then heated to 121°0160 minutes and cooled to room temperature.

In each case, the following xi-i to Xl1-
6. Specified microorganisms and antibiotics (if added) in the amounts specified in Table 6 were added to the blood in the first and second series of tubes fi 7.51 nl x the blood in the third series of tubes 8 m
Add l K tA. The blood is then placed in each lysis-centrifuge tube and the tubes are centrifuged as described in the specification of O. The same plate of each microbial pathogen-antibiotic combination is plated on large and small Petri plates as described in Example M.

The results are shown in Tables ni to Xl-6 below.

S brep Shiococcus pHeumoni
ae Original treatment liquid No chemicals 92. 70 94. 81 Ampicillin 43. 0084 NT NT
Seraaxitin 97. 39 100. 1
6 Penicillin 76. 024NT
NTNT = No test, no drug 97 1.42 Tatami Tatami Chloramphenicol 98. 91 + Tatami dopramycin 100. 028 98. 94
No need for tatami large plate medium test Discontinuation of production of liquid tubes (drying results only) Table XI-3 Table 84. 76 95 1.00 96. 88
69. 97B1. 52 95. 25 6
5. 60 85. 8899. 51 10
0. 27 99. 28 99. 7787
．． 57 93. 43 (52) ・78(
52) ・87th Seal X-Table 4--Tatami Tatami 98 1.20, Appetizer 89 1.6
97. 93 80 1.15 Tatami bones 94. 7
093 1.07 75. As can be seen from the above data, the dissolution-centrially separator constructed in accordance with the present invention containing the dry granular powder sample I + transport system of the present invention has a sample transport system in an aqueous bath in snow. works at least as well as a system according to the invention containing. Both systems clearly outperform the original system described in the examples.

EXAMPLE EXAMPLE Increasing Retention Time of Blood Samples A first series of original lysis-centrifuge devices is assembled as described in Example 1. The second series of lysis-centrifuges is the same as the third series of lysis-centrifuges used to obtain the data shown in Tables Xl-1 to Xl-6 of Example 2. Assembled by method.

In each case, the stated amounts of the specific microorganisms shown in Table xIv-1 below are added to 7.5 d of blood in the first series of tubes and 8 Tnl of blood in the second series of tubes. . The blood is then placed in each lysis-centrifuge tube and the tubes are injected for 2 hours as stated in Table X[V-1 below.
Maintain at 1°C. The tube is centrifuged and the compressed contents are applied to a growth medium as described herein.

j Microorganisms CFU TNTC = Too many to count Table XIV-1 Blood Size Time Reconstitution Test 86 91. 8±, 2. 2±, 193 7
6. 8±, 1. 5±, 399.7 89
．． 8±, 3. 4±, 1ioo 100.
9±, 6. 3±, 199 TNTC2, 2±, 8
TNTC95941,2±,44. 4±2.
699 100. 8±, 05. 03±, 0
0498 97 1.0±, 3 1.5±,
698TNTC1,7±,5 TNTCloo
100. 9±, 11. 0±, 493 9
8 1.0±, 3. 8±, 0490 99 1
．． 0±, 1 1.1±, 298 1.2±
, 5 TNTC99-, 9±, 2 TNTC' As can be seen from the data presented in Table XIV-1, the bacteria in the ridges growing or dying in the original tube when kept for 24 hours. For C, these same species do not substantially grow or die in the second series of new tubes containing the test #+ transport system. Although it is not recommended to hold centrifuge tubes for long periods of time, it has been found that such tubes are held in hospital environments for periods of time prior to processing. The data show that 2 of many species in the new tube
Although no substantial growth was shown at 4 hours, it is believed that tubes should be processed as early as possible and certainly before the 12 hour holding time.

Furthermore, species of bacteria such as Enterobacter
(To ensure that there is no growth of E. loacae, sodium chloride can be added, for example, to urine samples as described in Example 2 below.
- IJum is approximately 0 for final processing solutions and blood samples.
．． It may be present from 1 to 10 wb%, preferably about 1 to 5 wt%, most preferably about 3 wt%.

Example Xv Use of fermentation components A series of lysis-centrifuge devices is assembled similarly to a second series containing a sample transport system, such as in Example 1. Into each tube was 842 CFU of E. coli and 20 μg lM% of the antibiotic cefotaxime as well as part xv-i.
8 ml of blood containing the stated amount of β-lactamase enzyme shown in the table is weighed. The β-lactamase used was Calb
iochem-Behring Corporation
n LaJolla, Califoruia Lot No. 203435, Order No. 426205 β
- Lactamase (Bacillus cereus). Note: For each unit of β-lactamase ■-activity, β-
Lactamase I - 13 units of activity. Then lyse each blood
Place in a centrifuge tube and centrifuge as described in the specification.

The same amount of microbial pathogen-antibiotic-enzyme combination was used in Example X.
Plate onto small Petri plates as described in t. The results are shown in Table xv-i below.

Table XV-1 E, coli - Cefotaxime 20 μg/m/[158
,02 ,01100,011 0,1100,052 1,099,33 2,099L17 4.0 99 . 825.
0 99.8. 95th xv
- As can be seen from the table, β-lactamase as an essential part of the sample transport system inhibits the activity of antibiotics and prevents the killing of microbial pathogens while contained in the lysis-centrifuge tube. Works effectively to prevent

As a comparison, a second series of tubes was assembled as described in Example I, and the tubes contained E. coli 765 CFU, 20 μg/ml of cefotaxime, and β in the units shown in Table XV-2.
-Add lactamase and blood 7.5-. The material is then centrifuged and the results are reported in Section XV-2.

Table XV-2 0 93 , 08 1 90 , 04 5 95 . 155 Comparing the results in Table XV-2 with the results in Table
-1* is not improved.

Additionally, a first series of lysis-centrifuge tubes prepared similarly to those in Table XV-2 above, but containing no sample transport system, and identical to those used in Table XV-1 above, but a second series of lysis-centrifuge tubes containing no enzyme;
Same as the second series of tubes, but below Nos.
A third series of lysis-centrifuge tubes containing the indicated amounts of β-lactamase enzyme shown in Table V-8 are tested and compared. Blood containing 200-1000 CFU of the indicated bacteria was added to the tube, the tube was processed as described in this example, and
The results are shown in Tables XV-3 to XV-8 below.

The data shown in Tables XV-3 to XV-8 show that the addition of enzymes to the sample transport factor system of the present invention effectively neutralizes the antibiotics of the groups indicated above. It shows that it increases.

Example Assemble the lysis-centrifuge tube. To the first series of these tubes is added the amount of β-lactamase enzyme stated in Table XVI-1.

This tube is then subjected to cobalt sterilization, after which
Blood 8- containing the microbial pathogens and antibiotics shown in Table VI-1 is added and processed as described in Example X. A second series of these tubes was steam sterilized and then the indicated amount of β-lactamase enzyme was added to it, and then the blood with the indicated amount of microbial pathogen and antibiotic was added to it. is added and the tube is centrifuged and processed as described in Example XV. The results of these tests are in Section XVI.
-1 and XvI-2 tables.

As can be seen by comparing the data in Table XVI-1 with the data in Table XVI-2, the loss of enzyme activity due to cobalt disinfection ranges from 20 to 80
% range. However, Table XVI-1 clearly states that
The cobalt sterilization method can be used effectively and, when used, explains that increased amounts of enzyme should be added to the tube prior to sterilization. It should be noted that other chemical agents are expected to be used in the sample transport system, depending somewhat on the type of antimicrobial agent expected to be present in the sample. For example, other groups of antimicrobial agents include water-soluble compounds such as sodium hypochlorite, heavy metals such as sodium bisulfite sulfhydryl. As described, the sample transport system of the present invention is described, for example, in U.S. Pat. No. 4,131;
512 and 4.212,948 have found particular utility in lysis-centrifuge tubes. Furthermore, the sample transport system is disclosed in U.S. Pat. No. 4,164,44.
A lysis-centrifuge tube such as that described in No. 9 is useful.

Moreover, the sample transport system of the present invention provides blood 1-2II
Il! Can be used with neonatal blood processing tubes, including standard single-stop vacuum tubes designed to pull out. The tube contains no other substances than the sample transport system of the invention and, if desired, saponin. Blood can be injected into the tube and applied directly onto the growth medium.

The foregoing examples illustrate the beneficial effects of the sample transport system of the present invention when used in lysis bicentrifuge tubes for the analysis of microbial pathogens in blood samples. However, the sample transport system of the present invention also finds benefit in protecting microorganisms in sample fluids other than blood that are collected and subsequently analyzed for the presence of microbial pathogens. For example, the sample transport system of the present invention can protect microorganisms present in swabs, urine, sputum, spinal fluid, and other body fluids during transport. It is well known that these fluids also contain body fluids and chemical antimicrobial agents (if the patient has been treated with antibiotics). When it comes to urine samples, the concentration of antibiotics may actually exceed the concentration in serum. An example of a modified sample delivery system for neutralizing antibiotics in urine is in Example X■ below.

EXAMPLE Test the ability to hold stable.

Place the following dry mixture into each of a series of tubes.

Sodium polyanetholesulfonate 0.03 g Thioglycolate 0.005 g lCN free base cysteine 0.1 g Sodium bicarbonate
o, i 9. A tube containing the urine cocktail of the sample transport system and the urine containing antibiotics of the types listed in Tables X-1 to X-6 below, specified therein and at a certain concentration. Add to the same number of tubes and without the cocktail. Sterile urine 5- is then added to all tubes and the tubes are mixed vigorously. Control tubes contain either urine alone or urine plus the sample transport system urine cocktail described above. No antibiotics are added to control tubes.

The microorganisms listed in Tables X-1 to X-6 are McFa.
Adjust to rlin O-5 and dilute 1:100 with sterile culture medium. A 0.111 Ll aliquot of a single microorganism is added to each urine-containing tube and the resulting mixture is stirred vigorously. A 10 μt aliquot from the 6 tubes containing the resulting mixture is directly inoculated onto tryptic soy plates and spread with a sterile diffuser. The inoculated plate medium is
Incubate overnight in an environment and temperature appropriate for the microorganism used. The tubes are then left at room temperature for 24 hours. Additional 10 .mu.t aliquots are applied as described above at time intervals shown in Tables XI-1 to XVII-6. All platings were performed four times, and the S-factor was calculated as the average of the four platings.
Record in Table 1-X-6.

Table XvII-3 - Time not included in the reconstruction Sample transport system Urine cocktail present Bone The number in parentheses represents the final concentration of antibiotic in the urine (μp'go) TNTC = too many to count Table XVri-4 - Reconstitution, time not included in composition Sample transport system Urine cocktail present
) representing TNTC = too many to count, Hyotam Tables X■-1 to X■-6 clearly indicate that the sample transport system urine cocktail inhibits conventional therapeutic antimicrobial agents in urine. However, in the absence of an antimicrobial agent, it has shown the ability to maintain a relatively constant microbial population.

It should be noted that with respect to normal urine without antibiotics, normal pathogenic time will develop. Therefore, false positive results can occur if the urine sample is not analyzed promptly. In the presence of average urine concentrations of antibiotics (10 times that of blood serum), sensitive pathogenic microorganisms die rapidly. This means that in the laboratory, the sample is actually collected in a considerable amount (105)
of pathogens, it can be concluded that the sample does not contain significant amounts of pathogenic microorganisms.

In other words, if 2 or more times elapse between collection and laboratory processing, the number of bacteria obtained is 1
If it is as low as 03, it may be considered that there is no problem.

This urine sample transport system achieved a major improvement in 25). Namely: 1. It can effectively block the bactericidal effect of antibiotics for at least 6 hours, and often up to 24 hours.

2. Maintain the number of microorganisms present at time 0 constant for at least 6 hours in the presence or absence of antibiotics.

In conclusion, the unique features of this urine sample transport system are:
It is an object of the present invention to enable a urine sample to be maintained for up to 24 hours before processing without having a detrimental effect on the microbial susceptibility of the sample.

No refrigeration is required and the system is effective in the presence or absence of antibiotics.

As can be seen from the examples above, sample transport systems within the scope of the present invention have many uses.

The ability of this sample transport system to maintain a constant microbial population allows detection of important microbial species that would otherwise be masked by overgrowth of rapidly dividing microorganisms.

Example
Increasing hypertonicity to create a statis effect The following dry mixture is placed in a series of 6 tubes.

Sodium polyanetholesulfonate 0.03 g thioglycollate 0.0059 ICN free base cysteine 0.1 g Sodium bicarbonate 0.
Up to 1 g of sodium chloride at an inoculum weight percent as shown in Table XI-1 is placed into individual tubes containing the sample transport system urine cocktail described above. Then add 5 m/ml of sterile urine aliquot to all tubes containing the sample transport system urine cocktail and the same number of tubes without urine cocktail. Enterobacter cloa
A culture medium containing S. cae ATCυ1344-2,
Culture medium 11nI! McFarlin o,s, representing approximately 5 x 108 microorganisms, was adjusted to 1 in sterile culture medium.
: Dilute to 100. Diluted microbial aliquot) 0.1
m/ is added to all tubes containing urine and the resulting mixture is stirred vigorously. 10μt aliquot of the mixture
are plated on agar plates as described in Example ①.

The remaining mixture is 243! Allow to stand at room temperature for a period of 30 minutes, then apply a second 10 .mu.t aliquot as described in Example 3 above.

All applications are done in duplicate and the survival index (S-factor) is calculated for each as described in the examples. The average S-factor for each hour is determined and recorded in Table X-1 below.

1 -+-i, QQ TNTC-ゝ2 +-1
,001,72 3+ 1 1.00 1.274
+ 2 1.00 -805 +
, 4 1.0[] 1.036
+8 1.00 0.69+S
TS Sample transport system urine cocktail Aochi N
Percentage of aC1 wt tsit ts TNTC too many to count Sample transport system In the absence of urine cocktail, part X-1
See sample 1 of the table. The microorganisms present in the urine multiply rapidly, which prevents the clinician from determining the exact number of microorganisms per ILl of urine.

The results shown in Table X4-2 above show that although the sample transport system urine cocktail can reduce the replication rate of microorganisms, the addition of salts, such as sodium chloride,
It has been shown that the effect of stably maintaining the number of bacteria in urine for 24 hours is increased. As determined from the results shown in Table X4-2 above, the preferred range of added salt is 2
．． 5-4, OWt%. From the results of the above salt titration experiment, it was found that adding approximately 3 wt% to the urine cocktail of the sample transport system can reduce Enterobacter cloacae in the urine.
It is concluded that the overgrowth of the following can be prevented for 24 hours.

To substantiate this conclusion, the sample transport system urine cocktail prepared as described above is added to a series of tubes.

- A second series of tubes is prepared by adding the same cocktail of 3 wt % IJum plus an equivalent of 0.159 sodium chloride. A third series of tubes is assembled without cocktail. Then add 5 ml of sterile urine to 6 tubes, including the tube without cocktail. The four strains of Enterobacter cloacae identified in Table 40 below are grown individually and adjusted to McFarlin 0.5. Each strain was then divided 1:10 in sterile medium.
0 and aliquots of 1 ml each are added to individual urine tubes as identified in Table Xl-2 below. After vigorous mixing, a 10 μt aliquot from the tube is spread onto the agar medium described in Example X■. The mixture is then allowed to stand at room temperature and at the time intervals shown in Table X4-2 below further 10 .mu.t aliquots are applied as described in Example 1. The hourly results given in Table X-2 below represent the average survival index (S-factor) for four applications.

The results presented in Table X-2 below confirm that approximately 3 wt % sodium chloride addition to the sample delivery system cocktail increases the stabilization of colony formation for all strains.

Hypertonicity may also be increased using other salts, carbohydrates or sugars. It is anticipated that a suitable concentration approximating the effect of sodium chloride in this example may be calculated using the knowledge disclosed herein.

Example Explain the effect of the sample transport system on

Place the following dry mixture into a series of sterile tubes.

Sodium polyanetholesulfonate 0.03 g Thioglycolate 0.00511 ICN free base cysteine 0.1 g Sodium bicarbonate
0.1g Sodium chloride 0.1
59 Table X1l (-1 to Then aseptically add 5 mA' to all tubes and mix vigorously. Control tubes contain urine alone or urine with the sample transport system urine cocktail described above. Control tubes contain no antibiotics. No.The following XD(-1
~XIK-3 Microorganisms listed in Table 74 McFarli
Adjust to n O-5 and dilute 1:100 with sterile culture medium. A 0.1 ml aliquot of a single microorganism is added to each urine-containing tube and the resulting mixture is vortexed vigorously. 10 .mu.t directly from the tube containing the resulting mixture is applied as described in Example X.sub.2 above. Then the tube 24
Leave at room temperature for an hour. Additionally, a further aliquot of 10μ
t is applied as described in Example XSU at the times indicated in Table The reported S-factor represents the average of four applications.

The results, described in Tables XI-1 to XIK-3, indicate that the salt-containing sample transport system urine cocktail is used for many antibiotic pyo
This shows that the number of colonies of Genes can be maintained relatively stably.

Example XX For microbiological integrity preservation of antibiotic-containing urine samples:
Effective Concentration of Sodium Polyanetholesulfonate (5PS) for the Sample Transport System A dry mixture of the following is placed into a series of sterile tubes.

Thioglycolate 0.005 gIC'
N free base cysteine 0.19 Sodium bicarbonate 0.1 g Sodium chloride
Add varying amounts of 0.15 g sps as indicated in Table XX-1 1 cwt% to the tubes containing the sample transport system urine cocktail described above and to the control tubes. Add the various antibiotics listed in Section XX-1 below to half of the tubes containing the sample transport system urine cocktail. Then add 5+nl aliquots of sterile urine to all tubes. Control tubes were set up containing urine but no antibiotics, half of which contained the sample transport system urine cocktail and various amounts of sps as shown in Table XX-1 below. Sta,ph
ylococcus aureus ATCC≠259
23 was grown and prepared as described in Example X■ above, and aliquots thereof were added to all tubes. All pipes are
At the time shown in Table XX-1 below, the above-mentioned Example 17
).

0 0 0 0 0 Z To N column 〒- To N F 1 bottle *+1 The results set forth in Table XX-1 above clarify the appropriate range of amounts for SPS in the sample transport system urine cocktail. , it is about 0.6-2. It's OWt.

Although the invention has been described in relation to its preferred embodiments, it is to be understood that various modifications thereof will be apparent to those skilled in the art upon reading this specification. It is intended to cover such modifications as come within the scope of the appended claims.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view of a centrifuge article that can be used to practice the present invention. 2-9 are explanatory diagrams showing the steps of a microbial pathogen detection method in which the present invention can be used. FIG. 10 is an illustration of another embodiment of the invention that includes an apparatus for collection and transport of body secretion samples. FIG. 11 graphically illustrates the preservation of microbial susceptibility of samples in the presence of antibiotics within one hour using the present invention as compared to conventional systems (Example V). (details)
It is a line diagram. Figures 12-16 are diagrams (described in detail in Example ■) that graphically illustrate the preservation of microbial integrity of samples in the presence of antibiotics over a 4-hour period compared to conventional systems. be. FIG. 17 is a cross-sectional view of one embodiment for sample collection and transport. FIG. 18 is a cross-sectional view of the embodiment of FIG. 17 after the sample has been introduced. FIG. 19 is a cross-sectional view of an embodiment of the invention incorporating a pierceable closure member useful with the embodiment of FIG. 17. FIG. 20 is a cross-sectional view of the sample collection and transport embodiment showing alternative piston faces and shaft disengagement. FIG. 21 is a cross-sectional view of the embodiment of FIG. 17 when used to draw a liquid sample from a sample receptacle. FIG. 22 is a cross-sectional view of another embodiment of sample collection and transport when used as a vacuum sample drawer. FIG. 23 is a cross-sectional view of another embodiment of sample collection and transport showing a powdered sample transport system. FIG. 24 is a cross-sectional view of the embodiment of FIG. 20 showing an alternative method of fixation. FIG. 25 is a sectional view taken along line A-A in FIG. 24. Procedural amendment (flag) April 11, 1991

Claims (1)

Claims: (1) The interior is closed with an injectable closure means containing an empty space maintained at a pressure below atmospheric pressure and into which a predetermined weight of sample liquid is drawn. A sample comprising an enclosed centrifuge container and a processing liquid that includes a lysing agent as well as a sample transport system that is effective in maintaining the microbial integrity of the sample liquid and is not lethal to microbial pathogens. A device used for the collection and treatment of microorganisms in (2) The device according to (1) above, wherein the sample transport system includes an effective amount of a drug selected from the group consisting of sodium polyanetholesulfonate and sodium amylosulfate. (3) The sample transport system further includes an effective amount of a sulfhydryl group-containing substance that is nontoxic to microorganisms, as described in (2) above.
The device described in. (4) The device according to item (2) above, wherein the sample transport system further includes a hypertonicity enhancer effective in increasing the bacteriostatic effect. (5) The sample transport system further includes a substance effective in inhibiting the replication of Gram-positive microorganisms,
). (6) The sample transport system further adjusts the pH of the sample to approximately 6.5.
7.5. The device according to item (2) above, comprising a substance effective to buffer the temperature to 7.5. (7) The apparatus of paragraph (2), wherein the sample transport system further includes a growth substrate effective to maintain the general nutritional requirements of the microorganism. (8) The device according to item (2) above, wherein the sample transport system includes a sulfa drug inhibitor. (9) The device according to item (2) above, wherein the sample transport system includes an antibiotic-specific enzyme. (10) a generally elongated container having a first end and a second end; a piston slidably mounted within the container; and a piston attached to the piston and extending through the first end; The shaft, the removable cap, the cap, the container, and the piston form a sealed chamber in which the pressure may be reduced by moving the piston toward the first end to remove the sample. The cap can be pierced for loading and is effective in preserving the microbial integrity of the sample, including the composition placed in the container, and the microorganisms in the sample. equipment for the collection and processing of (11) The composition includes a sample transport system (
10). (12) The device further includes a stop that engages the piston to prevent movement of the piston within the container.
). (13) Where the shaft includes a thin section that allows it to break at the thin section when the shaft is bent,
11). (14) The device further includes a stop that engages the piston to prevent movement of the piston within the container.
13). (15) The device according to item (11), wherein the shaft is removably connected to the piston. (16) The device further includes a stop that engages the piston to prevent movement of the piston within the container.
15). (17) The device according to item (11), wherein the sample transport system includes an effective amount of a drug selected from the group consisting of sodium polyanetholesulfonate and sodium amylosulfate. (18) The sample transport system is also non-toxic to microorganisms.
The device according to the preceding item (17), which includes a sulfhydryl group-containing substance. (19) The device according to the preceding item (17), wherein the sample transport system further includes a hypertonicity enhancer effective in suppressing an increase in the bacteriostatic effect. (20) The sample transport system further includes a substance effective to inhibit the replication of Gram-positive microorganisms,
). (21) a generally elongated container having a first end and a second end; a piston slidably mounted within the container; and a shaft attached to the piston and extending through the first end. and a removable cap, the cap, the container, and the piston form a sealed chamber, the pressure therein may be reduced by moving the piston toward the first end, and the cap is compressed through the cap. for the collection and treatment of microorganisms in a sample, including a nozzle providing a passageway, and a composition effective to preserve the microbial integrity of the sample and placed in the container. equipment. (22) The composition includes a sample transport system (
21). (23) The device further includes a stop that engages the piston to prevent movement of the piston within the container.
21). (24) The device of paragraph (21), wherein the shaft includes a thin section such that the thin section breaks when it is bent. (25) Where the device further includes a stop that engages the piston to prevent movement of the piston within the container.
24). (26) The device according to item (21), wherein the shaft is removably connected to the piston. (27) Where the device further includes a stop that engages the piston to prevent movement of the piston within the container.
26). (28) The device further includes a stop that engages the piston to prevent movement of the piston within the container.
27). (29) introducing a sample into a container having a sample transport system effective to preserve the microbial integrity of the sample, the container having a first end and a second end; , generally elongated in shape, further comprising a piston slidably mounted in the container, a shaft attached to the piston and extending through a first end, and a removable cap. the cap, the container and the piston form a sealed chamber in which the pressure may be reduced by moving the piston towards the first end, the cap for loading the sample; Method of collecting and transporting pierceable liquid samples suspected of containing microorganisms. (30) The method according to the preceding item (29), wherein the sample transport system includes an effective amount of a drug selected from the group consisting of sodium polyanetholesulfonate and sodium amylosulfate. (31) The method according to item (30), wherein the sample transport system further includes an effective amount of a sulfhydryl group-containing substance that is nontoxic to microorganisms. (32) The sample transport system further includes a hypertonicity enhancer effective in suppressing an increase in the bacteriostatic effect (3) above.
0). (33) The sample transport system further includes a substance having the effect of inhibiting the replication of Gram-positive microorganisms.
). (34) The sample transport system further adjusts the pH of the sample to about 6.
5 to 7.5;
The method described in the preceding section (30). (35) The method according to item (30), wherein the sample transport system further includes a growth substrate effective in maintaining the general nutritional requirements of the microorganism. (36) The method according to the preceding item (30), wherein the sample transport system includes a sulfa-based inhibitor. (37) The method according to item (30), wherein the sample transport system further includes an antibiotic-specific enzyme. (38) The method of (29) above, wherein the device further includes a stop that engages the piston to prevent movement of the piston within the container. (39) When the shaft is bent, the thin part is included so that the shaft breaks at the thin part. (29)
). (40) The method of paragraph (39), wherein the device further includes a stop that engages the piston to prevent movement of the piston within the container. (41) The method according to the preceding item (39), wherein the shaft is removably connected to the piston. (42) The method of claim 41, wherein the device further includes a stop that engages the piston to prevent movement of the piston within the container. (43) incorporating the sample into a container having a sample transport system effective to preserve the microbial integrity of the sample, the container having a first end and a second end; The container is generally elongate in shape and further comprises a piston slidably mounted in the container, a shaft attached to the piston extending through a first end, and a removable cap. , the cap, the container and the piston form a sealed chamber in which the pressure may be reduced by moving the piston towards the first end, the cap providing a passage through the cap. A method for collecting and transporting a liquid sample suspected of containing microorganisms containing a nozzle. (44) The method according to the preceding item (43), wherein the sample transport system includes an effective amount of a drug selected from the group consisting of sodium polyanetholesulfonate and sodium amylosulfate. (45) The sample transport system further includes an effective amount of a sulfhydryl group-containing substance that is non-toxic to microorganisms.
The method described in 4). (46) The sample transport system further includes a hypertonicity enhancer effective for suppressing an increase in the bacteriostatic effect (45).
The method described in. (47) Where the sample transport system further includes a substance effective in inhibiting the replication of Gram-positive microorganisms,
45). (48) The sample transport system further adjusts the pH of the sample to about 6.
5 to 7.5;
The method described in the preceding section (45). (49) The method according to item (45), wherein the sample transport system further includes a growth substrate effective in maintaining the general nutritional requirements of the microorganism. (50) The method according to the preceding item (45), wherein the sample transport system includes a sulfa drug inhibitor. (51) The method according to (45), wherein the sample transport system further includes an antibiotic-specific enzyme. (52) The container includes a stop that engages the piston to prevent movement of the piston within the container.
The method described in. (53) The method according to the preceding item (44), which includes a thin portion that allows the shaft to break at the thin portion when bent. (54) Where the container includes a stop that engages the piston to prevent movement of the piston within the container.
53). (55) The method according to the preceding item (44), wherein the shaft is removably connected to the piston. (56) The method of (55) above, wherein the container includes a stop that engages the piston to prevent movement of the piston within the container.