GB2177419A - Film solution - Google Patents

Abstract

A diluent solution for making normally dry, self-contained ready-to-use bacteria culture medium film specific for selective growth of lactic acid bacteria by applying as a diluent a solution containing a gram negative organism inhibitor, water soluble anti-fungal agent, and a water soluble nitrite salt, with the solution having a pH within the range of from about 6.5 to about 7.5.

Description

SPECIFICATION Dried medium culture film solution and method for making medium lactobacillacea selective Recent times have seen the development of dry medium culture plates, a typical example of which is the one sold by 3M Company (Medical Products Division, St. Paul, Minnesota 55144, underthe trademark Petrifilm), whose productstypicallyconsist of a dry, self-contained, ready-to-use bacterial culture medium coated onto a film base and overlaid with, for example, a polyethylene film. The base carried Standard Method nutrients and a cold water soluble gelling agent. The overlay film is also coated withthe gelling agent, and in addition, 2,3,5-triphenyltetrazolium chloride indicator dye, in ordertofacilitate counting.Grids (each square 1 cm x 1 cm) outlined on the bottom film also aid the counting process. The overall dimension of a single Petrifilm plate is 20 cm sq.

Such dry medium culture plates, as those previously described, are often advantageous over the more conventional Petri dishes and agar plates, commonly used for inoculation and bacterial growth. For example, the older, more conventional use of Petri dishes and nutrient agar media involves considerable bulk, weight, and space. This makes transport oforganism samples from the field to the laboratory setting difficult, at best. On the other hand, the dry medium culture film which become supportive of bacterial growth simply bythe addition of an aqueous sample would represent a more efficient way of doing viable bacterial counts than present conventional methods.

It is a fact, however,thatthe available dry medium culture films only contain a Standard Methods culture medium coated onto the film base. Put another way, the presently used culture medium is one suitable for growing al/types of organisms and is not selective for growth of any particulartype. Thus, a bacteriologist seeking to grow a specific organism, in the past has had to abandon the possibility of using the very desirableculture media films and return to useof tedious conventional agar plates, with specifically tailored culturemedia known to enhance the growth ofthe desired specific organisms, which at the same time inhibit growth of undesirable ones.Thus, because bacteriologists and microbiologists are often desirous of growing only a specific organism for further isolation, study, evaluation and use, the Standard Medium containing dry media culture films have not been useful in many instances because of their non-selective medium.

Accordingly, it is a primary object of the present invention to develop a method and meansformaking normally dry, self-contained, ready-to-use medium culture film specific for selective growth of certain bacteria,while atthesametime inhibiting the growth of others.

Anotherobjectofthe present invention isthe development of a method, means and technique of making 3M Petrofilm medium culture film specific for the growth of Lactobacillaceae organisms, or in other words the lactic acid bacteria.

Another specific object ofthe present invention is to develop a diluent solution for use in preparing samples for inoculation into self-contained, ready-touse medium culture films, which makethefilm specific for lactic acid bacteria, while atthe sametime inhibiting the growth of undesired bacterial organisms, yeasts and molds.

The method and meansforaccomplishing each of the above objectives, as well as others, will become appa rent from the detailed description ofthe invention.

Normally dry, self-contained, ready-to-use bacterial medium culture films are made specific for selctive growth of lactic acid bacteria, under anaerobic conditions. This is done with a water based diluent solution, or diluent, which contains a gram negative organism inhibitor, in combinationwith awatersolubleantifungal agent, and a water soluble nitrite salt. Especially preferred is sodium nitrite. The solution has a pH within the range of about 6.5 to about 7.5, preferably neutral. The solution, when used as a diluent or carrier for bacterial inoculation,functionsto make the otherwise Standard Methods culture medium, coated onto the base film, Lactobacillaceae specific.

Dry medium culture film containing Standard Methods culture medium will become supportive of bacterial growth, simply by the addition of an aqueous sample. In accordance with this invention, it has been discovered that if the aqueous sample is inoculated onto the culture film by use of a specific solution, hereinafter described, then, and only then, will the film become organism specific for Lactobacillaceae.

There are numerous reasons why bacteriologists and microbiologists may desire to grow a specific organism, to the exclusion of others. Carrier solution ordiluentsolution developed in accordance with this invention is especially designed for use with culture films to make them Lactobaciltaceaespecific.Thus, it allows convenient use of the desirable advantages of culture medium films such asfield collection, trans- port and study, even when specifically culturing only the Lactobacitlaceae organism.

Typical lactic acid organisms which can be selectively grown on the culture films previously described, when oneusesthe diluent ofthepresent invention, are streptococci, pediococci, lactobacilli and leuconostocs.

The diluentfor use in this invention is athree component system, and is water based. The solution contains in combination a gram negative organism inhibitor, an antifungal agent, and a water soluble nitrite salt. The solution also must have a pH within the range offrom about 6.5 to about 7.5, and is preferably neutral. When these conditions are met, it allows for a diluent which, when placed on a dry medium culture film, makes the film organism specific for Lactobacillaceae.

The gram negative organism inhibitorcan bea Polymixin antibiotic and should have a concentration within the range of 24 units/ml of diluent solution to about 40 units/m I ofdiluentsolutionwith 24units/ml functionally very satisfactory for Polymixin B sulfate.

The preferred antibiotic is Polymixin and Polymixin B sulfate is the preferred Polymixin.

The inoculating solution also contains a water soluble anti-fungal agent, in a concentration within the range of 10 micrograms/ml to about 250 micro grams/ml,with about 10 microgramslmltoabout20 micrograms/ml being preferred. A preferred antifungal agent is cycloheximide. However, in addition to cycloheximide, one may use otheranti-fungal agents and preservatives such as potassium sorbate, buty lated hydroxyanisole (BHA) and burylated hydroxyto luene (BHT).

The third component of the carrier solution, or diluent, is water soluble nitrite salt at a concentration within the range of 600 micrograms/ml to about 1800 micrograms/ml. The nitrite salt may be any water soluble metal nitrite salt, but ispreferably a Group I metal nitrite salt and is most preferably either sodium nitrite or potassium nitrite. Potassium nitrite is most preferred. A nitrite salt, when used within this range of concentrations has been found to inhibit growth of organisms other than lactic acid bacteria.

Thecarriersolution musthavea pH within the range of 6.5 to about 7.5, preferably from about 6.5 to about 7.0. Ideally, the diluent solution is neutral. It has been found critical that in making the culture film Lactobacillaceae specificthatthe pH remains generally within the range of neutral, i.e. from about 6.5 to about 7.5.

This is so because the commercially available films, such asthe Petrifilm sold by 3M Company, contain a tetrazolium dye for dying the cultured bacteria red, to enhance color counting. However, the tetrazolium dye becomestoxicto all growing organisms, lithe pH becomes more acid, such as for example at a level of pH 5.55. Thus, the carrier solution ofthis invention must have a pH within the range specified, which avoids the adverse reaction with tetrazolium dye.

in orderto maximize the inhibitory affect of the growth of other organisms it is also essential forthis invention thatthe inoculated film be incubated under anaerobic conditions. Put another way, the diluent solution is organism specific to Lactobacillaceae only under anaerobic conditions. This anaerobic environment may be induced in many conventional ways, such as use of Gas Pack anaerobic generators, manufactured by BBL Microbiology Systems of Cokeysville, Maryland.

The previous description has been given in connection with the use of Polymixin anti-biotics as the gram negative organism inhibitor. An additional formulation has been developed which is suitable for many purposes, and in some instances performs even better than the previously described formulation. It is based upon use of 2-phenylethanol as the gram negative organism inhibitor. When 2-phenylethanol is used a preferred composition is one having the following description. The concentration of 2-phenylethanol shouldbefromaboutl mg/mito about4mg/ml.The concentration ofthe anti-fungal agent should be from about 10 micrograms/ml to about 250 micrograms/ml and the concentration of the water soluble nitrite salt should be from about 600 micrograms/ml to about 800 micrograms/ml.The pHwill thus bewithinthe range offrom about 6.5 to about 7.5, preferably from about 6.5 to about 7.0.

The use ofthe dry medium coated film with the carrier solutions of thins invention is a conventional technique known to microbiologists and bacteriologists. Basically, the use involves the following techni- que.Thefilm is placed on aflatsurface,thetop transparentfilm layer lifted so that a 1 millilitersample of the diluent carrying the organism can be placed on the bottom film.Thetopfilm is then closed carefully down over the bottom film. A plastic sample spreader is then lightly pressed down over the top film to spread the carrier solution across the bottom medium containing fiim. Thereafter, the film is left undisturbed forapproximately a minute to allowgelling.Thefllm is then incubated in a horizontal position with the clear film side up at32 Cfor up to 48 hours. The organisms will reducethetetrazolium indicator dye in the bottom film making the organism colonies on thefilm appear as red dots, which can be counted.

The following examples are offered to further illustrate but not limitthe process,technique and product composition ofthe present invention.

Examples In each ofthe examples demonstrated hereinafter, the dry medium culture film was the Petrifilm"' plate developed by the 3M Company (Medical Products Division, St. Paul, Minnesota 55144). This Petrifilm"' consisted of a dry self-contained, ready-to-use bacte rial culture medium coated onto a film base and overlaid with a polyethylene film. The base carried Standard Method nutrients andacoldwatersoluble gelling agent. The overlay film, which was also coated with a gelling agent, also contained 2,3,5-triphenyltet razolium chloride indicator dye to facilitate organism counting. A grid of 1 cmx 1 cm squares was outlined on the bottom film to also aid in counting. The overall dimensions ofthe Petrifilm"' plate was 20 square cm.

The basic procedure involved preparation of stock olutions of the diluent, inoculating the Petrifllms"', followed by incubation at32 Cfor48 hours under anaerobic conditions, and thereafter counting organ isms (cfu/ml). Asoil samplefrom a greenhousewas usedas the test culture and another sample desig nated S/80, known to be a Lactobacillus culture was used as a control. The solid sample was known to contain many organisms other than Lactobacillus.

The diluent stock solutions were prepared at the following levels of sodium nitrite, by percentweightl volume: 1% weight"volume, 6% weight'volume, 12% weight'vctlume, and 18% weight'volume. Cyclohex imide was used at percentweightSvolume levels of: 0.1%, 0.5%, 1% and 2.5% weightlvolume. Polymyxin B, in this first series of examples, since they were designed to study the variations in concentration of sodium nitrite and cycloheximide, was used at a constant 0.03% weightivolume.

The stock solutions were filter sterilized and the final diluentwas prepared by using 97 ml of sterilized distilled water and adding 1 ml per 100 ml of water of ) each ofthethree components, i.e. sodium nitrite, cycloheximide and Polymixin B all within the ranges previously specified.Sterile mixtures were then aseptically delivered to test tubes at a rate of 9 ml/tube.Thefollowing mixtureswereprepared: Final Concentration oflngredients Control Butterfield's* buffer plus 0.1% Tweed 80 Variation 1 1001lg/ml nitrite 10,ug/ml cycloheximide 24 units/ml Polymixin B Variation 2 600 g/ml nitrite 10 ug/mI cycloheximide 24units/ml Polymixin B Variation 3 1200 g/ml nitrite 101lg/mi cycloheximide 24 units/ml Polymixin B Variation 4 1800,ug/ml nitrite 10,ug/ml cycloheximide 24units/ml Polymixin B Variation 5 600 pg/ml nitrite 50 g/ml cycloheximide 24unitslml Polymixin B Variation 6 600 g/ml nitrite 1001lg/ml cycloheximide 24units/ml Polymixin B Variation 7 600 pg/ml nitrite 2501lg/ml cycloheximide 24units/ml Polymixin B * Butterfield's buffer is a conventional potassium phosphate buffering solution in food microbiology.

Standard Petrifilm"' dilution procedures were followed in plating the S/80 control, in particular 1 ml of an overnight culture was serially diluted using the test solution and 1 ml volumes were delivered to the plate. An 11 gram sample of greenhouse soil was initially diluted in 100 ml of Butterfield's buffer.-This preparation was then serially diluted using the test solution.

1.0 ml volumes of 10.6, IO and 1 c}8 diluti6ns of S/80 were delivered to Petrifilm"' plating medium.

1 ml volumes of 10-4,10-5 and 104 dilutions of soil weredetiveredto Petrifilm"' plating medium. Plates samples were incubated anaerobically in jars containing Gas Pack0' generators (BBL). They were incubated at32 Cfor48 h.

Table t below shows the resulting organism count, using the diluent solution herein specified.

TABLE I Sample Colony forming Units/ml (cfu/ml) Soil: Control 5x107 Variation 1 3.3 x 106 Variation 2 2.3 x 107 Variation 3 5x104 Variation4 0x104 Variation 5 7.0 x 106 Variation 6 7.1 x 107 Variation 7 4.0 x 107 r/80: Control 2.4 x 109 Variation 1 7.5 x 108 Variation 2 1.3 x 109 Variation 3 1.3 x 109 Variation 4 1.3 x 109 Variation 5 9.6 x 108 Variation 6 1.2 x 109 Variation 7 1.0 x 109 As can be seen, there is a significant increase in the inhibitory effect of the diluent on soil micro-organisms as the concentration of the nitrite was increased from 600 g/m to 1200 g/ml. The S/80 was unaffected by the variation of either the nitrite or cycloheximide concentration overthe ranges of 100 tig/ml to 1800 g/ml and 10 g/ml to 250 g/ml, respectively.It can also be seen that the diluent allowed the culture film to be organism specific for the Lactobaclllus organism, and inhibited the growth of other organisms. Similar experiments to those shown in variations 1-7were done varying the Polymixin B concentration which was shown to have no effect on the S/80 organism over a range of 24-40 units/ml. For soil organisms levels of below 24 units/ml or Polymixin B decreased the inhibitory effect.

Examples Showing Replacement of Polymixin B Sulfate With 2-Phenylethanol 2-Phenylethanol (PEA) purchased from Sigma Scientific as a liquid (1 ml = 1.02 grams) was used in the diluent. The PEA is added to obtain the concentra tions specified below,to a givenvolume of distilled water, and autoclaved. Filter sterilized 6% weight/ volume sodium nitrite and 0.1 % weightlvolume cycloheximide solutions were used to complete the below listed mixtures by adding 1 ml per 100 ml of diluent prepared.

TABLE II Mixtures Prepared % By Wt./Vol.

8 600 Mg/mt nitrite 10 g/ml cycloheximide 0.05% PEA 9 600 Krg/ml nitrite 10pg/ml cycloheximide 0.10% PEA 10 6001lg/ml nitrite 10 g/ml cycloheximide 0.20% PEA 11 600 g/ml nitrite 10 tig/ml cycloheximide 0.4% PEA Inoculation and technique was as in the previously described examples. An S/80 solution and soil were the tested samples. The S/80 was serially diluted from an overnight culture through the diluent mixtures, and plated on Petrifilm"' at 107 and 108. The soil was initially diluted 1:10 with 99 ml modified Butterfield's buffer and then serially diluted in the given diluent.

The 10-4, 10-5 and 16-6 dilutions of soil were delivered to Petrifilm. Films were incubated anaerobically using BBLGas Packs# for 48 hours at32 C. The results are shown in Table Ill.

Table 111 Results Inoculating Solutions # cfu/ml Soil: Variation 8 6.2 x 105 Variation 9 5 x Variation 10 103 Variation 11 0x103 S/80: Variation 11 9.4 x 108 As can be seen, the phenylethyl alcohol substitution in thefomulation forthe Polymixin B sulfate antibiotic appears quite effective in inhibiting the soil microorganisms.

The following examples demonstrate various modification oftheformulations using phenylethyl alcohol (PEA) as the gram negative inhibitor.

TABLE Formulations Phenylethyl Mixtures Sodium Nitrate Cycloheximide Alcohol Prepared Concentration Concentration Concentration Control (Butterfield's Buffer) 12 800 ugiml 10 Mg/ml 0.1% 13 800 tig/ml 10 tig/ml 0.2% 14 600 llg/ml 10 ,ug/mt 0.1% 15 600 tig/ml 10 ,ug/ml 0.2% 16* 800 tig/ml 10 llg/ml 0.1% 17* 600 llg/ml 10 ,ug/ml 0.1% * Includes Polymixin B ata level of 24 units/ml.

S/80 and soil were test samples. S/80 dilutions were plated at 10-7 and 10.8. Soil was diluted initially in 99 ml (1:10) of modified Butterfield's buffer and plated at 10-3 orthrough 10-6. Petrifilms were incubated anaerobically using BBL Gas Packs"' at 32"C for 48 hours, as similarly described in earlier examples. The results are indicated below.

TABLE V- Results Sample Mixture cfu/ml Control: Control 9.8 x 108 S/80 12 8.7 x 108 13 8.5 x 108 14 8.7 x 108 15 1.0 x 109 16 7.4 x 108 17 1.1 x 109 Control: -- 2.7 x 107 Soil: 12 1.8 x 104 13 3.0 x 102 14 3.4 x 106 15 3.3 x 104 16 4.2 x 104 17 9.0 x 104 As can be seen, solution #13 gave the best inhibitory results with soil,without adversely affecting the growth of S/80.

It can be seen from each ofthe above examples as presented in the various tables that effective diluent compositions can make media films lactic acid bacteria specific and therefore effectively accomplish their intended purposes.

Claims (13)

1. Adiluentsolutionformaking normallydry, self-contained, ready-to-use bacterial culture films specifisforselectivegrowth of lacticacid bacteria under anaerobic conditions, comprising a diluent solution having a concentration offrom about 1.0 mg/ml to about 4.0 mg/ml of diluent of 2-phenylethanol, from about 10 micrograms/ml to about 250 micrograms/mt of a water solubie anti-fungal agent and from about 600 micrograms/ml to about 800 micrograms/ml of watersoiuble nitrite salt, the solution having a ptOwithin the range of from about 6.5 to about 7.5.

2. A diluent solution as claimed in claim 1, in which the anti-fungal agent is selected fromthe group consisting of cycloheximide, potassium sorbate, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA).

3. A diluent solution as claimed in claim 2, in which,the anti-fungal agent is cycloheximide.

4. A diluent solution asclaimed in claim 1,2 or3 in which the pH is from about 6.5 to about 7.0.

5. A diluent solution as claimed in any of claims 1 to 4, in which the nitrite is a group I metal nitrite.

6. A diluent solution as claimed in any preceding claim in which the nitrite is sodium nitrite.

7. A normally dry self-contained, ready-to-use bacterial culture medium film combined with a diluent formu lated to make the ready-to-use cultured media film specific for selective growth of lactic acid bacteria under anaerobic conditions.

8. A combination as claimed in claim 7, in which the normally dry, self-contained, ready-to-use bacte rial culture film comprises a bacterial culture medium coated on to a film base and overlaid with a transparentfilm,the film base carrying standard method nutrients, and a cold water soluble gelling agent, the overlay being coated with a gelling agent and 2,3,5-triphenyltetrazolium chloride indicator dye to facilitate organism counting.

9. Acombination as claimed in claim 7, in which the diluentsolution is a watersolution containing from about 24 units/mt of diiuentto about 40 units/ml of diluent of a Polymixin antibiotic, from about 10 micrograms/mi of diluentto about 250 micrograms/ ml of diluent of a watersoluble anti-fungai agent, and from about 600 micrograms/ml to about 1800 micro grams/ml of diluent of a nitrite salt, the solution having a pH of from about 6.5to about 7.0.

10. Acombination as claimed in claim 7, in which media solution is a solution comprising from about 1.0 mg/mt to about 4.0 mg/ml of 2-phenylethanol, from about 10 micrograms/ml to about 250 micro grams/ml of cycloheximide and from about 600 micrograms/ml to about 800 micrograms/ml of sodium nitrite.

11. A method of anaerobically culturing lactic acid bacteria, which are shelf-stablefor up to one year, consisting essentially of obtaining a biological film plate containing standard methods culture medium, preparing an aqueous diluent solution which contains in combination from about 24 units/ml of diluent solution to about 40 units/ml of diluent solution of a gram negativeorganism inhibitor, from about 10 micrograms/mI of solution to about 250 micrograms/ ml of solution of a water soluble anti-fungal agent, from about 600 micrograms/ml to about 1800 micro- grams/mi of a water soluble nitrite salt, the solution having a pH within the range of from about 6.5 to about 7.5 and applying a small but effective amount ofthesolutiontothefilm platein conjunctionwith a sample containing lactic acid organisms and incubating the film under anaerobic conditions to provide a shelf-stable culture.

12. Adiluentsolutionformaking normallydry self-contained ready-to-use bacterial culture films specific for selective growth of lactic acid bacteria substantially as herein described.

13. A method of anaerobicatly culturing lactic acid bacteria substantially as herein described.