GB2077760A

GB2077760A - Medium for Growing Bacteria to a Predetermined Concentration

Info

Publication number: GB2077760A
Application number: GB8019237A
Authority: GB
Original assignee: Minnesota Mining and Manufacturing Co
Current assignee: 3M Co
Priority date: 1977-06-21
Filing date: 1978-06-20
Publication date: 1981-12-23
Also published as: SE443799B; SE7806946L; SE8305617L; DE2858341C2; JPS548787A; SE8305616L; SE8305617D0; DE2827484C2; DE2827484A1; DE2858340C2; GB2000187A; SE452020B; JPS632591B2; GB2077760B; SE8305616D0; GB2000187B; FR2395312B1; FR2395312A1; SE458367B

Abstract

The present invention provides an aqueous medium capable of growing a species of aerobic, pathogenic, rapidly growing bacteria from a beginning population to a determined ending population in the range 6 x 10<7> to 3 x 10<8> CFU/ml, at which said growth of said bacteria substantially subsides due to the lack of nutrient in the medium and wherein said bacteria remain viable. The medium comprises an aqueous solution which has either 0.42 to 0.70 milligrams of carbon per millilitre of medium and 0.09 to 0.15 milligrams of nitrogen per millilitre of medium in the form of peptone, or 0.16 to 0.27 milligrams of carbon per millilitre of medium and 0.035 to 0.056 milligrams of nitrogen per millilitre of medium in the form of proteose peptone together with vitamins and minerals of sufficient quantity to provide said growth and in a form usable by said bacteria for said growth. The beginning concentration can range as low as 1 CFU but will normally be, and is preferably, at least 5 x 10<8> CFU/ml.

Description

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1 GB 2 077 760 A 1 .DTD:

SPECIFICATION .DTD:

Bacteria Growing Device This invention relates to a method for growing bacteria. Specifically, this invention relates to a method for growing bacteria from an initial population to a final predetermined population.

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Growth limiting media according to the invention are useful in a number of procedures utilised to 5 identify bacteria or to determine the susceptibility of bacteria to certain antibiotics. In such procedures it is necessary to have bacteria at the beginning of the test procedure in a certain concentration range (colony forming units per millilitre (CFU/ml)), or the final result will not be accurate. For example, in an article entitled "Antibiotics Susceptibility Testing by Standardised Single-Disc Method", The American Journal of Clinical Pathology, Vol. 45, No: 4, April, 1966, Pages293to296andinthe"Performance 10 Standards for antimicrobial Disc Susceptibility Test", ASM-2, promulgated by the National Committee for Clinical Laboratory Standards of the United States of America, the Kirby- Bauer procedure for determining the susceptibility of rapidly growing bacteria to antibiotics and chemotherapy agents is described.

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The Kirby-Bauer procedure normally involves growing colonies of bacteria obtained from a 15 patient on an agar plate. 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 millilitres,of soybean casein digest broth. The tubes are the incubated for 2 to 8 hours to produce a bacterial suspension of moderate cloudiness. The suspension is then diluted, if necessary, with saline solution or like broth to a density visually equivalent to that of a standard prepared by adding 0.5 milliliters of 1% BaCI2 to 99.5 milliliters of 1% H2SO4 (0.36 N) (0.5 20 McFarland standard hereinafter referred to as the McFarland standard). A plate containing Mueller- Hinton agar is then streaked with the bacterial broth suspension using a cotton swab. After the inoculum has dried, a paper disc containing an antibiotic or chemotherapeutic agent is applied to the agar. The plates are incubated overnight, and the area ground each disc wherein there is an absence of bacterial growth is measured. This is known as the zone of inhibition and is used to determine which 25 antibiotic will be useful in combating the particular bacteria.

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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 visual comparison with the McFarland standard described above. The time period to reach this concentration of bacteria may vary from 2 to 8 hours depending on the bacteria. If the 30 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.

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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 the 35 preparation of a series of concentrations of an antibiotic, either in a liquid or solid medium, which will support the growth of the 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 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 40 or absence of growth of each antibiotic concentration is observed. In this way the minimum inhibitory concentration 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. However, this mehod 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 in a certain concentration range, i.e., normally 105 45 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.

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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 Kirby-Bauer test 50 or to inoculate the diluted antibiotics in the case of MIC test. This is required in order for the test to be accurate. If a lesser concentration of bacteria is utilised in the test, the result would indicate that the bacteria are more susceptible to the antibiotic than it would be as an actual fact. On the other hand, if 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 55 indications would be erroneous.

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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 to 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 the " concentration of bacteria is equivalent to the McFarland standard. From a visual examination of the medium, one will find that some cultures of bacteria have not grown to the proper concentration while others have grown beyond the appropriate concentration. The former requires that the technician allow 2 GB 2 077 760 A 2 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.

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We have developed a method by which bacteria can grow but which limits the concentration level to which the bacteria will indeed grow. According to the present invention there is provided a method of growing at least one species from two different genera of aerobic, pathogenic, rapidly 5 I_ growing bacteria from a beginning population to a determined ending population in the range 6x 107 to 3 x 108 CFU/ml, at which said growth of said bacteria substantially subsides due to the lack of nutrient in the medium and wherein said bacteria remain viable, the method comprising innoculating a growth- limiting medium with at least an amount of said bacteria equivalent to 1 CFU/ml of the growth-limiting medium, the medium comprising an aqueous solution which has either 0.42 to 0.70 milligrams of 10 carbon per millilitre of medium and 0.09 to 0.15 milligrams of nitrogen per millilitre of medium ir the form of peptone, or 0.16 to 0.27 milligrams of carbon per millilitre of medium and 0.035 to 0.056 milligrams of nitrogen per millilitre of medium in the form of proteose peptone together with vitamins and minerals of sufficient quantity to provide said growth and in a form usable by said bacteria for said growth. The beginning concentration can range as low as 1 CFU/ml but will normally be, and is 15 preferably, at least 5x 106 CFU/mI.

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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.

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The medium is useful with both gram-negative as well as gram-positive aerobic, pathogenic, 20 rapidly growing bacteria. However, the type add amount of the various ingredients in the medium are normally different for the gram-positive than for the gram-negative bacteria, and the time period required to obtain the requisite concentration from the gram-positive tends to be longer than that for the gram-negative bacteria.

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The growth medium will grow at least one species from two different genera of aerobic, 25 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 is performed. These are Staphylococcus and Streptococcus. If the medium will grow species from each of these two genera then it allows one to 30 merely test the bacteria to determine whether it is gram-positive or negative using a gram stain test. If the bacteria is gram-positive the medium which grow the gram-positive bacteria in use and the medium will grow the bacteria to the level desired such as to the equivalent of the McFarland standard.

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If the bacteria is determined to be gram-negative using the gram stain test, the bacteria could be from a much larger number of genera. Sixteen genera represent the bacteria found to be the cause of 35 99% of illnesses caused by gram-negative aerobic bacteria. Those gram- negative genera include:

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Escherichia, Shigella, Edwardsiella, Salmonella, Arizona, Citrobacter, Klebsiella, Enterobacter, Serratia, Proteus, Providencia, Yersinia, Pseudomonas, Acinetobacter, Moraxella and Pasteurella.

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Starting with a low initial population of concentration will now cause the medium to grow the bacteria to a significantly different final population or concentration than with a higher starting 40 concentration but will affect the time it takes for the final concentration 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.

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As above discussed the time to reach a concentration 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 45 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 reaches the stationary phase in 2 hours, it 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. 50 When the stationary phase or final concentration is reached the growth of the bacteria substantially subsides. This is due to exhaustion of at least one nutrient critical to the continuous growth of the bacteria and not to the formation of toxic byproducts by the bacteria which stop growth and can cause the population to decrease substantially. With the standard media used prior to the present invention and described in the Kirby-Bauer procedure, the population of bacteria which will be 55 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.

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The final concentration or maximum stationary phase is obtained because of exhaustion of one or more 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 60 various tests to be performed thereon such as those described above. Normally the time period of such viability is at least 18 hours.

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The final desired concentration for most bacteria will be between 6 x 107 to 3.0 x 108 CFU/ml.

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Thus is equivalent to the 0.5 McFarland standard. The medium can be modified to reach different desired concentration levels. 65 3 GB 2 077 760 A 3 The medium will contain different types and amounts of ingredients depending upon the final concentration of bacteria desired and the type of 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 growth.

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Normally, vitamins and minerals are also present. However, as noted, the amount of one or more of these ingredients is limited to cause the bacteria to reach a final predetermined concentration and substantially cease growing.

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For the gram-negative bacteria the medium may comprise 0.42 to 0.70 milligram of carbon per millilitre of medium, the carbon is in a form useful by the bacteria for growth which form has been found to be peptone. The medium also comprises 0.09 to O. 15 milligram of nitrogen per millilitre of medium in the form of peptone. Preferably, the medium has a pH from about 7 to 8. Another suitable 10 medium comprises proteose peptone such that the range of carbon is from 0. 16 to 0.27 milligram of carbon per millilitre of medium and the nitrogen is 0.035 to 0.056 milligram of nitrogen per millilitre of medium. A typical analysis of the peptone is set forth below in which the figures are expressed as percent by weight.

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Proteose 15 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 20 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 25 Tryptophane 0.29 0.51 Tryosine 0.98 2.51 Crystine 0.22 0.56 Organic Sulfur 0.33 0.60 Inorganic Sulfur 0.29 0.04 30 Phosphorus 0.22 0.47 Chlorine 0.27 3.95 Sodium 1.08 2.84 Potassium 0.22 0.70 Calcium 0.058 O. 137 35 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 40 Reaction, pH 7.0 6.8 pH 1% solution is distilled water after autoclaving 15 minutes at 121 C.

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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 the gram-negative bacteria to a final concentration of from 6x 107 to 3 x 108 CFU/ml in less than 5 hours 45 comprise a mixture of 1000 ml of water containing 0.8 gram peptone or 0.3 gram proteose peptone, 0.03 gram dextrose, 2.5 gram dipotassium phosphate, 1.25 gram monopotassium phosphate and 5.0 grams sodium chloride. The phosphates are added as a buffer material to maintain the composition at a pH of approximately 7.0. 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, 50 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 5x 106 CFU/ml.

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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 McFarland standard wfthin the same time frame 55 and, do not provide the appropriate nutrients to grow some gram-negative bacteria to any significant degree. Therefore, these materials are useful for more limited numbers of bacteria. However, combinations of such materials with peptone or proteose peptone can be used to provide a medium useful with a larger number of bacteria.

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A preferred medium for use with the gram-positive bacteria comprises a solution of 1000 ml of 60 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.

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- All of the various media are used by innoculating the medium with the bacteria and incubating the medium for 2 to 8 hours.

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A device has been designed which utilizes the above-described growth limiting medium and 65 4 GB 2 077 760 A 4 allows one to obtain a certain predetermined amount of bacteria from colonies of bacteria with which to inoculate the growth limiting media. 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 the gram-negative bacteria, the beginning population must be at least about 5 x 10e CFU/ml.

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Reference is directed to our copending Application No. 27368/78 allotted serial No. 2,000,187 which 5 described and claims a device suitable for growing bacteria using a medium according to the present invention.

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The invention will now be described by way of examples in which following materials and bacteria are referenced. Their source is also set forth below. In the examples, "growing device" means a device having dimensions as described in Application No. 27368/78, alloted serial No. 2,000,187. 10 Tryptone, an enzymatic hydrolysate of casein, Difco, Inc., Detroit, Michigan, U.S.A.

Peptone, an enzymatic hydrolysate of casin, 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. 15 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 Collection, (ATCC) No. 19028.

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Shigella Sonnei, (ATCC 25331).

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Enterobacter cloacae (ATCC 23355) and St. Paul Ramsey Hospital, St. Paul, Minnesota, U.S.A. 20 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. 25 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.

Arizona, University of Minnesota, Minneapolis, Minnesota, U.S.A.

Yersinia, University of Minnesota, Minneapolis, Minnesota, U.S.A. 30 Pseudomonas aeuruginosa (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. 35 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.

0.8 gram Peptone 45 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. 50 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 concentrations were determined and are as follows in CFU/mI. The results given are the mean of 15 samples, i.e., 5 samples of each of the 3 formulations of each medium.

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Proteose 55 Peptone Peptone Tryptone Neopeptone Polypeptone i Escherichia coil 1.8x 107 2.1 xl07 3.6x 107 -- 1.4x 107 Shigella 3.0x107 6.0x107 __ __ 2.3x107 Klebsiella 2.7x107 3.7x107 5.3x107 __ 3.3x107 Enterobacter 4.5x 107 4.0x 107 8.8 x 107 -- -- 60 Providencia 7.5x 107 7.0x 107 3.6x 107 --- Example 1 40 .DTD:

Devices having the specification described in Application No. 27368/78 (allotted Serial No.

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2,000,187) were inoculated with various bacteria using the tapered groove of the wand of the device.

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An initial concentration of bacteria was noted. The growth limiting media included within the ampoule of each device contains the following:

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GB 2 077 760 A 5 Proteose Peptone Peptone Tryptone Neopeptone Polypeptone Proteus Mirabilis 4.8x 107 5.6x 107 2.9x 107 -- __ Salmonella 2.8x107 4.0x107 1.5x107 2.9x107 1.8x107 Pseudomonas 0.8x 107 1.9x107 3.1 xlO7 -6.4x107 Citrobacter 4.5x107 4.5x107 2.9x107 8.0x107 23.9x107 Arizona 1.5x107 2.4x107 -- 3.0x107 __ Edwardsiella 2.4x 107....

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Yersinia 3.3x107 5.1x107 2.8x107 5.7x107 4.4x107 Serratia 1.3x107 7.5x107 3.9x107 3.9x107 4.4x107 Proteus vulgaris 1.2x 107 -- -- 2.7x 107 2.6x 107 Species of bacteria are as above listed.

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Where there are blanks the inoculum was in error.

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Example 2 .DTD:

The following materials were dissolved in 1000 milliliters of deionized water and steam sterilized at 121 C. for 15 minutes:

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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 preparecJ. 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 35 C. Viable bacteria counts were performed at O, 4, 5 and 6 hours. The results are set forth in the table below:

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Bacteria Escherichia coil Shigella sonnei Klebsiella pneumoniae Table 2 .DTD:

Time 0 hours 4 hours 5 hours 6 hours Count (x 107 CFU/ml) 0.2 0.8 g 1.6 g 1.6 1.44 2.4 0.4 5.2 11.6 3.7 10.2 14.8 1.3 7.6 15.4 Enterobacter cloacae 0 hours 4 hours 5 hours 6 hours 4.2 3.62 1.2 5.7 14.4 15.8 8.1 17.0 19.8, 6.0 12.2 21.0 Providencia species 0 hours 2.6 2.86 2.6 4 hours 5.3 13.4 11.6 hours 4.9 13.6 13.6 6 hours 4.9 12.0 16.0 0 hours 5.3 4.4 3.9 4 hours 9.4 17.0 17.4 hours 9.2 19.0 26.0 6 hours 9.4 19.6 38.6 0 hours 7.3 6.14 9.1 4 hours 11.6 22.2 30.2 hours 13.2 27.4 38.6 6 hours 13.2 22.8 39.8 Proteus mirabilis 0 4 hours hours hours hours 4.2 4.66 5.4 8.9 21.4 21.4 8.6 19.8 33.2 9.7 24.0 37.2 6 GB 2 077 760 A 6 Bacteria Salmonella typhimurium Table 2 (cont.) Count (x 107 CFU/ml) Time 0.2 g 0.8 g 1.6 g 0 hours 2.4 2.32 3.8 4 hours 6.6 16.4 27.8 hours 7.1 16.4 31.0 6 hours 6.8 19.0 33.2 Pseudomonas aeruginosa 0 hours 1.0 0.7 0.8 4 hours 5.6 14.6 11.4 hours 3.6 16.6 18.6 6 hours 5.3 26.4 29.2 Citrobacter species 0 hours 3.8 31.0 6.6 4 hours 9.2 20.8 21.0 hours 9.3 20.8 30.6 6 hours 12.6 31.4 32.6 Arizona 0 hours 1.1 1.46 2.0 4 hours 4.1 15.2 16.0 hours 4.9 16.0 21.8 6 hours 4.9 17.6 26.2 Edwardsiella 0 hours 2.2 2.98 1.9 4 hours 2.3 6.04 8.3 hours 2.5 6.02 8.8 6 hours 2.4 5.9 10.5 Yersinia 0 hours 3.3 3.02 3.7 4 hours 5.0 9.9 16.0 hours 5.4 11.5 19.2 6 hours 6.7 13.0 21.2 Serrati marcescens 0 hours 1.1 1.62 1.1 4 hours 5.2 10.0 14.0 hours 6.3 16.8 17.8 6 hours 6.8 26.3 23.4 Proteus vulgaris 0 hours 1.7 1.36 0.5 4 hours 6.5 19.36 19.2 hours 6.3 23.2 31.4 6 hours 7.3 22.0 34.5 Example 3 .DTD:

Example 2 was repeated except that polypeptone was substi=ted for peptone. The results are set forth in the table below:

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Bacteria Escherichia coil Table 3 .DTD:

Shigella Sonnei Klebsiella pneumoniae Count (x 107 CFU/ml) 40 Time 0.2 g 0.8 g 1.6 g 0 hours 1.4 0.8 1.1 4 hours 7.8 9.8 6.8 hours 6.0 5.5 5.5 6 hours 6.7 7.9 7.9 45 1.7 1.68 2.8 7.1 9.98 10.6 6.4 9.88 11.4 8.0 11.4 11.0 0 hours 4 hours 5 hours 6 hours 0 hours 3.0 3.64 3.3 4 hours 6.9 4.78 7.1 hours 7.0 7.3 8.0 6 hours 7.8 12.6 9.5 7 GB 2 077 760 A 7 Bacteria Enterobacter cloacae Providencia species (inoculum error) Proteus mirabilis Salmonella typhimurium Pseudomonas aeruginosa Citrobacter species Arizona Edwardsiella Yersinia Serratia marcescens Proteus vulgaris Table 3 (cont.) Time 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours 0 hours 4 hours 5 hours 6 hours / Count (x 107 CFU/ml) 0.2 g 0.8 g 1.6 g 4.2 3.2 1.8 16.2 19.0 8.1 15.8 12.8 13.2 11.6 13.6 16.8 0.46 2.7 7.6 7.5 0.46 3.86 14.6 10.6 0.34 3.6 13.0 11.4 1.3 6.8 10.0 9.0 1.4 6.9 11.8 14.1 0.91 7.4 10.7 11.0 5.8 8.0 7.5 7.7 8.3 22.2 20.3 5.6 11.0 24.8 23.6 15.4 16.6 14.8 15.2 21.6 22.4 3114 27.2 34.2 22.2 25.6 30.4 3.7 5.3 6.6 7.0 4.0 6.6 13.2 20.8 3.2 5.6 12.5 17.2 4.4 4.4 8.0 8.6 No growth No growth No growth No growth 2.85 5.0 9.73 11.0 5.7 10.7 17.2 18.6 4.4 13.6 15.2 15.4 4.5 6.9 15.5 16.5 4.6 11.1 14.4 15.0 2.64 7.92 15.4 18.0 4.1 8.8 10.1 13.6 1.4 4.2 11.3 15.3 8 GB 2 077 760 A 8 Example 4 .DTD:

Example 2 was repeated except that neopeptone was substituted for the peptone. The results are set forth in the table below:

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Bacteria Escherichia coli Table 4 .DTD:

Count (x 107 CFU/ml) 5 Time 0.2 g 0.8 g 1.6g 0 hours 0.84 1.0 0.55 4 hours 0.54 0.6 0.38 hours 1.8 1.96 1.1 6 hours 1.3 4.0 3.0 10 Shigella sonnei 0 hours 1.8 0.8 0.79 4 hours 3.3 2.3 1.0 hours 4.6 6.2 4.2 6 hours 6.3 6.1 4.0 Klebsiella pneumoniae 0 hours 0.2 0.13 0.1 4 hours 3.7 2.5 4.0 hours 7.2 2.5 8.0 6 hours 5.3 5.8 8.2 Enterobacter cloacae hours hours hours hours No growth No growth No growth No growth Providencia species hours hours hours hours 0.4 0.7 1.4 1.9 0.2 0.6 0.7 1.8 Proteus mirabilis 0 hours 2.0 1.0 4.2 4 hours 6.3 8.5 6.8 hours 10.0 17.6 16.0 6 hours 10.5 21.8 22.2 Salmonella typhimurium 0 hours 3.3 2.48 2.6 4 hours 7.2 8.92 8.0 hours 13.2 17.2 19.0 6 hours 12.6 16.6 18.0 Pseudomonas aeruginosa Citrobacter species 0 hours 0.2 O. 16 4 hours 1.8 5.9 hours 5.3 9.5 6 hours 7.2 18.0 0 hours 5.4 7.62 4 hours 7.4 7.44 hours 12.2 24.6 6 hours 15.6 30.2 0.24 4.9 9.6 16.0 13.0 27.2 31.4 Arizona 0 hours 3.4 3.0 2.6 4 hours 6.3 6.6 7.6 hours 7.9 13.0 11.8 6 hours 9.8 16.4 17.4 Yersinia 0 4 hours hours hours hours 4.9 6.24 6.0 6.0 10.7 10.8 9.7 15.5 16.0 0.4 20.6 21.0 Edwardsiella 0 hours 4 hours 5 hours 6 hours No data No data No data No data 9 GB 2 077 760 A 9 Table 4 (cont.) Count (x 107 CFU/ml) Bacteria Time 0.2 g 0,8 g 1.6 g Serratia Marcescens 0 hours 3,5 3.16 5.1 4 hours 8.7 10.1 9.8 hours 11,3 11.6 12.3 6 hours 9,7 12.7 12.8 Proteus vulgaris 0 hours 2,4 3.56 2.0 4 hours 5.5 12.5 8.1 hours 8.6 23.8 16.4 6 hours 10,7 28,4 23.2 Example 5 .DTD:

Example 2 was repeated except that tryptone was substituted for peptone. The results are set forth in the table below:

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Bacteria Escherichia coil Shigella sonnef Klebsiella pneumoniae Enterobacter cloacae Povidencia species Proteus mirabilis Salmonella typhimurium Table 5 15 .DTD:

Count (x 10 CFU/ml) Time 0.2 g 0.B g 1.6 E 0 hours 3.5 3.9 2.1 4 hours 12.2 17.8 13.5 hours 13.2 22.8 18.4 20 6 hours 13.0 25.2 23.2 0 hours 1.O 0.4 0.33 4 hours 6.3 11.0 10.0 hours 7.1 16.0 16.0 6 hours 8.2 21.3 22.3 25 0 hours 5.9 5.32 4.7 4 hours 13.2 14.2 16.2 hours 14.0 15.8 15.4 6 hours 13.0 19.6 19.4 Pseudomonas aeruginosa 0 hours 8.7 7.08 10.6 4 hours 15.4 29.0 24.2 hours 15.2 34.2 33.4 6 hours 17.4 40.6 39.4 Citrobacter species 0 hours 3.2 3.62 3.9 4 hours 8,2 16.4 17.0 hours 8,2 22,4 22.2 6 hours 5.8 24.6 27.4 0 hours 3.3 2.08 3.3 4 hours 11.8 16.2 19.8 hours 11.121.6 19.0 6 hours 13.0 26,2 24.0 0 hours 1.1 1.56 1.0 4 hours 7.5 15.6 11.6 hours 8.0 21.4 13,8 6 hours 10.6 27.6 19.2 Arizona 0 hours 3.4 2.52 2.9 4 hours 10.0 11.0 11.6 hours 11.0 10.4 13.0 6 hours O hours 3.3 2.66 2.4 4 hours 16.6 15.8 17.2 hours 16.8 25.4 20.4 6 hours 17.0 34.4 27.7 0 hours 1.4 1.0 0.66 4 hours 5.5 6.2 6.3 hours 5.7 12.0 10.2 6 hours 6.4 17.3 14.2 GB 2 077 760 A 10 Bacteria Edwardsiella Table 5 (cont.) Count (x 107 CFU/ml) Time 0.2 g 0.8 g 1.6 g 0 hours 0.78 0.85 1.6 4 hours 2.3 1.9 3.4 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 hours 6.1 8.42 13.6 6 hours 7.1 12.8 18.3 Serratia marcescens 0 hours 3.2 4.98 3.0 4 hours 16.6 20.8 15.6 hours 19.2 25.4 21.4 6 hours 23.2 29.8 25.8 Proteus vulgaris 0 hours 1.46 1.1 1.0 4 hours 8.0 16.2 13.3 hours 10.0 16.5 18.0 6 hours 10.0 25.7 22.3 Example 6 .DTD:

Example 2 was repeated except that proteose peptone was substituted for the peptone. The results are set forth in the table below:

.DTD:

Bacteria Escherichia coli Table 6 .DTD:

Time 0 hours 4 hours 5 hours 6 hours Count (x 107 CFU/ml) 0.2 g 0.8 g 1.6 g 2.2 1.78 1.9 10.0 15.4 14.5 7.4 22.0 22.0 7.6 20.4 29.0 Shigella sonnei 0 hours 6.9 6.38 4.6 4 hours 14.0 23.2 20.6 hours 13.4 20.8 28.8 6 hours 13.8 25.4 33.4 Klebsiella pneumoniae 0 hours 4.1 3.52 3.5 4 hours 12.6 15.2 15.4 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 hours 13.0 28.3 27.0 6 hours -27.0 35.2 Providencia species 0 hours 6.4 6.8 7.7 4 hours 19.2 33.2 32.0 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 hours 18.8 38.2 38.0 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 hours 15.0 34.2 30.6 6 hours 17.8 35.2 38.4 11 G8 2 077 760 A 11 Bacteria Pseudomonas aeruginosa Table 6 (cont.) Time 0 hours 4 hours 5 hours 6 hours Count (x 107 CFU/m/) 0,2 g 0.8 g 1.6 g 1.8 1.78 2.2 5.4 7.98 11.3 9,3 14,2 21.6 8.7 15.8 19.0 Citrobacter species 0 hours 4.4 3.82 5.2 4 hours 15.8 23.2 26.8 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 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 hours 3.4 8.3 12.8 6 hours 3.9 10.0 15.8 Yersinia 0 hours 6.4 3.0 5.9 4 hours 11.4 12.2 13,2 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 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 hours 11.2 29.4 27.2 6 hours 1:.6 33.2 33.6 Example 7 .DTD:

The results obtained with the medium of the present invention were compared with the results obtained utilizing a conventional broth medium, i.e., tryptic soy broth and standard techniques. One hundred growing devices were prepared which contained the same medium as set forth in Example 2. One hundred clinical isolates of bacteria that were received from patients were run using these growing devices and the standard growing technique established for the Kirby-Bauer test. The bacteria tested included:

.DTD:

Escherichia coil Klebsiella pneumoniae Pseudomonae aeruginosa Acinetobacter calocoaceticus Proteus mJrabJlJs Proteus morganii Enteroba cter aerogenes Enterobacter cloacae Serratia marcescens 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 35 C 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 12 GB 2 077 760 A 12 completed according to the National Clinical Committee for Laboratory Standards (NCCLS) of the United States of America, Antibiotics Susceptibility Standard set forth above. A comparison was made between the antibiotic susceptibility results obtained using the growth media device of the present invention versus the standard technique for growing bacteria. The results were comparable.

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Example 8 .DTD:

The following materials were dissolved in 1000 milliliters of deionized water and steam sterilized at 121 C for 15 minutes:

.DTD:

1.7 gram Trypticase 0.3 gram Phytone 0.25 gram Dextrose 10 0.5 gram NaCl 0.25 gram K2HPO4 The growing devices utilized in this example were polypropylene sleeves as described in our copending application No. 27368/78, allotted Serial No. 2,000,187 with the medium placed directly therein. The sleeves were capped with a plastic cap containing a "Tyvec" filter. The medium was 15 inoculated using a wire loop with Staphyloccocus aureus bacteria obtained from 4 to 5 colonies of said bacteria on an agar plate. The growing devices were vortexed, i.e., mixed for 10 seconds and then incubated at 35 C. Viable bacteria counts were performed at O, 1,2-1/2, 4- 1/2, 5-1/2, 6-1/2, 11-1/2, 13-1/2, 23-1/2 and 31 hours. The results show that the count initially was 1 x 104 and at 11-1/2 hours the count had increased to about 1.7 to 1.9 x 108 CFU/ml and did not substantially decrease therefrom 20 through the remainder of the aforesaid time/count intervals.

.DTD:

.CLME:

Claims

Claims .CLME:

1. A method of growing a species of aerobic, pathogenic, rapidly growing bacteria from a beginning population to a predetermined ending population in the range 6x 107 to 3x 108 CFU/ml at which said growth of said bacteria substantially subsides due to the lack of nutrient in said medium and 25 wherein said bacteria remain viable, the method comprising innoculating a growth-limiting medium with at least an amount of said bacteria equivalent to 1 CFU/ml of the growth-limiting medium, the medium comprising an aqueous solution having 0.42 to 0.70 milligrams of carbon per millilitre of medium and 0.09 to 0.15 milligrams of nitrogen per millilitre of medium in the form of peptone; and vitamins and minerals of sufficient quantity to provide said growth and in a form usable by said 30 bacteria for said growth.

.CLME:

2. A method of growing a species of aerobic, pathogenic, rapidly growing bacteria from a beginning population to a predetermined ending population in the range 6x 107 to

3 x 108 CFU/ml at which said growth of said bacteria substantially subsides due to the lack of nutrient in said medium and wherein said bacteria remain viable, the method comprising innoculating a growth-limiting medium 35 with at least an amount of said bacteria equivalent to 1 CFU/ml of the growth-limiting medium, the medium comprising an aqueous solution having 0.16 to 0.27 milligrams of carbon per millilitre of medium and 0.035 to 0.056 milligrams of nitrogen per millilitre of medium in the form of proteose peptone; and vitamins and minerals of sufficient quantity to provide said growth and in a form usable by said bacteria for said growth. 40 3. A method as claimed in Claim 1 in which the bacteria are gram-negative and the medium comprises an aqueous solution of peptone which is buffered to ma;,.tain a pH of from 7 to 8.

.CLME:

4. A method as claimed in Claim 2 in which the bacteria are gram-negative and the medium comprises an aqueous solution of proteose peptone which is buffered to maintain a pH of from 7 to 8.

.CLME:

5. A method as claimed in Claim 1 or Claim 2 in which the bacteria are gram-positive and the 45 medium comprises an aqueous solution of an enzymatic hydrolysate of protein.

.CLME:

6. A method as claimed in any preceding claim in which the bacteria have a generation time of less than 50 minutes.

.CLME:

7. A method as claimed in any preceding claim in which the medium is innoculated with an amount of bacteria equivalent to at least 5x 106 CFU/ml of medium which grow to a population in the 50 range 6x 107 to 3x 108 CFU/ml in not more than 5 hours.

.CLME:

8. A method as claimed in Claim 1 or claim 2 substantially as herein described with reference to any one of the Examples.

.CLME:

Printed for Her Majesty's Stationery Office by the Courier Press, Leamington Spa, 1981. Published by the Patent Office, Southampton Buildings, London, WC2A lAY, from which copies may be obtained.

.CLME: