CA1099654A - Bacteria growing device - Google Patents
Bacteria growing deviceInfo
- Publication number
- CA1099654A CA1099654A CA303,712A CA303712A CA1099654A CA 1099654 A CA1099654 A CA 1099654A CA 303712 A CA303712 A CA 303712A CA 1099654 A CA1099654 A CA 1099654A
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- bacteria
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Abstract
913,812 ABSTRACT
A device for growing bacteria from a pre-determined initial population to a predetermined final population comprising a means for obtaining the pre-determined initial population and a medium to obtain growth of the bacteria to the predetermined final population.
A device for growing bacteria from a pre-determined initial population to a predetermined final population comprising a means for obtaining the pre-determined initial population and a medium to obtain growth of the bacteria to the predetermined final population.
Description
-~ -1- lU99654 913, 812 BACTERIA GROWING DEVICE
This invention relates to a device for growing bacteria. Specifically, this invention relates to a device for growing bacteria from an initial population to a final predetermined population, said device including a medium for such growth and a means for obtaining a predetermined population for inoculation of said medium.
In Canadian patent application serial No.
303,737 filed concurrently with this application growth ; 10 limiting media are described and claimed. These media are described as being useful in a number of procedures utilized to identify the bacteria or to determine the susceptibility of the bacteria to certain antibiotics. In such procedures it is necessary to have the bacteria at the beginning of the test procedure in a certain concentration range (colony forming units per milliliter (CFU/ml)) or the final result will not be accurate. For example, in an artlcle entitled "Antibiotics Susceptibility Testing by Standardized Single-Disc Method", The American Journal of Clinical Pathology, Vol.
45, No. 4, April, 1966, Pages 293-296 and in the "Performance Standards for Antimicrobial Disc Susceptibility Test", ASM-2, promulgated by the National Committee for Clinical Laboratory Standards of the United States, the Kirby-Bauer procedure for determining the susceptibility of rapidly growing bacteria to antibiotics and chemotherapy agents is described.
The Kirby-Bauer procedure normally involves ~` growing on an agar plate colonies of bacteria obtained from a patient. A wire loop is used to pick from 4 to 5 colonies of the bacteria and introduce them into test ; tubes containing 4 to 5 milliliters of soybean casein digest broth. The tubes are then incubated for 2 to 8 hours to produ¢e a bacterial suspension of moderate .~
' '.
' .
: ' ' cloudiness. The suspension is then diluted, if necessary, with saline solution or like broth to a density visually equivalent of that of a standard prepared by adding 0.5 milliliter of 1% BaC12 to 99.5 milliliters of 1% H2SO4 (0.36 N) (0.5 McFarland Standard hereinafter referred to as the McFarland standard). A plate containing Mueller-Hinton agar is then stréaked with the bacterial broth suspension using a cotton swab. After the inculum has dried, a paper disc containing an antibiotic or chemotherapeutic agent is applied to the agar. The plates are incubated. After overnight incubation the area around each disc wherein there is an absence of bacteria growth is measured. This is known as the zone of inhibition and is used to determine which antibiotic will be useful in combating the particular bacteria.
In order for the Kirby Bauer technique to be accurate there must be approximately 1 x 108 CFU/ml included in the medium which is streaked onto the agar plate. Usually the level of growth is determined by using the visual comparison with the McFarland standard described above. The time period to reach this concentra-tion of bacteria may vary from 2 to 8 hours depending on the bacteria. If the bacteria are allowed to grow in excess of 1 x 108 CFU/milliliter and become more turbid than the McFarland standard, the medium must be diluted in order to be equivalent to the standard.
Another method of determining the susceptibility of bacteria to various antibiotics is called the MIC or Minimum Inhibitory Concentration test. This test is discussed in Current Techniques for Antibiotics Susceptibility Testing, Albert Balows, ~ 1974, pages 77-87. This method involves preparation of a series of concentrations of an antibiotic, either in a liquid or solid medium which will support the growth of a bacteria to be tested. Liquid media are conveniently dispensed in test tubes and solid media are usually poured into petri dishes. It is common practice to prepare a range of _3_ 1 ~9 g6 S 4 antibiotic concentrations as a series of two-fold dilutions in order to carry out the test. Each tube or petri dish is inoculated with the bacteria in question.
After a period of incubation, the bacterial growth or absence of growth of each antibiotic concentration is observed. In this way, the minimum inhibitory concentra-tion of the antibiotic is determined to the nearest dilution when used in a series. This is the most accurate method of determining the inhibitory concentration.
~owever, this method did not gain popularity until recently when the laborious effort of making the dilutions was simplified. The diluted antibiotic is inoculated in the MIC test with bacteria at a certain concentration, i.e., normally 105 to 106 CFU/ml. Broth containing bacteria grown to the equivalent of the McFarland standard, i.e., approximately 1 x 108 CFU/ml, is diluted to obtain this concentration.
The aforesaid susceptibility tests as well as other tests for determining the types of bacteria or susceptibility thereof to antibiotics require that a certain predetermined amount of bacteria be utilized in the test to inoculate the plates upon which the paper disc will be placed in the case of the Kirby-Bauer test or to inoculate the diluted antibiotics in the case of the MIC
test~ This is required in order for the test to be accurate. If a lesser concentration of bacteria is utilized in the test, the result would indicate that the bacteria is more susceptible to the antibiotic than it would be as an actual fact. On the other hand, if the bacteria are present in a higher concentration, the test result would indicate that a higher concentration of the antibiotic would be required in order to inhibit the growth of the bacteria. Both indications would be erroneous.
In order for the aforesaid tests or tests similar thereto to be performed, it is necessary for the laboratory technician to take a sample of bacteria from 4 lV99654 or 5 colonies of bacteria from the agar plate upon which the bacteria have been growing and place it in a broth growing medium such as above described for 2 to 8 hours.
The medium is checked periodically to determine whether or not a sufficient concentration of bacteria has grown to be equivalent to the McFarland standard. From a visual examination of the medium, one will find that some medium cultures of bacteria have not grown to the proper concen-tration while others have grown beyond the appropriate concentration. The former requires that the technicianallow the bacteria to grow longer, whereas the latter requires a dilution to bring the concentration back to that of the standard. All of these measures are tedious and time consuming.
The above-mentioned Canadian patent application serial No. 303,737 filed concurrently herewith describes a growth-limiting medium which will grow bacteria to a certain predetermined concentration or population level.
That medium is capable of growing at least one species from two different genera of aerobic, pathogenic, rapidly growing bacteria from a beginning population to a determined ending population at which growth of the bacteria substantially subsides due to the lack of nutrient in the medium and wherein bacteria remain viable for at least 18 hours; the medium comprising an aqueous solution comprising a carbon source, a nitrogen source, vitamins and minerals of sufficient quantity to provide growth and in a form usable by the bacteria for growth.
The general description of that medium will be described herein for purposes of completeness.
The bacteria upon which the medium is useful are aerobic bacteria, i.e., those which use oxygen to grow.
The bacteria are also pathogenic in that they cause diseases and are rapidly growing in that they have a generation time of 50 minutes or less.
The medium is useful with both gram-negative as well as gram-positive aerobic, pathogenic, rapidly growing .
_5_ 1~99~54 bacteria. However, the type and amount of the various ingredients in the medium are normally different for gram-positive than for gram-negative bacteria, and the time period required to obtain the requisite concen-tration for gram-positive tends to be longer than that for gram-negative bacteria.
The growth medium will grow at least one species from two different genera of aerobic, pathogenic bacteria.
Normally, the medium will grow at least one species from two genera of gram-positive bacteria or at least one species from at least two genera of gram-negative bacteria. Within gram-positive, aerobic bacteria, there are two genera which include the bacteria that cause most diseases for which normal bacteria and susceptibility testing are performed. These are Staphylococcus and Streptococcus. If the medium will grow species from each of these two genera, then it allows one to merely test the bacteria to determine whether they are gram-positive or negative using a gram stain test. If the bacteria are gram-positive, medium which grows the gram-positive bacteria is used, and the medium will grow the bacteria to the level desired such as to the equivalent of the McFarland standard.
If the bacteria are determined to be gram-negative using the gram stain test, the bacteria could befrom a much larger number of genera. Sixteen genera represent the bacteria found to be the cause of 99% of illnesses caused by gram-negative aerobic bacteria. These gram-negative genera include: Escherichia, Shigella Edwardsiella, Salmonella, Arizona, Citrobacter, Klebsiella, Enterobacter, Serratia, Proteus, Providencia, Yersinia, Pseudomonas, Acinetobacter, Moraxella and Pasteurella.
The medium grows the bacteria from a certain population which will normally be described in terms of CFU as above defined and will normally be referenced to the population in a certain volume of medium, i.e., ' ", ' ' ~ . :
' ' .
~ 6 1~9~6~
, CFU/ml. The beginning concentration can range as low as l CFU but will normally be, and is preferably, at least 5 x 106 CFU/ml.
Starting with a lower initial population or concentration will not cause the medium to grow the bacteria to a significantly different final population or concentration than with a higher starting concentration, but will affect the time it takes for the final concentra-tion to be reached. Thus, if one is to control the incubation time, the beginning concentration must be controlled. The final concentration to which the bacteria grow is referred to as the stationary phase.
As above discussed the time to reach a concentra-tion equivalent to the McFarland standard varies according to the test procedure from 2 to 8 hours with most bacteria taking at least 5 to 6 hours to reach that concentration.
With the growth-limiting medium, the bacteria preferably reach the final concentration or stationary phase within 5 hours. With the growth-limiting medium, if the bacteria reach the stationary phase in 2 hours, they will remain there even if the technician does not check the medium for 5 hours, and no dilution will be required to obtain a concentration equivalent to the McFarland standard.
When the stationary phase or final concentration - 25 is reached, the growth of the bacteria substantially subsides. This is due to exhaustion of at least one nutrient critical to the continued growth of the bacteria and not to the formation of toxic byproducts by the bacteria which stop growth and can cause the population to - 30 decrease substantially. With the standard media used prior to the growth-limiting medium and described in the Kirby-Bauer procedure, the population of bacteria which will be reached in order for growth to subside would be determined by toxic byproducts of the bacteria. This population of bacteria is above the McFarland standard.
The final concentration or maximum stationary phase is obtained because of exhaustion of one or more , . . .
.
.' :. ' .
1(~99ti5~
nutrients. At that point the viable CFU/ml count levels off and remains substantially unchanged for at least about 18 hours. The bacteria remain viable for a period of time useful for carrying out the various tests to be performed thereon, for example, those described above. Normally the time period for such viability is at least 18 hours.
The final concentration which is desired to be reached with most bacteria will be between 6 x 107 to 3.0 x 108 CFU/ml. This is equivalent to the 0.5 McFarland standard. The medium can be modified to reach different desired concentration levels.
The medium will contain different types and amounts of ingredients depending upon the final concen-tration of bacteria desired and depending upon the type of - 15 bacteria being grown. In all cases a carbon and nitrogen source are present which provide carbon and nitrogen in a form useful by the bacteria for growth. Normally, vita-mins and minerals are also present. However, as noted, the amount of one or more of these ingredients is limited to cauge the bacteria to reach a final predetermined concentration and substantially cease growing.
For the gram-negative bacteria, a preferred medium comprises about 0.42 to about 0.70 milligram of carbon per milliliter of medium. The carbon is in a form useful by the bacteria for growth. This form has been found to be that form in which carbon is present in peptone or a similar form. The preferred medium also comprises 0.09 to 0.15 milligram of nitrogen per milliliter of medium in a form similar to the nitrogen present in peptone. The preferred medium has a pH from about 7 to 8. With proteose peptone, the range for carbon is from about 0.16 to about 0.27 milligram of carbon per milliliter of medium, the nitrogen is 0.035 to 0.056 milli-gram of nitrogen per milliliter of medium, and the carbon and nitrogen are as found in proteose peptone or a form similar thereto. A typical analysis of the peptone is set forth below.
.
~ -8- 1~996S~
Percent _ Peptone Peptone Total Nitrogen 16.16 14.37 Primary Proteose N 0.06 0.60 Secondary Proteose N 0.68 4.03 Peptone N 15.38 9.74 Ammonia N 0.04 0.00 Free Amino 3.20 2.66 Amide N 0.49 0.94 Mono-amino N 9.42 7.61 Di-amino N 4.07 4.51 Tryptophane 0.29 0.51 Tr~osine 0.98 2.51 Crystine 0.22 0.56 Organic Sulfur 0.33 0.60 Inorganic Sulfur 0.29 0.04 Phosphorus 0.22 0.47 Chlorine 0.27 3.95 Sodium 1.08 2.84 Potassium 0.22 0.70 Calcium 0.058 0.137 Magnesium 0.056 0.118 Manganese nil 0.0002 Iron 0.0033 0.0056 Ash 3.53 9.61 Ether Soluble Extract 0.37 0.32 Reaction, pH 7.0 6.8 - p~ 1~ solution is distilled water after autoclaving 15 minutes at 121C.
1a~99654 g The preferred formulation contains the vitamins and minerals found in peptone or proteose peptone. Two specifically preferred formulations which have been found to be useful in growing gram-negative bacteria to a final concentration of from 6 x 107 to 3 x 108 CFU/ml in less than 5 hours comprises a mixture of 1000 ml of water containing 0.8 gram peptone or 0.3 gram proteose peptone, 0.03 gram dextrose, 2.5 grams dipotassium phosphate, 1.25 grams monopotassium phosphate and 5.0 grams sodium chloride. The phosphates are added as a buffer material to main~ain the composition at a pH of approximately 7Ø
Buffering is necessary with certain of the bacteria. The aforesaid two formulations provide a medium upon which species from most of the genera of the gram-negative, aerobic, pathogenic bacteria can grow to the above described CFU/ml ranges within 5 hours if the initial concentration of bacteria is sufficient, i.e., at least about 5 x 106 CFU/ml.
As noted the preferred carbon and nitrogen sources are peptone and proteose peptone for the gram-negative bacteria. Neopeptone, tryptone and polypeptone can also be used but it has been found that these do not produce growth to the levels of the ~cFarland standard within the same time frame, and with some of the bacteria within this group, they do not provide the appropriate nutrients to grow the bacteria to any signi-ficant degree. Therefore, these materials are useful for more limited numbers of bacteria. However, combinations of such materials with peptone and proteose peptone can be used to provide a medium useful with a larger number of bacteria.
A preferred medium for use with gram-positive bacteria comprises a solution of 1000 ml of water containing 1.7 grams trypticase, 0.3 gram phytone, 0.25 gram dextrose, 0.5 gram sodium chloride and 0.25 gram dipotassium phosphate.
.
Applicants have found a device which utilizes the above described growth-limiting medium and allows one to obtain from colonies of bacteria a certain predetermined amount of bacteria so that the growth-limiting media is inoculated with a predetermined numberof bacteria. This allows the time period for growth of the bacteria to be predetermined as well. In order for the requisite growth to occur within the preferred time of within 5 hours for gram-negative bacteria, the beginning population must be at least about 5 x 106 CFU/ml.
Applicants have, therefore, discovered a device for growing bacteria from an initial population to a determined ending population at which the growth of the bacteria substantially subsides due to the lack of nutrient comprising: (a) a vessel containing a supply of growth medium capable of growing the bacteria from the beginning population to the determined ending population, the vessel containing an opening; (b) means within the vessel for obtaining a known quantity of bacteria from at least one growth colony of the bacteria external to the vessel and for inoculating the growth medium; and (c) removable means for covering the opening in said vessel to preserve the sterility of the inside of the vessel, for permitting the means for obtaining said bacteria to be removed to obtain the known quantity of bacteria from the colony of the bacteria external of the vessel, which bacteria are used to inoculate the medium and for closing the vessel during the incubation of the inoculated medium.
The device will be described in more detail with reference to the following drawings in which Figure 1 is an exploded sectional view of the device of the present invention; Figure 2 is a prospective view of the device of the present invention with parts in section; Figure 3 is an exploded view of a portion of the wand of the device of the present invention Figure 4 is the wand of Figure 3 showing the inclusion therein of bacteria; Figure 5 is a sectional view of the device of the present invention just after inoculation of the growth medium with the bacteria;
Figure 6 depicts a section of the device of the present invention as shown in Figure 5 taken along line 6-6;
Figure 7 is a section of the device of the present invention after inoculation and incubation to the maximum stationary phase or the predetermined growth stage; and Figure 8 depicts a section of the device of Figure 7 taken along line 8-8.
The device of the present invention comprises a vessel or sleeve 1 which is made of a transparent deformable material such as polypropylene, polyamide, cellulose acetate butyrate or various polyesters.
Contained within the sleeve 1 is glass ampoule 2 which contains within it the growth limiting medium 3 as above described. The glass ampoule, as will be discussed later, is frangible, i.e., it breaks when the deformable sleeve 1 is squeezed. In juxtaposition to the ampoule 2 of the device is wand 4 which contains tapered groove 5 which will be described in more detail with reference to Figures 3 and 4. Wand 4 is used to pick bacteria from the various colonies of bacteria and is made to injest a predetermined amount of bacteria from the colonies into the tapered groove 5. Wand 4 is affixed to cap 6. Both cap 6 and wand 4 are made from plastic material such as poly-propylene. Interior to cap 6 are two ridges 7 and 8 whichallow for an aseptic seal between the cap 6 and sleeve 1.
This prevents other microorganisms from entering the sleeve 1 prior to the time that the device is inoculated as well as during incubation. Also contained within cap 6 are three prongs 9, two of which are shown in Figure 1.
These prongs protect hole 10 in cap 6 from being blocked by broken glass from ampoule 2 (caused when the device is activated) during the time when deformable sleeve 1 is being deformed to express or exude the bacteria and medium 3 via hole 10. Hole 10 is covered with pressure sensitive adhesive tape 11 containing tab 12 during the time prior to use of the device and up until incubation of the device '' . ' ' ' ' - , ' . ~ ..
.
' ' -12- 1~9965~
is complete. At that time, tape ll is removed exposing hole 10. This allows for the medium 3 and bacteria to be exuded or expressed from the sleeve 1.
Figure 2 shows the device in its normal form prior to use except the pressure sensitive adhesive tape ll is not present. As can be seen, wand 4 is adjacent to ampoule 2 and rims 7 and 8 are in juxtaposition to the upper portion of sleeve l. In this case hole 10 is exposed to show it from a prospective view and pressure sensitive adhesive tape 11 is not shown. Prongs 9 are not visible in this view. Glass ampoule 2 is ~ormally 35.6 millimeters by 6.3 millimeters in dimensions and contains approximately 0.6 millimeters of growth media. The sleeve 1 is normally 43.0 millimeters long and 8.6 millimeters in diameter.
An important feature of the device of the present invention is the wand 4 containing tapered groove 5. This groove 5 is shown in more detail in Figure 3. As can be seen, the groove 5 is tapered. This groove i8 designed to provide a capillary action by which the bacteria are pushed into the groove 5. As the bacteria are pu~hed by means of pushing the wand 4 perpendicular to the surface of the colony so that the larger end of tapered groove 5 first contacts the colony, the bacteria begin to move up the groove and air is exuded from the tapered end of the groove. At a certain predetermined level, no further bacteria can be pushed into the groove because bacteria which are continually forced into the groove 5 move out of the top of the groove 5 rather than moving up to the tapered end of the groove 5. Figure 4 shows schematically bacteria 13 in the groove 5 at the normal filled groove level. The groove 5 is designed to allow a certain population of bacteria to enter the groove. This is normally equivalent to at least 5 x lO6 bacteria per milliliter when the bacteria are placed in the medium of the device of the present invention. A
tapered groove about 0.43 millimeter deep, 0.25 millimeter . ~
1(~99654 wide at its largest end and 3.1 millimeters long will allow for the aforesaid pick-up. The groove can be modified to accomplish different desired populations of bacteria.
The use of the device will be described now with reference to Figures 5 through 8. Figure 5 illustrates schematically, with the device in section, the device of the present invention just after inoculation of the device using wand 4. Cap 6 has been removed from the sleeve 1, and wand 4, via tapered groove 5, has picked up bacteria from 4 to 5 colonies of bacteria, normally at least 5 x 106 bacteria. Cap 6 has been replaced, and the bacteria, via wand 4 and groove 5, were placed adjacent to the ampoule 2. As shown in Figure 5, the ampoule 2 has been broken by deforming sleeve 1 and, the bacteria 13 are now shown schematically in the growth-limiting media 3.
The device in this form will be incubated at approximately 35C for normally from 2 to 8 hours. With the preferred growth media, approximately 5 hours will be sufficient to obtain the predetermined preferred amount of bacteria, i,e., from 6 x 107 to 3 x 108 CFU/ml. Figure 6 depicts in section the device of Figure 5 at this beginning stage of incubation and shows the sleeve 1 containing broken ampoule 2, growth media 3 and bacteria 13.
After incubation the device is in the form shown in Figure 7. In this case, all of the device is just as it was before except, as can be seen, the population of bacteria 13 has increased significantly. Figure 8 is a section of the device of Figure 7 at this stage. After incubation, the device of Figure 7 shows that the tape 11 has been removed and the device is ready now for exuding of the growth bacteria via hole 10. The device is merely held with hole 10 in a downward direction, sleeve 1 is squeezed and deformed, and the bacteria 13 and media 3 are exuded via hole 10. Broken glass from ampoule 2 is precluded from plugging the hole via prongs 9.
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.
.
1~39~65~
The tapered groove 5 in the wand 4 can be changed to affectuate a different size inoculum of bacteria so the population of bacteria picked up is greater than or less than that above described. Other configurations than a tapered groove could be used in the present invention as long as there is an ability of the wand or pick-up device to pick-up, on a repeatable basis, a predetermined amount of bacteria. The device then in combination will allow the bacteria to grow from a certain predetermined level which is determined by the pick-up device to a final level which is determined by the growth-limiting media in a certain period of time, which time period is determined by the beginning population, the growth limiting medium formulation and type of bacteria.
For use in a Kirby-Bauer test or MIC test, it is preferred to have the device grow the bacteria within 5 hours to a concentration of from about 6 x 107 to 3 x 108 CFU/ml.
The device as shown contains the growth limiting media 3 in a glass ampoule 2. Other modi~ications can be made to affectuate the same results. For example, the device can merely be a threaded sleeve with a screw-on cap which has attached to it the wand In this case the growth-limiting medium is within the sleeve and is inoculated directly when the cap was removed. The bacteria are picked up with the wand and the cap screwed back on placing the wand in the medium. Other modifica-tions will be apparent to one skilled in the art and are included within the claims.
The device depicted in Figure 1 is made by molding and forming the various glass and plastic components. Approximately 0.6 milliliter of medium 3 is placed in glass ampoule 2, and the glass ampoule 2 is heat - sealed. The ampoule 2 is steam sterilized for 10 minutes at 121C. The sleeve 1, cap 6 and tape 11 are gas sterilized with ethylene oxide for 3 hours at 100F and then aerated for 8 hours. The sealed and sterilized glass ampoule 2 is asceptically added to the sleeve 1; the cap 6 1~99tiS4 is attached and the tap 11 is pressed into place.
In the following examples the following materials and bacteria are referenced. Their source is set forth below. In the examples growing device means a device as described above with the dimensions and prepared as described above.
Tryptone, an enzymatic hydrolysate of casein, Difco, Inc., Detroit, Michigan, U.S.A.
Peptone, an enzymatic hydrolysate of casein, Difco, Inc., Detroit, Michigan, U.S.A.
Dextrose, Difco, Inc., Detroit, Michigan, U.S.A.
Polypeptone, an enzymatic hydrolysate of casein and animal tissue, Bioquest, Inc., Baltimore, Maryland, U.S.A.
Neopeptone, an enzymatic hydrolysate of protein, Difco, Inc., Detroit, Michigan, U.S.A.
Proteose peptone, an enzymatic hydrolysate of protein, Difco, Inc., Detroit, Michigan, U.S.A.
Salmonella typhimurium, American Type Culture 20 Collection, (ATCC) No. 19028 Shigella Sonnei, ~ATCC 25331) Enterobacter cloacae (ATCC 23355) and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Klebsiella pneumoniae, (ATCC 23357) and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Proteus vulgaris (ATCC 6380) Proteus mirabilis, St. John's Hospital, St.
Paul, Minnesota and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Serratia marcescens (ATCC 8100) and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Providencia species, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Citrobacter species, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Edwardsiella, University of Minnesota, Minneapolis, Minnesota, U.S.A.
1~99654 , .
Arizona, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Yersinia, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Pseudomonas aeruginosa (ATCC 27853) St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Escherichia coli (ATCC 25922) and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Acinetobacter calcoaceticus, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Proteus morganii, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Enterobacter aerogenes, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Pasteurella (species), St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
CDC Group II F, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Moraxella, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Citrobacter freundii, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Example 1 Devices having the specifications described above were inoculated with various bacteria using the tapered groove of the wand of the device. An initial concentration of bacteria was noted. The growth limiting media included within the ampoule of each device contains the following:
0.8 gram Peptone 0.03 gram Dextrose
This invention relates to a device for growing bacteria. Specifically, this invention relates to a device for growing bacteria from an initial population to a final predetermined population, said device including a medium for such growth and a means for obtaining a predetermined population for inoculation of said medium.
In Canadian patent application serial No.
303,737 filed concurrently with this application growth ; 10 limiting media are described and claimed. These media are described as being useful in a number of procedures utilized to identify the bacteria or to determine the susceptibility of the bacteria to certain antibiotics. In such procedures it is necessary to have the bacteria at the beginning of the test procedure in a certain concentration range (colony forming units per milliliter (CFU/ml)) or the final result will not be accurate. For example, in an artlcle entitled "Antibiotics Susceptibility Testing by Standardized Single-Disc Method", The American Journal of Clinical Pathology, Vol.
45, No. 4, April, 1966, Pages 293-296 and in the "Performance Standards for Antimicrobial Disc Susceptibility Test", ASM-2, promulgated by the National Committee for Clinical Laboratory Standards of the United States, the Kirby-Bauer procedure for determining the susceptibility of rapidly growing bacteria to antibiotics and chemotherapy agents is described.
The Kirby-Bauer procedure normally involves ~` growing on an agar plate colonies of bacteria obtained from a patient. A wire loop is used to pick from 4 to 5 colonies of the bacteria and introduce them into test ; tubes containing 4 to 5 milliliters of soybean casein digest broth. The tubes are then incubated for 2 to 8 hours to produ¢e a bacterial suspension of moderate .~
' '.
' .
: ' ' cloudiness. The suspension is then diluted, if necessary, with saline solution or like broth to a density visually equivalent of that of a standard prepared by adding 0.5 milliliter of 1% BaC12 to 99.5 milliliters of 1% H2SO4 (0.36 N) (0.5 McFarland Standard hereinafter referred to as the McFarland standard). A plate containing Mueller-Hinton agar is then stréaked with the bacterial broth suspension using a cotton swab. After the inculum has dried, a paper disc containing an antibiotic or chemotherapeutic agent is applied to the agar. The plates are incubated. After overnight incubation the area around each disc wherein there is an absence of bacteria growth is measured. This is known as the zone of inhibition and is used to determine which antibiotic will be useful in combating the particular bacteria.
In order for the Kirby Bauer technique to be accurate there must be approximately 1 x 108 CFU/ml included in the medium which is streaked onto the agar plate. Usually the level of growth is determined by using the visual comparison with the McFarland standard described above. The time period to reach this concentra-tion of bacteria may vary from 2 to 8 hours depending on the bacteria. If the bacteria are allowed to grow in excess of 1 x 108 CFU/milliliter and become more turbid than the McFarland standard, the medium must be diluted in order to be equivalent to the standard.
Another method of determining the susceptibility of bacteria to various antibiotics is called the MIC or Minimum Inhibitory Concentration test. This test is discussed in Current Techniques for Antibiotics Susceptibility Testing, Albert Balows, ~ 1974, pages 77-87. This method involves preparation of a series of concentrations of an antibiotic, either in a liquid or solid medium which will support the growth of a bacteria to be tested. Liquid media are conveniently dispensed in test tubes and solid media are usually poured into petri dishes. It is common practice to prepare a range of _3_ 1 ~9 g6 S 4 antibiotic concentrations as a series of two-fold dilutions in order to carry out the test. Each tube or petri dish is inoculated with the bacteria in question.
After a period of incubation, the bacterial growth or absence of growth of each antibiotic concentration is observed. In this way, the minimum inhibitory concentra-tion of the antibiotic is determined to the nearest dilution when used in a series. This is the most accurate method of determining the inhibitory concentration.
~owever, this method did not gain popularity until recently when the laborious effort of making the dilutions was simplified. The diluted antibiotic is inoculated in the MIC test with bacteria at a certain concentration, i.e., normally 105 to 106 CFU/ml. Broth containing bacteria grown to the equivalent of the McFarland standard, i.e., approximately 1 x 108 CFU/ml, is diluted to obtain this concentration.
The aforesaid susceptibility tests as well as other tests for determining the types of bacteria or susceptibility thereof to antibiotics require that a certain predetermined amount of bacteria be utilized in the test to inoculate the plates upon which the paper disc will be placed in the case of the Kirby-Bauer test or to inoculate the diluted antibiotics in the case of the MIC
test~ This is required in order for the test to be accurate. If a lesser concentration of bacteria is utilized in the test, the result would indicate that the bacteria is more susceptible to the antibiotic than it would be as an actual fact. On the other hand, if the bacteria are present in a higher concentration, the test result would indicate that a higher concentration of the antibiotic would be required in order to inhibit the growth of the bacteria. Both indications would be erroneous.
In order for the aforesaid tests or tests similar thereto to be performed, it is necessary for the laboratory technician to take a sample of bacteria from 4 lV99654 or 5 colonies of bacteria from the agar plate upon which the bacteria have been growing and place it in a broth growing medium such as above described for 2 to 8 hours.
The medium is checked periodically to determine whether or not a sufficient concentration of bacteria has grown to be equivalent to the McFarland standard. From a visual examination of the medium, one will find that some medium cultures of bacteria have not grown to the proper concen-tration while others have grown beyond the appropriate concentration. The former requires that the technicianallow the bacteria to grow longer, whereas the latter requires a dilution to bring the concentration back to that of the standard. All of these measures are tedious and time consuming.
The above-mentioned Canadian patent application serial No. 303,737 filed concurrently herewith describes a growth-limiting medium which will grow bacteria to a certain predetermined concentration or population level.
That medium is capable of growing at least one species from two different genera of aerobic, pathogenic, rapidly growing bacteria from a beginning population to a determined ending population at which growth of the bacteria substantially subsides due to the lack of nutrient in the medium and wherein bacteria remain viable for at least 18 hours; the medium comprising an aqueous solution comprising a carbon source, a nitrogen source, vitamins and minerals of sufficient quantity to provide growth and in a form usable by the bacteria for growth.
The general description of that medium will be described herein for purposes of completeness.
The bacteria upon which the medium is useful are aerobic bacteria, i.e., those which use oxygen to grow.
The bacteria are also pathogenic in that they cause diseases and are rapidly growing in that they have a generation time of 50 minutes or less.
The medium is useful with both gram-negative as well as gram-positive aerobic, pathogenic, rapidly growing .
_5_ 1~99~54 bacteria. However, the type and amount of the various ingredients in the medium are normally different for gram-positive than for gram-negative bacteria, and the time period required to obtain the requisite concen-tration for gram-positive tends to be longer than that for gram-negative bacteria.
The growth medium will grow at least one species from two different genera of aerobic, pathogenic bacteria.
Normally, the medium will grow at least one species from two genera of gram-positive bacteria or at least one species from at least two genera of gram-negative bacteria. Within gram-positive, aerobic bacteria, there are two genera which include the bacteria that cause most diseases for which normal bacteria and susceptibility testing are performed. These are Staphylococcus and Streptococcus. If the medium will grow species from each of these two genera, then it allows one to merely test the bacteria to determine whether they are gram-positive or negative using a gram stain test. If the bacteria are gram-positive, medium which grows the gram-positive bacteria is used, and the medium will grow the bacteria to the level desired such as to the equivalent of the McFarland standard.
If the bacteria are determined to be gram-negative using the gram stain test, the bacteria could befrom a much larger number of genera. Sixteen genera represent the bacteria found to be the cause of 99% of illnesses caused by gram-negative aerobic bacteria. These gram-negative genera include: Escherichia, Shigella Edwardsiella, Salmonella, Arizona, Citrobacter, Klebsiella, Enterobacter, Serratia, Proteus, Providencia, Yersinia, Pseudomonas, Acinetobacter, Moraxella and Pasteurella.
The medium grows the bacteria from a certain population which will normally be described in terms of CFU as above defined and will normally be referenced to the population in a certain volume of medium, i.e., ' ", ' ' ~ . :
' ' .
~ 6 1~9~6~
, CFU/ml. The beginning concentration can range as low as l CFU but will normally be, and is preferably, at least 5 x 106 CFU/ml.
Starting with a lower initial population or concentration will not cause the medium to grow the bacteria to a significantly different final population or concentration than with a higher starting concentration, but will affect the time it takes for the final concentra-tion to be reached. Thus, if one is to control the incubation time, the beginning concentration must be controlled. The final concentration to which the bacteria grow is referred to as the stationary phase.
As above discussed the time to reach a concentra-tion equivalent to the McFarland standard varies according to the test procedure from 2 to 8 hours with most bacteria taking at least 5 to 6 hours to reach that concentration.
With the growth-limiting medium, the bacteria preferably reach the final concentration or stationary phase within 5 hours. With the growth-limiting medium, if the bacteria reach the stationary phase in 2 hours, they will remain there even if the technician does not check the medium for 5 hours, and no dilution will be required to obtain a concentration equivalent to the McFarland standard.
When the stationary phase or final concentration - 25 is reached, the growth of the bacteria substantially subsides. This is due to exhaustion of at least one nutrient critical to the continued growth of the bacteria and not to the formation of toxic byproducts by the bacteria which stop growth and can cause the population to - 30 decrease substantially. With the standard media used prior to the growth-limiting medium and described in the Kirby-Bauer procedure, the population of bacteria which will be reached in order for growth to subside would be determined by toxic byproducts of the bacteria. This population of bacteria is above the McFarland standard.
The final concentration or maximum stationary phase is obtained because of exhaustion of one or more , . . .
.
.' :. ' .
1(~99ti5~
nutrients. At that point the viable CFU/ml count levels off and remains substantially unchanged for at least about 18 hours. The bacteria remain viable for a period of time useful for carrying out the various tests to be performed thereon, for example, those described above. Normally the time period for such viability is at least 18 hours.
The final concentration which is desired to be reached with most bacteria will be between 6 x 107 to 3.0 x 108 CFU/ml. This is equivalent to the 0.5 McFarland standard. The medium can be modified to reach different desired concentration levels.
The medium will contain different types and amounts of ingredients depending upon the final concen-tration of bacteria desired and depending upon the type of - 15 bacteria being grown. In all cases a carbon and nitrogen source are present which provide carbon and nitrogen in a form useful by the bacteria for growth. Normally, vita-mins and minerals are also present. However, as noted, the amount of one or more of these ingredients is limited to cauge the bacteria to reach a final predetermined concentration and substantially cease growing.
For the gram-negative bacteria, a preferred medium comprises about 0.42 to about 0.70 milligram of carbon per milliliter of medium. The carbon is in a form useful by the bacteria for growth. This form has been found to be that form in which carbon is present in peptone or a similar form. The preferred medium also comprises 0.09 to 0.15 milligram of nitrogen per milliliter of medium in a form similar to the nitrogen present in peptone. The preferred medium has a pH from about 7 to 8. With proteose peptone, the range for carbon is from about 0.16 to about 0.27 milligram of carbon per milliliter of medium, the nitrogen is 0.035 to 0.056 milli-gram of nitrogen per milliliter of medium, and the carbon and nitrogen are as found in proteose peptone or a form similar thereto. A typical analysis of the peptone is set forth below.
.
~ -8- 1~996S~
Percent _ Peptone Peptone Total Nitrogen 16.16 14.37 Primary Proteose N 0.06 0.60 Secondary Proteose N 0.68 4.03 Peptone N 15.38 9.74 Ammonia N 0.04 0.00 Free Amino 3.20 2.66 Amide N 0.49 0.94 Mono-amino N 9.42 7.61 Di-amino N 4.07 4.51 Tryptophane 0.29 0.51 Tr~osine 0.98 2.51 Crystine 0.22 0.56 Organic Sulfur 0.33 0.60 Inorganic Sulfur 0.29 0.04 Phosphorus 0.22 0.47 Chlorine 0.27 3.95 Sodium 1.08 2.84 Potassium 0.22 0.70 Calcium 0.058 0.137 Magnesium 0.056 0.118 Manganese nil 0.0002 Iron 0.0033 0.0056 Ash 3.53 9.61 Ether Soluble Extract 0.37 0.32 Reaction, pH 7.0 6.8 - p~ 1~ solution is distilled water after autoclaving 15 minutes at 121C.
1a~99654 g The preferred formulation contains the vitamins and minerals found in peptone or proteose peptone. Two specifically preferred formulations which have been found to be useful in growing gram-negative bacteria to a final concentration of from 6 x 107 to 3 x 108 CFU/ml in less than 5 hours comprises a mixture of 1000 ml of water containing 0.8 gram peptone or 0.3 gram proteose peptone, 0.03 gram dextrose, 2.5 grams dipotassium phosphate, 1.25 grams monopotassium phosphate and 5.0 grams sodium chloride. The phosphates are added as a buffer material to main~ain the composition at a pH of approximately 7Ø
Buffering is necessary with certain of the bacteria. The aforesaid two formulations provide a medium upon which species from most of the genera of the gram-negative, aerobic, pathogenic bacteria can grow to the above described CFU/ml ranges within 5 hours if the initial concentration of bacteria is sufficient, i.e., at least about 5 x 106 CFU/ml.
As noted the preferred carbon and nitrogen sources are peptone and proteose peptone for the gram-negative bacteria. Neopeptone, tryptone and polypeptone can also be used but it has been found that these do not produce growth to the levels of the ~cFarland standard within the same time frame, and with some of the bacteria within this group, they do not provide the appropriate nutrients to grow the bacteria to any signi-ficant degree. Therefore, these materials are useful for more limited numbers of bacteria. However, combinations of such materials with peptone and proteose peptone can be used to provide a medium useful with a larger number of bacteria.
A preferred medium for use with gram-positive bacteria comprises a solution of 1000 ml of water containing 1.7 grams trypticase, 0.3 gram phytone, 0.25 gram dextrose, 0.5 gram sodium chloride and 0.25 gram dipotassium phosphate.
.
Applicants have found a device which utilizes the above described growth-limiting medium and allows one to obtain from colonies of bacteria a certain predetermined amount of bacteria so that the growth-limiting media is inoculated with a predetermined numberof bacteria. This allows the time period for growth of the bacteria to be predetermined as well. In order for the requisite growth to occur within the preferred time of within 5 hours for gram-negative bacteria, the beginning population must be at least about 5 x 106 CFU/ml.
Applicants have, therefore, discovered a device for growing bacteria from an initial population to a determined ending population at which the growth of the bacteria substantially subsides due to the lack of nutrient comprising: (a) a vessel containing a supply of growth medium capable of growing the bacteria from the beginning population to the determined ending population, the vessel containing an opening; (b) means within the vessel for obtaining a known quantity of bacteria from at least one growth colony of the bacteria external to the vessel and for inoculating the growth medium; and (c) removable means for covering the opening in said vessel to preserve the sterility of the inside of the vessel, for permitting the means for obtaining said bacteria to be removed to obtain the known quantity of bacteria from the colony of the bacteria external of the vessel, which bacteria are used to inoculate the medium and for closing the vessel during the incubation of the inoculated medium.
The device will be described in more detail with reference to the following drawings in which Figure 1 is an exploded sectional view of the device of the present invention; Figure 2 is a prospective view of the device of the present invention with parts in section; Figure 3 is an exploded view of a portion of the wand of the device of the present invention Figure 4 is the wand of Figure 3 showing the inclusion therein of bacteria; Figure 5 is a sectional view of the device of the present invention just after inoculation of the growth medium with the bacteria;
Figure 6 depicts a section of the device of the present invention as shown in Figure 5 taken along line 6-6;
Figure 7 is a section of the device of the present invention after inoculation and incubation to the maximum stationary phase or the predetermined growth stage; and Figure 8 depicts a section of the device of Figure 7 taken along line 8-8.
The device of the present invention comprises a vessel or sleeve 1 which is made of a transparent deformable material such as polypropylene, polyamide, cellulose acetate butyrate or various polyesters.
Contained within the sleeve 1 is glass ampoule 2 which contains within it the growth limiting medium 3 as above described. The glass ampoule, as will be discussed later, is frangible, i.e., it breaks when the deformable sleeve 1 is squeezed. In juxtaposition to the ampoule 2 of the device is wand 4 which contains tapered groove 5 which will be described in more detail with reference to Figures 3 and 4. Wand 4 is used to pick bacteria from the various colonies of bacteria and is made to injest a predetermined amount of bacteria from the colonies into the tapered groove 5. Wand 4 is affixed to cap 6. Both cap 6 and wand 4 are made from plastic material such as poly-propylene. Interior to cap 6 are two ridges 7 and 8 whichallow for an aseptic seal between the cap 6 and sleeve 1.
This prevents other microorganisms from entering the sleeve 1 prior to the time that the device is inoculated as well as during incubation. Also contained within cap 6 are three prongs 9, two of which are shown in Figure 1.
These prongs protect hole 10 in cap 6 from being blocked by broken glass from ampoule 2 (caused when the device is activated) during the time when deformable sleeve 1 is being deformed to express or exude the bacteria and medium 3 via hole 10. Hole 10 is covered with pressure sensitive adhesive tape 11 containing tab 12 during the time prior to use of the device and up until incubation of the device '' . ' ' ' ' - , ' . ~ ..
.
' ' -12- 1~9965~
is complete. At that time, tape ll is removed exposing hole 10. This allows for the medium 3 and bacteria to be exuded or expressed from the sleeve 1.
Figure 2 shows the device in its normal form prior to use except the pressure sensitive adhesive tape ll is not present. As can be seen, wand 4 is adjacent to ampoule 2 and rims 7 and 8 are in juxtaposition to the upper portion of sleeve l. In this case hole 10 is exposed to show it from a prospective view and pressure sensitive adhesive tape 11 is not shown. Prongs 9 are not visible in this view. Glass ampoule 2 is ~ormally 35.6 millimeters by 6.3 millimeters in dimensions and contains approximately 0.6 millimeters of growth media. The sleeve 1 is normally 43.0 millimeters long and 8.6 millimeters in diameter.
An important feature of the device of the present invention is the wand 4 containing tapered groove 5. This groove 5 is shown in more detail in Figure 3. As can be seen, the groove 5 is tapered. This groove i8 designed to provide a capillary action by which the bacteria are pushed into the groove 5. As the bacteria are pu~hed by means of pushing the wand 4 perpendicular to the surface of the colony so that the larger end of tapered groove 5 first contacts the colony, the bacteria begin to move up the groove and air is exuded from the tapered end of the groove. At a certain predetermined level, no further bacteria can be pushed into the groove because bacteria which are continually forced into the groove 5 move out of the top of the groove 5 rather than moving up to the tapered end of the groove 5. Figure 4 shows schematically bacteria 13 in the groove 5 at the normal filled groove level. The groove 5 is designed to allow a certain population of bacteria to enter the groove. This is normally equivalent to at least 5 x lO6 bacteria per milliliter when the bacteria are placed in the medium of the device of the present invention. A
tapered groove about 0.43 millimeter deep, 0.25 millimeter . ~
1(~99654 wide at its largest end and 3.1 millimeters long will allow for the aforesaid pick-up. The groove can be modified to accomplish different desired populations of bacteria.
The use of the device will be described now with reference to Figures 5 through 8. Figure 5 illustrates schematically, with the device in section, the device of the present invention just after inoculation of the device using wand 4. Cap 6 has been removed from the sleeve 1, and wand 4, via tapered groove 5, has picked up bacteria from 4 to 5 colonies of bacteria, normally at least 5 x 106 bacteria. Cap 6 has been replaced, and the bacteria, via wand 4 and groove 5, were placed adjacent to the ampoule 2. As shown in Figure 5, the ampoule 2 has been broken by deforming sleeve 1 and, the bacteria 13 are now shown schematically in the growth-limiting media 3.
The device in this form will be incubated at approximately 35C for normally from 2 to 8 hours. With the preferred growth media, approximately 5 hours will be sufficient to obtain the predetermined preferred amount of bacteria, i,e., from 6 x 107 to 3 x 108 CFU/ml. Figure 6 depicts in section the device of Figure 5 at this beginning stage of incubation and shows the sleeve 1 containing broken ampoule 2, growth media 3 and bacteria 13.
After incubation the device is in the form shown in Figure 7. In this case, all of the device is just as it was before except, as can be seen, the population of bacteria 13 has increased significantly. Figure 8 is a section of the device of Figure 7 at this stage. After incubation, the device of Figure 7 shows that the tape 11 has been removed and the device is ready now for exuding of the growth bacteria via hole 10. The device is merely held with hole 10 in a downward direction, sleeve 1 is squeezed and deformed, and the bacteria 13 and media 3 are exuded via hole 10. Broken glass from ampoule 2 is precluded from plugging the hole via prongs 9.
.
.
.
1~39~65~
The tapered groove 5 in the wand 4 can be changed to affectuate a different size inoculum of bacteria so the population of bacteria picked up is greater than or less than that above described. Other configurations than a tapered groove could be used in the present invention as long as there is an ability of the wand or pick-up device to pick-up, on a repeatable basis, a predetermined amount of bacteria. The device then in combination will allow the bacteria to grow from a certain predetermined level which is determined by the pick-up device to a final level which is determined by the growth-limiting media in a certain period of time, which time period is determined by the beginning population, the growth limiting medium formulation and type of bacteria.
For use in a Kirby-Bauer test or MIC test, it is preferred to have the device grow the bacteria within 5 hours to a concentration of from about 6 x 107 to 3 x 108 CFU/ml.
The device as shown contains the growth limiting media 3 in a glass ampoule 2. Other modi~ications can be made to affectuate the same results. For example, the device can merely be a threaded sleeve with a screw-on cap which has attached to it the wand In this case the growth-limiting medium is within the sleeve and is inoculated directly when the cap was removed. The bacteria are picked up with the wand and the cap screwed back on placing the wand in the medium. Other modifica-tions will be apparent to one skilled in the art and are included within the claims.
The device depicted in Figure 1 is made by molding and forming the various glass and plastic components. Approximately 0.6 milliliter of medium 3 is placed in glass ampoule 2, and the glass ampoule 2 is heat - sealed. The ampoule 2 is steam sterilized for 10 minutes at 121C. The sleeve 1, cap 6 and tape 11 are gas sterilized with ethylene oxide for 3 hours at 100F and then aerated for 8 hours. The sealed and sterilized glass ampoule 2 is asceptically added to the sleeve 1; the cap 6 1~99tiS4 is attached and the tap 11 is pressed into place.
In the following examples the following materials and bacteria are referenced. Their source is set forth below. In the examples growing device means a device as described above with the dimensions and prepared as described above.
Tryptone, an enzymatic hydrolysate of casein, Difco, Inc., Detroit, Michigan, U.S.A.
Peptone, an enzymatic hydrolysate of casein, Difco, Inc., Detroit, Michigan, U.S.A.
Dextrose, Difco, Inc., Detroit, Michigan, U.S.A.
Polypeptone, an enzymatic hydrolysate of casein and animal tissue, Bioquest, Inc., Baltimore, Maryland, U.S.A.
Neopeptone, an enzymatic hydrolysate of protein, Difco, Inc., Detroit, Michigan, U.S.A.
Proteose peptone, an enzymatic hydrolysate of protein, Difco, Inc., Detroit, Michigan, U.S.A.
Salmonella typhimurium, American Type Culture 20 Collection, (ATCC) No. 19028 Shigella Sonnei, ~ATCC 25331) Enterobacter cloacae (ATCC 23355) and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Klebsiella pneumoniae, (ATCC 23357) and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Proteus vulgaris (ATCC 6380) Proteus mirabilis, St. John's Hospital, St.
Paul, Minnesota and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Serratia marcescens (ATCC 8100) and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Providencia species, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Citrobacter species, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Edwardsiella, University of Minnesota, Minneapolis, Minnesota, U.S.A.
1~99654 , .
Arizona, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Yersinia, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Pseudomonas aeruginosa (ATCC 27853) St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Escherichia coli (ATCC 25922) and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Acinetobacter calcoaceticus, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Proteus morganii, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Enterobacter aerogenes, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Pasteurella (species), St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
CDC Group II F, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Moraxella, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Citrobacter freundii, St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Example 1 Devices having the specifications described above were inoculated with various bacteria using the tapered groove of the wand of the device. An initial concentration of bacteria was noted. The growth limiting media included within the ampoule of each device contains the following:
0.8 gram Peptone 0.03 gram Dextrose
2.5 grams Dipotassium phosphate 1.25 grams Monopotassium phosphate 5.0 grams Sodium chloride Solutions of media containing 0.2 gram peptone and 1.6 grams peptone were also prepared.
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Four other growth limiting media were used which include proteose peptone, tryptone, neopeptone and polypeptone. These were substituted for the peptone of the above formulation. The resulting initial concentra-tions were determined and are as follows in CFU/ml. Theresults given are the mean of 15 samples, i.e., 5 samples of each of the 3 formulations of each medium.
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Example 2 The following materials were dissolved in 1000 milliliters of deionized water and steam sterilized at 121C for 15 minutes:
0.8 gram Peptone 0.03 gram Dextrose 2.5 grams Dipotassium phosphate 1.25 grams Monopotassium phosphate 5.0 grams Sodium chloride Solutions of media containing 0.2 gram peptone and 1.6 grams peptone were also prepared. Growing devices were then prepared using each of the media. Utilizing the wand 4 of the growing device bacteria were picked from 4 to 5, 18 to 24 hour old bacterial colonies of the various bacteria set forth in the table below. Five growing devices were used for each bacteria to obtain a mean of 5 samples for each bacteria. Fourteen different bacteria were tested; thus, there were 70 growing devices utilized in the test for each of the 3 media. Each growing device was vortexed, i.e., mixed for 10 seconds and incubated at 35C. Viable bacteria counts were performed at 0, 4, 5, and 6 hours. The results are set forth in the table below:
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Table 2 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g Escherichia coli 0 hours 1.6 1.44 2.4 4 hours 0.4 5.2 11.6 5 hours 3.7 10.2 14.8 6 hours 1.3 7.6 15.4 Shigella sonnei 0 hours 4.2 3.62 1.2 4 hours 5.7 14.4 15.8 5 hours 8.1 17.0 19.8 6 hours 6.0 12.2 21.0 Klebsiella pneumoniae0 hours 2.6 2.86 2.6 4 hours 5.3 13.4 11.6 5 hours 4.9 13.6 13.6 6 hours 4.9 12.0 16.0 Enterobacter cloacae 0 hours 5 3 4.4 3.9 4 hours 9.4 17.0 17.4 5 hours 9.2 19.0 26.0 6 hours 9.4 19.6 38.6 20 Providencia species 0 hours 7.3 6.14 9.1 4 hours 11.6 22.2 30.2 5 hours 13.2 27.4 38.6 6 hours 13.2 22.8 39.8 Proteus mirabilis 0 hours 4.2 4.66 5.4 4 hours 8.9 21.4 21.4 5 hours 8.6 19.8 33.2 6 hours 9.7 24.0 37.2 9g654 Count (x 107 CFU/ml) Bacteria _ Time 0.2 g 0.8 g 1.6 g Salmonella typhimurium 0 hours 2.42.32 3.8 4 hours 6.616.4 27.8 5 hours 7.116.4 31.0 6 hours 6.819.0 33.2 Pseudomonas aeruginosa 0 hours 1.0 0.7 0.8 4 hours 5.614.6 11 4 5 hours 3.616.6 18.6 6 hours 5.326.4 29.2 Citrobacter species 0 hours 3.831.0 6.6 4 hours 9.220.8 21.0 5 hours 9.320.8 30.6 6 hours 12.631.4 32.6 15 AriZona 0 hours 1.11.46 2.0 4 hours 4.115.2 16.0 5 hours 4.916.0 21.8 6 hours 4.917.6 26.22 Edwardsiella 0 hours 2.22.98 1.9 4 hours 2.36.04 8.3 5 hours 2.56.02 8.8 6 hours 2.4 5.9 10.5 Yersinia 0 hours 3.33.02 3.7 4 hours 5.0 9.9 16.0 5 hours 5~411.5 19.2 6 hours 6.713.0 21.2 Serrati marcescens 0 hours 1.11.62 1.1 4 hours 5.210.0 14.0 5 hours 6.316.8 17.8 6 hours 6.826.3 23.4 - .
-22- ~ 09 96 54 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g Proteus vulgaris0 hours 1.71.36 0.5 4 hours 6.519.3619.2 5 hours 6.323.2 31.4 6 hours 7.322.0 34.5 Example 3 ~xample 2 was repeated except that polypeptone wag substituted for peptone. The results are set forth in the table below:
Table 3 Count (x 107 CFU/ml) BacteriaTime 0.2 g 0.8 g 1._ g 15 Escherichia coli0 hours 1.4 0.8 1.1 4 hours 7.8 9.8 6.8 5 hours 6.0 5.5 5.5 6 hours 6.7 7.9 7.9 Shigella sonnei0 hours 1.71.68 2.8 4 hours 7.19.98 10.6 5 hours 6.49.88 11.4 6 hours 8.011.4 11.0 Klebsiella pneumoniae 0 hours3.03.64 3.3 4 hours 6.94.78 7.1 5 hours 7.0 7.3 8.0 6 hours 7.812.6 9.5 Enterobacter cloacae 0 hours4.2 3.2 1.8 4 hours 16.219.0 8.1 5 hours 15.812.8 13.2 6 hours 11.613.6 16.8 1~99654 Count (x 107 CFU/ml) Bacteria Time0.2 g0.8 9 1.6 Providencia species 0 hours - - -(inoculum error) 4 hours - - -5 hours 6 hours - - -Proteus mirabilis 0 hours 0.46 0.46 0.34 4 hours 2.7 3.86 3.6 5 hours 7.6 14.6 13.0 6 hours 7.5 10.6 11.4 Salmonella typhimurium 0 hours 1.3 1.4 0.91 4 hours 6.8 6.9 7.4 5 hours 10.0 11.8 10.7 6 hours 9.0 14.1 11.0 Pseudomonas aeruginosa 0 hours 5.8 7.7 5.6 4 hours 8.0 8.3 11.0 5 hours - 22.2 24.8 6 hours 7.5 20.3 23.6 Citrobacter species 0 hours 15.4 21.6 34.2 4 hours 16.6 22.4 22.2 5 hours 14.8 31.4 25.6 6 hours 15.2 27.2 30.4 Arizona 0 hours 3.7 4.0 3.2 4 hours 5.3 6.6 5.6 ;~: 25 5 hours 6.6 13.2 12.5 6 hours 7.0 20.8 17.2 `~ Edwardsiella 0 hours No growth 4 hours No growth 5 hours No growth 6 hour~ No growth ~, . ~- -.
' -24- 1~99654 Count (x 107 CFU/ml) _ Bacteria Time 0.2_g 0.8 g 1.6 g Yersinia 0 hours 4.42 85 5.7 4 hours 4.45.0 10.7 5 hours 8.09.73 17.2 6 hours 8.611.0 18.6 Serratia marcescens 0 hours 4.44.6 4.1 4 hours 13.611.1 8.8 5 hours 15.214.4 10.1 6 hours 15.415.0 13.6 Proteus vulgaris 0 hours 4.52.64 1.4 4 hours 6.97.92 4.2 5 hours 15.515.4 11.3 6 hours 16.518.0 15.3 Example 4 Example 2 was repeated except that neopeptone was substituted for the peptone. The results are set forth in the table below:
Table 4 Count (x 107 CFU/ml) Bacteria Time0.2 g0.8 g1.6 9 Escherichia coli 0 hours 0.841.0 0.55 4 hours 0.540.6 0.38 5 hours 1.81.96 1.1 6 hours 1.34.0 3.0 Shigella sonnei 0 hours 1.80.8 0.79 4 hours 3 32.3 1.0 5 hours 4.66.2 4.2 6 hours 6.36.1 4.0 Count (x 107 CFU/ml) Bacteria Time 0~2 g 0.8 g 1.6 g Klebsiella pneumoniae 0 hours 0.2 0.13 0.1 4 hours3.7 2.5 4.0 5 hours7.2 2.5 8.0 6 hours5.3 5.8 8.2 Enterobacter cloacae 0 hours No growth 4 hours No growth 5 hours No growth 6 hours No growth Providencia species 0 hours0.4 - 0.2 4 hours0.7 - 0.6 5 hours1.4 - 0.7 6 hours1.9 - 1.8 Proteus mirabilis 0 hours2.0 1.0 4.2 4 hours6.3 8.5 6.8 5 hours10.017.6 16.0 6 hours10.521.8 22.2 Salmonella typhimurium0 hours 3.3 2.48 2.6 4 hours7.28.92 8.0 5 hours13.217.2 19.0 `~ 6 hours12.616.6 18.0 Pseudomonas aeruginosa0 hours 0.2 0.16 0.24 4 hours1.8 5.9 4.9 ~: 25 5 hours5.3 9.5 9.6 6 hours7.218.0 16.0 - Citrobacter species 0 hours5.47.62 13.0 4 hours7.47.44 5 hours12.224.6 27.2 6 hours15.630.2 31.4 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 ~ 1.6 g Arizona 0 hours 3.4 3.0 2.6 4 hours 6.3 6.6 7.6 5 hours 7.913.0 11.8 6 hours 9.816.4 17.4 Yersinia 0 hours 4.96.24 6.0 4 hours 6.010.7 10.8 5 hours 9.715.5 16.0 6 hours 10.420.6 21.0 Edwardsiella 0 hours No data 4 hours No data 5 hours No data 6 hours No data 15 Serratia marcescens 0 hours 3.53.16 5.1 4 hours 8.710.1 9.8 5 hours 11.311.6 12.3 6 hours 9.712.7 12.8 Proteus vulgaris 0 hours 2.43.56 2.0 4 hours 5.512.5 8.1 5 hours 8.623.8 16.4 6 hours 10.728.4 23.2 Example 5 Example 2 was repeated except that tryptone was substituted for peptone. The results are set forth in the table below:
$
Table 5 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g .. .. _ Escherichia coli 0 hours3.5 3.9 2.1 4 hours12.2 17.8 13.5 5 hours13.2 22.8 18.4 6 hours13.0 25.2 23.2 Shigella sonnei 0 hours1.0 0.4 0.33 4 hours6.3 11.0 10.0 5 hours7.1 16.0 16.0 6 hours8.2 21.3 22.3 Klebsiella pneumoniae 0 hours5.9 5.32 4.7 4 hours13.2 14.2 16.2 5 hours14.0 15.8 15.4 6 hours13.0 19.6 19.4 Enterobacter cloacae 0 hours8.7 7.08 10.6 4 hour.s 15.4 29.0 24.2 5 hours15.2 34.2 33.4 6 hours17.4 40.6 39.4 Providencia species 0 hours3.2 3.62 3.9 4 hours8.2 16.4 17.0 5 hours8.2 22.4 22.2 6 hours5.8 24.6 27.4 : Proteus mirabilis 0 hours3.3 2.08 3.3 4 hours11.8 16.2 19.8 5 hours11.1 21.6 19.0 6 hours13.0 26.2 24.0 -28- ~99~S4 Count (x 107 CFU/ml) Bacteria Time 0.2 ~ 0.8 g 1.6 g Salmonella typhimurium0 hours 1.1 1.56 1.0 4 hours 7.515.6 11.6 5 hours 8.021.4 13.8 6 hours 10.627.6 19.2 Pseudomonas aeruginosa0 hours 3.4 2.52 2.9 4 hours 10.011.0 11.6 5 hours 11.010.4 13.0 6 hours Citrobacter species0 hours 3.3 2.66 2.4 4 hours 16.615.8 17.2 5 hours 16.825.4 20.4 6 hours 17.034.4 27.7 AriZona 0 hours 1.4 1.0 0.66 4 hours 5.5 6.2 6.3 5 hours 5.712.0 10.2 6 hours 6.417.3 14.2 Edwardsiella 0 hours 0.78 0.85 1.6 4 hours 2.3 1.9 3.4 5 hours 3 1 2.3 3.9 6 hours 2.9 2.8 4.8 Yersinia 0 hours 2.0 1.58 2.7 4 hours 5.1 5.8 10.0 5 hours 6.1 8.42 13.6 ` 6 hours 7.112.8 18.3 Serratia marcescens0 hours 3.2 4.98 3.0 4 hours 16.620.8 15.6 5 hours 19.225.4 21.4 6 hours 23.229.8 25.8 - 1~9965~
Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g Proteus vulgaris 0 hcurs 1.46 1.1 1.0 4 hours 8.0 16.2 13.3 5 hours 10.0 16.5 18.0 6 hours 10.0 25.7 22.3 Example 6 Example 2 was repeated except that proteose peptone was substituted for the peptone. The results are set forth in the table below:
Table 6 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 ~ 1.6 g Escherichia coli 0 hours 2.2 1.78 1.9 4 hours 10.0 15.4 14.5 5 hours 7.4 22.0 22.0 6 hours 7.6 20.4 29.0 Shigella sonnei 0 hours 6.9 6.38 4.6 4 hours 14.0 23.2 20.6 5 hours 13.4 20.8 28.8 6 hours 13.8 25.4 33.4 Klebsiella pneumoniae0 hours 4.1 3.52 3.5 4 hours 12.6 15.2 15.4 5 hours 12.8 26.0 23.4 6 hours 13.4 21.2 17.8 Enterobacter cloacae 0 hours 4.2 0.8 4.3 4 hours 12.2 24.2 17.6 5 hours 13.0 28.3 27.0 6 hours - 27.0 35.2 1(~99654~
Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g . .
Providencia species 0 hours 6.4 6.8 7.7 4 hours 19.2 33.2 32.0 5 5 hours 22.8 44.8 41.8 6 hours 24.6 49.6 44.6 Proteus mirabilis 0 hours 5.6 5.98 5.3 4 hours 18.8 38.2 32.4 5 hours 18.8 38.2 38.0 10 6 hours 18.4 39.6 48.6 Salmonella typhimurium 0 hours 3.0 3.86 5.0 4 hours 15.0 28.0 25.2 5 hours 15.0 34.2 30.6 6 hours 17.8 35.2 38.4 15Pseudomonas aeruginosa 0 hours 1.8 1.78 2.2 4 hours 5.4 7.98 11.3 5 hours 9.3 14.2 21.6 6 hours 8.7 15.8 19.0 Citrobacter species 0 hours 4.4 3.82 5.2 20 4 hours 15.8 23.2 26.8 5 hours 16.6 27.6 28.2 6 hours 16.0 32.2 34.8 Arizona 0 hours 2.5 1.92 2.7 4 hours 10.8 14.2 15.0 25 5 hours 11.2 22.4 17.8 6 hours 11.6 25.6 24.0 Edwardsiella 0 hours 1.6 1.3 1.3 4 hours 3.6 5.9 10.3 5 hours 3.4 8.3 12.8 30 6 hours 3.9 10.0 15.8 ~(~99654 Count tx 107 CFU/ml) Bac~eria Time 0.2 g 0.8 g 1.6 g Yersinia 0 hours 6.4 3.0 5.9 4 hours 11.4 12.2 13.2 5 hours 13.4 18.0 19.6 6 hours 13.4 22.4 26.4 Serratia marcescens 0 hours 9.9 6.14 6.5 4 hours 28.0 26.2 25.8 5 hours 27.6 30.2 27.6 6 hours 33.8 37.8 35.8 Proteus vulgaris 0 hours 5.2 7.6 7.4 4 hours 12.6 19.6 18.4 5 hours 11.2 29.4 27.2 6 hours 13.6 33.2 33.6 Example 7 In order to compare the mediu~ of the present invention with the results obtained utilizing a standard broth growth technique, l.e., tryptic soy broth prior to placing the bacteria onto discs for use in the Kirby-Bauer procedure, 100 growing devices were prepared which contained the ~ame medium as set forth in Example 2. One hundred clinical isolates of bacteria that were received from patients were run using both the growing device and the standard growing technique of the standard set forth 5 for the Kirby-Bauer test. The bacteria tested included:
Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Acinetobacter calocoaceticus Proteus mirabilis Proteus morganii Enterobacter aerogenes Enterobacter cloacae Serratia marcescens - ' .
1(;199654 Pasteurella (species) CDC Group II F
Moraxella Citrobacter freundii Specifically, 4 to 5 isolated colonies were touched with the wand from the growing device and the wand was used to inoculate the growing medium in the growing device. The units were incubated at 35C in a 3M brand incubator Model 107 for 4 hours. The top tape seal was removed from the cap of the growing unit and 6 to 8 drops of bacterial suspension were dispensed onto a cotton swab. The swab was streaked in three directions over a Mueller-Hinton agar plate and the Kirby-Bauer test was completed according to the National Clinical Committee for Laboratory Standards (NCCLS) Antibiotics Susceptibility Standard set forth above. A comparison was made between the results obtained in respect to the susceptibility of the organism tested in using the growth media of the present invention versus the standard technique for growing bacteria. The results were comparable.
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Four other growth limiting media were used which include proteose peptone, tryptone, neopeptone and polypeptone. These were substituted for the peptone of the above formulation. The resulting initial concentra-tions were determined and are as follows in CFU/ml. Theresults given are the mean of 15 samples, i.e., 5 samples of each of the 3 formulations of each medium.
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Example 2 The following materials were dissolved in 1000 milliliters of deionized water and steam sterilized at 121C for 15 minutes:
0.8 gram Peptone 0.03 gram Dextrose 2.5 grams Dipotassium phosphate 1.25 grams Monopotassium phosphate 5.0 grams Sodium chloride Solutions of media containing 0.2 gram peptone and 1.6 grams peptone were also prepared. Growing devices were then prepared using each of the media. Utilizing the wand 4 of the growing device bacteria were picked from 4 to 5, 18 to 24 hour old bacterial colonies of the various bacteria set forth in the table below. Five growing devices were used for each bacteria to obtain a mean of 5 samples for each bacteria. Fourteen different bacteria were tested; thus, there were 70 growing devices utilized in the test for each of the 3 media. Each growing device was vortexed, i.e., mixed for 10 seconds and incubated at 35C. Viable bacteria counts were performed at 0, 4, 5, and 6 hours. The results are set forth in the table below:
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Table 2 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g Escherichia coli 0 hours 1.6 1.44 2.4 4 hours 0.4 5.2 11.6 5 hours 3.7 10.2 14.8 6 hours 1.3 7.6 15.4 Shigella sonnei 0 hours 4.2 3.62 1.2 4 hours 5.7 14.4 15.8 5 hours 8.1 17.0 19.8 6 hours 6.0 12.2 21.0 Klebsiella pneumoniae0 hours 2.6 2.86 2.6 4 hours 5.3 13.4 11.6 5 hours 4.9 13.6 13.6 6 hours 4.9 12.0 16.0 Enterobacter cloacae 0 hours 5 3 4.4 3.9 4 hours 9.4 17.0 17.4 5 hours 9.2 19.0 26.0 6 hours 9.4 19.6 38.6 20 Providencia species 0 hours 7.3 6.14 9.1 4 hours 11.6 22.2 30.2 5 hours 13.2 27.4 38.6 6 hours 13.2 22.8 39.8 Proteus mirabilis 0 hours 4.2 4.66 5.4 4 hours 8.9 21.4 21.4 5 hours 8.6 19.8 33.2 6 hours 9.7 24.0 37.2 9g654 Count (x 107 CFU/ml) Bacteria _ Time 0.2 g 0.8 g 1.6 g Salmonella typhimurium 0 hours 2.42.32 3.8 4 hours 6.616.4 27.8 5 hours 7.116.4 31.0 6 hours 6.819.0 33.2 Pseudomonas aeruginosa 0 hours 1.0 0.7 0.8 4 hours 5.614.6 11 4 5 hours 3.616.6 18.6 6 hours 5.326.4 29.2 Citrobacter species 0 hours 3.831.0 6.6 4 hours 9.220.8 21.0 5 hours 9.320.8 30.6 6 hours 12.631.4 32.6 15 AriZona 0 hours 1.11.46 2.0 4 hours 4.115.2 16.0 5 hours 4.916.0 21.8 6 hours 4.917.6 26.22 Edwardsiella 0 hours 2.22.98 1.9 4 hours 2.36.04 8.3 5 hours 2.56.02 8.8 6 hours 2.4 5.9 10.5 Yersinia 0 hours 3.33.02 3.7 4 hours 5.0 9.9 16.0 5 hours 5~411.5 19.2 6 hours 6.713.0 21.2 Serrati marcescens 0 hours 1.11.62 1.1 4 hours 5.210.0 14.0 5 hours 6.316.8 17.8 6 hours 6.826.3 23.4 - .
-22- ~ 09 96 54 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g Proteus vulgaris0 hours 1.71.36 0.5 4 hours 6.519.3619.2 5 hours 6.323.2 31.4 6 hours 7.322.0 34.5 Example 3 ~xample 2 was repeated except that polypeptone wag substituted for peptone. The results are set forth in the table below:
Table 3 Count (x 107 CFU/ml) BacteriaTime 0.2 g 0.8 g 1._ g 15 Escherichia coli0 hours 1.4 0.8 1.1 4 hours 7.8 9.8 6.8 5 hours 6.0 5.5 5.5 6 hours 6.7 7.9 7.9 Shigella sonnei0 hours 1.71.68 2.8 4 hours 7.19.98 10.6 5 hours 6.49.88 11.4 6 hours 8.011.4 11.0 Klebsiella pneumoniae 0 hours3.03.64 3.3 4 hours 6.94.78 7.1 5 hours 7.0 7.3 8.0 6 hours 7.812.6 9.5 Enterobacter cloacae 0 hours4.2 3.2 1.8 4 hours 16.219.0 8.1 5 hours 15.812.8 13.2 6 hours 11.613.6 16.8 1~99654 Count (x 107 CFU/ml) Bacteria Time0.2 g0.8 9 1.6 Providencia species 0 hours - - -(inoculum error) 4 hours - - -5 hours 6 hours - - -Proteus mirabilis 0 hours 0.46 0.46 0.34 4 hours 2.7 3.86 3.6 5 hours 7.6 14.6 13.0 6 hours 7.5 10.6 11.4 Salmonella typhimurium 0 hours 1.3 1.4 0.91 4 hours 6.8 6.9 7.4 5 hours 10.0 11.8 10.7 6 hours 9.0 14.1 11.0 Pseudomonas aeruginosa 0 hours 5.8 7.7 5.6 4 hours 8.0 8.3 11.0 5 hours - 22.2 24.8 6 hours 7.5 20.3 23.6 Citrobacter species 0 hours 15.4 21.6 34.2 4 hours 16.6 22.4 22.2 5 hours 14.8 31.4 25.6 6 hours 15.2 27.2 30.4 Arizona 0 hours 3.7 4.0 3.2 4 hours 5.3 6.6 5.6 ;~: 25 5 hours 6.6 13.2 12.5 6 hours 7.0 20.8 17.2 `~ Edwardsiella 0 hours No growth 4 hours No growth 5 hours No growth 6 hour~ No growth ~, . ~- -.
' -24- 1~99654 Count (x 107 CFU/ml) _ Bacteria Time 0.2_g 0.8 g 1.6 g Yersinia 0 hours 4.42 85 5.7 4 hours 4.45.0 10.7 5 hours 8.09.73 17.2 6 hours 8.611.0 18.6 Serratia marcescens 0 hours 4.44.6 4.1 4 hours 13.611.1 8.8 5 hours 15.214.4 10.1 6 hours 15.415.0 13.6 Proteus vulgaris 0 hours 4.52.64 1.4 4 hours 6.97.92 4.2 5 hours 15.515.4 11.3 6 hours 16.518.0 15.3 Example 4 Example 2 was repeated except that neopeptone was substituted for the peptone. The results are set forth in the table below:
Table 4 Count (x 107 CFU/ml) Bacteria Time0.2 g0.8 g1.6 9 Escherichia coli 0 hours 0.841.0 0.55 4 hours 0.540.6 0.38 5 hours 1.81.96 1.1 6 hours 1.34.0 3.0 Shigella sonnei 0 hours 1.80.8 0.79 4 hours 3 32.3 1.0 5 hours 4.66.2 4.2 6 hours 6.36.1 4.0 Count (x 107 CFU/ml) Bacteria Time 0~2 g 0.8 g 1.6 g Klebsiella pneumoniae 0 hours 0.2 0.13 0.1 4 hours3.7 2.5 4.0 5 hours7.2 2.5 8.0 6 hours5.3 5.8 8.2 Enterobacter cloacae 0 hours No growth 4 hours No growth 5 hours No growth 6 hours No growth Providencia species 0 hours0.4 - 0.2 4 hours0.7 - 0.6 5 hours1.4 - 0.7 6 hours1.9 - 1.8 Proteus mirabilis 0 hours2.0 1.0 4.2 4 hours6.3 8.5 6.8 5 hours10.017.6 16.0 6 hours10.521.8 22.2 Salmonella typhimurium0 hours 3.3 2.48 2.6 4 hours7.28.92 8.0 5 hours13.217.2 19.0 `~ 6 hours12.616.6 18.0 Pseudomonas aeruginosa0 hours 0.2 0.16 0.24 4 hours1.8 5.9 4.9 ~: 25 5 hours5.3 9.5 9.6 6 hours7.218.0 16.0 - Citrobacter species 0 hours5.47.62 13.0 4 hours7.47.44 5 hours12.224.6 27.2 6 hours15.630.2 31.4 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 ~ 1.6 g Arizona 0 hours 3.4 3.0 2.6 4 hours 6.3 6.6 7.6 5 hours 7.913.0 11.8 6 hours 9.816.4 17.4 Yersinia 0 hours 4.96.24 6.0 4 hours 6.010.7 10.8 5 hours 9.715.5 16.0 6 hours 10.420.6 21.0 Edwardsiella 0 hours No data 4 hours No data 5 hours No data 6 hours No data 15 Serratia marcescens 0 hours 3.53.16 5.1 4 hours 8.710.1 9.8 5 hours 11.311.6 12.3 6 hours 9.712.7 12.8 Proteus vulgaris 0 hours 2.43.56 2.0 4 hours 5.512.5 8.1 5 hours 8.623.8 16.4 6 hours 10.728.4 23.2 Example 5 Example 2 was repeated except that tryptone was substituted for peptone. The results are set forth in the table below:
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Table 5 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g .. .. _ Escherichia coli 0 hours3.5 3.9 2.1 4 hours12.2 17.8 13.5 5 hours13.2 22.8 18.4 6 hours13.0 25.2 23.2 Shigella sonnei 0 hours1.0 0.4 0.33 4 hours6.3 11.0 10.0 5 hours7.1 16.0 16.0 6 hours8.2 21.3 22.3 Klebsiella pneumoniae 0 hours5.9 5.32 4.7 4 hours13.2 14.2 16.2 5 hours14.0 15.8 15.4 6 hours13.0 19.6 19.4 Enterobacter cloacae 0 hours8.7 7.08 10.6 4 hour.s 15.4 29.0 24.2 5 hours15.2 34.2 33.4 6 hours17.4 40.6 39.4 Providencia species 0 hours3.2 3.62 3.9 4 hours8.2 16.4 17.0 5 hours8.2 22.4 22.2 6 hours5.8 24.6 27.4 : Proteus mirabilis 0 hours3.3 2.08 3.3 4 hours11.8 16.2 19.8 5 hours11.1 21.6 19.0 6 hours13.0 26.2 24.0 -28- ~99~S4 Count (x 107 CFU/ml) Bacteria Time 0.2 ~ 0.8 g 1.6 g Salmonella typhimurium0 hours 1.1 1.56 1.0 4 hours 7.515.6 11.6 5 hours 8.021.4 13.8 6 hours 10.627.6 19.2 Pseudomonas aeruginosa0 hours 3.4 2.52 2.9 4 hours 10.011.0 11.6 5 hours 11.010.4 13.0 6 hours Citrobacter species0 hours 3.3 2.66 2.4 4 hours 16.615.8 17.2 5 hours 16.825.4 20.4 6 hours 17.034.4 27.7 AriZona 0 hours 1.4 1.0 0.66 4 hours 5.5 6.2 6.3 5 hours 5.712.0 10.2 6 hours 6.417.3 14.2 Edwardsiella 0 hours 0.78 0.85 1.6 4 hours 2.3 1.9 3.4 5 hours 3 1 2.3 3.9 6 hours 2.9 2.8 4.8 Yersinia 0 hours 2.0 1.58 2.7 4 hours 5.1 5.8 10.0 5 hours 6.1 8.42 13.6 ` 6 hours 7.112.8 18.3 Serratia marcescens0 hours 3.2 4.98 3.0 4 hours 16.620.8 15.6 5 hours 19.225.4 21.4 6 hours 23.229.8 25.8 - 1~9965~
Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g Proteus vulgaris 0 hcurs 1.46 1.1 1.0 4 hours 8.0 16.2 13.3 5 hours 10.0 16.5 18.0 6 hours 10.0 25.7 22.3 Example 6 Example 2 was repeated except that proteose peptone was substituted for the peptone. The results are set forth in the table below:
Table 6 Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 ~ 1.6 g Escherichia coli 0 hours 2.2 1.78 1.9 4 hours 10.0 15.4 14.5 5 hours 7.4 22.0 22.0 6 hours 7.6 20.4 29.0 Shigella sonnei 0 hours 6.9 6.38 4.6 4 hours 14.0 23.2 20.6 5 hours 13.4 20.8 28.8 6 hours 13.8 25.4 33.4 Klebsiella pneumoniae0 hours 4.1 3.52 3.5 4 hours 12.6 15.2 15.4 5 hours 12.8 26.0 23.4 6 hours 13.4 21.2 17.8 Enterobacter cloacae 0 hours 4.2 0.8 4.3 4 hours 12.2 24.2 17.6 5 hours 13.0 28.3 27.0 6 hours - 27.0 35.2 1(~99654~
Count (x 107 CFU/ml) Bacteria Time 0.2 g 0.8 g 1.6 g . .
Providencia species 0 hours 6.4 6.8 7.7 4 hours 19.2 33.2 32.0 5 5 hours 22.8 44.8 41.8 6 hours 24.6 49.6 44.6 Proteus mirabilis 0 hours 5.6 5.98 5.3 4 hours 18.8 38.2 32.4 5 hours 18.8 38.2 38.0 10 6 hours 18.4 39.6 48.6 Salmonella typhimurium 0 hours 3.0 3.86 5.0 4 hours 15.0 28.0 25.2 5 hours 15.0 34.2 30.6 6 hours 17.8 35.2 38.4 15Pseudomonas aeruginosa 0 hours 1.8 1.78 2.2 4 hours 5.4 7.98 11.3 5 hours 9.3 14.2 21.6 6 hours 8.7 15.8 19.0 Citrobacter species 0 hours 4.4 3.82 5.2 20 4 hours 15.8 23.2 26.8 5 hours 16.6 27.6 28.2 6 hours 16.0 32.2 34.8 Arizona 0 hours 2.5 1.92 2.7 4 hours 10.8 14.2 15.0 25 5 hours 11.2 22.4 17.8 6 hours 11.6 25.6 24.0 Edwardsiella 0 hours 1.6 1.3 1.3 4 hours 3.6 5.9 10.3 5 hours 3.4 8.3 12.8 30 6 hours 3.9 10.0 15.8 ~(~99654 Count tx 107 CFU/ml) Bac~eria Time 0.2 g 0.8 g 1.6 g Yersinia 0 hours 6.4 3.0 5.9 4 hours 11.4 12.2 13.2 5 hours 13.4 18.0 19.6 6 hours 13.4 22.4 26.4 Serratia marcescens 0 hours 9.9 6.14 6.5 4 hours 28.0 26.2 25.8 5 hours 27.6 30.2 27.6 6 hours 33.8 37.8 35.8 Proteus vulgaris 0 hours 5.2 7.6 7.4 4 hours 12.6 19.6 18.4 5 hours 11.2 29.4 27.2 6 hours 13.6 33.2 33.6 Example 7 In order to compare the mediu~ of the present invention with the results obtained utilizing a standard broth growth technique, l.e., tryptic soy broth prior to placing the bacteria onto discs for use in the Kirby-Bauer procedure, 100 growing devices were prepared which contained the ~ame medium as set forth in Example 2. One hundred clinical isolates of bacteria that were received from patients were run using both the growing device and the standard growing technique of the standard set forth 5 for the Kirby-Bauer test. The bacteria tested included:
Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Acinetobacter calocoaceticus Proteus mirabilis Proteus morganii Enterobacter aerogenes Enterobacter cloacae Serratia marcescens - ' .
1(;199654 Pasteurella (species) CDC Group II F
Moraxella Citrobacter freundii Specifically, 4 to 5 isolated colonies were touched with the wand from the growing device and the wand was used to inoculate the growing medium in the growing device. The units were incubated at 35C in a 3M brand incubator Model 107 for 4 hours. The top tape seal was removed from the cap of the growing unit and 6 to 8 drops of bacterial suspension were dispensed onto a cotton swab. The swab was streaked in three directions over a Mueller-Hinton agar plate and the Kirby-Bauer test was completed according to the National Clinical Committee for Laboratory Standards (NCCLS) Antibiotics Susceptibility Standard set forth above. A comparison was made between the results obtained in respect to the susceptibility of the organism tested in using the growth media of the present invention versus the standard technique for growing bacteria. The results were comparable.
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Claims (10)
1. A device for growing bacteria from an initial population to a determined ending population at which the growth of said bacteria substantially subsides due to the lack of nutrient comprising: (a) a vessel containing a supply of growth medium capable of growing said bacteria from said beginning population to said determined ending population, said vessel containing an opening; (b) means within said vessel for obtaining a known quantity of bacteria from at least one growth colony of said bacteria external of said vessel and for inoculating said growth medium; and (c) removable means for covering said opening in said vessel, for permitting said means for obtaining said bacteria to be removed to obtain said known quantity of bacteria from said colony of said bacteria external of said vessel, which bacteria are used to inoculate said medium, and for closing said vessel during the incubation of said inoculated medium.
2. The device of claim 1 wherein said means for obtaining a known quantity of bacteria comprises a rod attached to said removable means with a capillary means on the end of said rod opposite to that attached to said removable means for picking up said bacteria by means of capillary means.
3. The device of claim 2 wherein said capillary means comprises a tapered groove.
4. The device of claim 2 wherein said removable means comprises a cap for said vessel containing a covered hole therein.
5. The device of claim 4 wherein said vessel is deformable and wherein said medium is contained within a frangible container within said vessel.
6. The device of claim 1 wherein said medium comprises an aqueous medium capable of growing at least one species from two different genera of aerobic, pathogenic, rapidly growing bacteria from a beginning population to a determined ending population at which said growth of said bacteria substantially subsides due to the lack of nutrient in said medium and wherein said bacteria remain viable, said medium comprising an aqueous solution comprising a carbon source, a nitrogen source, vitamins and minerals of sufficient quantity to provide said growth and in a form usable by said bacteria for said growth.
7. The device of claim 2 wherein said medium comprises an aqueous medium capable of growing at least one species from two different genera of aerobic, pathogenic, rapidly growing bacteria from a beginning population to a determined ending population at which said growth of said bacteria substantially subsides due to the lack of nutrient in said medium and wherein said bacteria remain viable, said medium comprising an aqueous solution comprising a carbon source, a nitrogen source, vitamins and minerals of sufficient quantity to provide said growth and in a form usable by said bacteria for said growth.
8. The device of claim 7 wherein said bacteria are gram-negative and wherein said medium comprises an aqueous solution of peptone which is buffered to maintain a pH of from about 7 to about 8.
9. The device of claim 7 wherein said bacteria are gram-negative and wherein said medium comprises an aqueous solution of proteose peptone which is buffered to maintain a pH of from about 7 to about 8.
10. A device for growing bacteria from an initial population to an ending population comprising:
(a) a vessel containing a supply of growth medium capable of growing said bacteria from said beginning population to said ending population, said vessel containing an opening;
(b) means within said vessel for obtaining a known quantity of bacteria from at least one growth colony of said bacteria external of said vessel and for inoculating said growth medium; and (c) removable means for covering said opening in said vessel, for permitting said means for obtaining said bacteria to be removed to obtain said known quantity of bacteria from said colony of said bacteria external of said vessel, which bacteria are used to inoculate said medium and for closing said vessel during the incubation of said inoculated medium.
(a) a vessel containing a supply of growth medium capable of growing said bacteria from said beginning population to said ending population, said vessel containing an opening;
(b) means within said vessel for obtaining a known quantity of bacteria from at least one growth colony of said bacteria external of said vessel and for inoculating said growth medium; and (c) removable means for covering said opening in said vessel, for permitting said means for obtaining said bacteria to be removed to obtain said known quantity of bacteria from said colony of said bacteria external of said vessel, which bacteria are used to inoculate said medium and for closing said vessel during the incubation of said inoculated medium.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US80845977A | 1977-06-21 | 1977-06-21 | |
| US808,459 | 1977-06-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1099654A true CA1099654A (en) | 1981-04-21 |
Family
ID=25198814
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA303,712A Expired CA1099654A (en) | 1977-06-21 | 1978-05-19 | Bacteria growing device |
| CA000479657A Expired CA1197802B (en) | 1977-06-21 | 1985-04-19 | Bacteria growing device |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000479657A Expired CA1197802B (en) | 1977-06-21 | 1985-04-19 | Bacteria growing device |
Country Status (1)
| Country | Link |
|---|---|
| CA (2) | CA1099654A (en) |
-
1978
- 1978-05-19 CA CA303,712A patent/CA1099654A/en not_active Expired
-
1985
- 1985-04-19 CA CA000479657A patent/CA1197802B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| CA1197802B (en) | 1985-12-10 |
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