CH636376A5 - Process for the identification of bacteria and culture medium for its implementation - Google Patents

Description

The present invention relates to the field of identification of bacteria, in particular enterobacteria, and relates to media for the identification of bacteria applicable to identification by micromethod.

Currently, the identification of enterobacteria is practiced daily in medical and veterinary biology laboratories, or in food bacteriology laboratories.

Generally, this identification is carried out by highlighting a set of biochemical properties of the bacterium using special culture media called identification media.

In the past, these media were complex agar media contained in glass tubes closed with a large screw. To inoculate the tubes, it was then necessary to have a pure culture strain obtained in 24 h by transplanting the isolated colony on isolation medium.

These identification media present, however, inter alia, the drawback of being bulky, impractical for mass production and expensive.

There are also other devices for identifying enterobacteria which make it possible to identify the bacterium from the isolation medium, and having as a corollary a saving of time and material.

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Such an identification device can be constituted by a compartmentalized cylinder containing different agar media, a pointed rod crossing the tube in its length, and allowing, by contact of its point with the colony to be identified, and by pulling on the rod, d 'inoculate each medium in passing.

Another device is in the form of a set of small cups of particular shapes placed on a support of synthetic material, and containing the identification media in dried forms. These wells are then filled using a suspension in water of the bacteria to be identified.

There are still other identification methods and devices, but all of them require the use of a special container and are of high cost price.

The present invention relates to the development of liquid media for the identification of bacteria applicable to identification by micromethod.

These media are characterized in that they have properties such that late reversals of the pH are avoided in the media for the study of sugar fermentations, that the addition of petrolatum oil to certain media (medium for sugar fermentations of enterobacteria, medium for the detection of urease, medium for the demonstration of the production of hydrogen sulphide, medium for the search for decarboxylases) is unnecessary, as the result of a biochemical reaction can be read, in general, directly thanks to the color of an internal indicator, without adding special developers at the end of incubation, that the reactions are rapid, which can be obtained in 3 to 18 h depending on the biochemical nature, and according to the bacteria studied, than the micromethod can be used in any usual containers in biology laboratories, such as hemolysis tubes or plate wells for microtitration, and in that the final reaction volume is from 150 to 650 (of which 100 to 500 | of identification broth.

Like others, the identification micromethod, applicable in particular to the identification of enterobacteria, uses a bacterial colony obtained on an isolation medium. The micromethod combines, to obtain the diagnosis of the genus and sometimes of species of enterobacterium, the following media: media for researching the fermentation of glucose, melibiose, rhamnose, medium for the production of acetoin, medium for the amino acid oxidase research and medium for the detection of indole production, medium for research of ß-galactosidase and nitrate reductase, medium for research of urease, lysine decarboxylase and ornithine decarboxylase, citrate medium, and medium for the demonstration of the production of hydrofluoric acid, the diagnosis being able, in certain cases, to be supplemented by the use of the medium for the research of arginine- dihydrolase, medium for the study of the use of malonate, and possibly other mediums for sweet fermentations, as well as medium for the detection of gelatinase.

According to a characteristic of the invention, the micromethod consists in suspending, in 2.5 ml of salt water containing 9 g / l of sodium chloride, a colony or colony fraction of enterobacterium to be studied, to be distributed then a drop of about 50 (it of this suspension in the identification broths previously distributed in the wells of the plate for microtitration and to close the wells with an adhesive paper, the wells containing the media for the search for acetoin, for the use of citrate or malonate and for the detection of amino acid oxidases, being closed by an adhesive paper pierced with a hole with a diameter of less than 1 mm, then placing the plate thus sown in an oven at 35 ° C for a period of 12 to 18 hours and, finally, to read the results.

According to a variant of the invention, the micromethod implements, with a view to biotyping the Cocci Gram (+) into a catalase (+) or catalase (-) cluster according to the classification of Baird Parker, with a colony of bacteria taken from selective media or on blood agar or ordinary agar, the following media: modified concentrated Koser medium, gallery broth, medium for the fermentation of glucose and mannitol, medium for the aerobic use of lactose, maltose, arabinose and mannitol, medium for the detection of acetoin without a pH indicator, medium for the search for "phosphatase, medium for the search for coagulase, medium for the demonstration of the reduction of nitrates.

According to a characteristic of the invention, the micromethod consists in suspending in 1 ml of gallery broth a colony of bacteria taken from selective media or from blood agar or ordinary agar, with a view to biotyping Cocci Gram (+) in cluster catalase (+) or catalase (-) according to the classification of Baird Parker, then to distribute a drop of suspension of approximately 50 ni in 100 μl of each of the identification media previously distributed in the wells of the plate for microtitration, to add three drops of sterile vaseline oil in the middle for the fermentation of glucose and mannitol, then close the cups with an adhesive paper, then put the plate in an oven at 35 ° C for a period of 16 to 20 h and, finally, to read the results.

In accordance with another alternative embodiment of the invention, the micromethod implements, with a view to identifying pseudo-domonadaceae (Gram (-) oxidase (+) bacilli or Gram (-) oxidase and nitrate (-) bacilli cultivating on selective lactose media or on ordinary agar), the following media: medium for researching the use of glucose aerobically, medium for researching the use of glucose by fermentation, medium for researching reduction nitrate at low and high nitrate concentration, medium for checking the use of citrate, medium for detecting gelatinase, and medium for researching arginine dihydrolase.

In accordance with a characteristic of the invention, the micromethod applied to pseudomonadaceae is implemented as follows:

A colony of bacteria to be identified (Gram (-) oxidase (+) bacilli or Gram (-) oxidase and nitrate (-) bacilli growing on selective lactose media or on ordinary agar), taken from an isolation medium, such as agar lactose or ordinary agar is suspended in 1 ml of gallery broth, then placed in an oven at 37 ° C for 2 h. Then 50 (11 of the suspension are distributed into 100 (there are identification broths previously distributed in the wells of the microtiter plate. To the well containing the medium for the study of the fermentation of glucose, three drops of petrolatum oil, and to the cup containing the medium for the detection of gelatinase, a small piece of photographic film is added, then the cups are closed using an adhesive paper, the adhesive paper of the citrate cup being provided with "A hole with a diameter of 1 mm. Finally, the microtiter plate and the suspension in the gallery broth are placed in the oven at 35 ° C. for 14 to 18 hours. After incubation, the adhesive paper is removed and read the results in the same way as for enterobacteria, except for the oxidation of glucose and the reduction of nitrates. In each of the two media for the demonstration of nitrate reductase, the Griess reagents. cose is oxidized, the medium turns yellow, while it remains blue otherwise.

Finally, a drop is removed from the gallery broth between slide and coverslip and the mobility is observed under a dark field microscope.

The media used in the micromethods subject of the invention are described below.

1. Basic medium for the study of sweet fermentations endowed with properties opposing late pH reversals

This medium is used to dilute 1/10 of the concentrated aqueous solutions of sugars to be studied, and contains a trihydroxymethyl-aminomethane / hydrochloric acid buffer which has been shown to be experimentally favorable for sweet use, and the molarity of which has been chosen such that very rapid results are obtained, namely in a time interval of between 3 and 12 hours depending on the sugar studied.

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It may happen that, with certain bacteria, the pH of the medium,

after having passed into an acidic zone under the influence of the fixed acid products formed from sugar, returns to an alkaline zone. This phenomenon is explained by the transformation of these acids into acetylmethylcarbinol, or by oxidative processes, that is to say loss of acidity, and is known as late pH reversion. To avoid this phenomenon, which disturbs the readability of the results, the basic medium according to the invention contains an enolase inhibitor, namely sodium fluoride, at a dose which does not prevent the transformation of the sugars, but which inhibits the transformation of the fixed acids formed, so that reversals of pH or non-specific alkalinizations are impossible within 24 to 48 h.

The inhibitory effect of fluoride is inversely proportional to the pH, so that the inhibitory action is gradually greater as the sweet fermentation progresses.

This basic medium for the study of sugar fermentations has the following preferred composition:

2.5 g yeast extract 1 to 2 mmol of sodium fluoride

1 ml of bromocresol purple solution at 16 g / 1 in ethanol or 100 mg of bromothymol blue 10 to 100 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2 distilled water q.s.p. 1000 ml.

The concentrated aqueous solutions of sugars, diluted before their use in the basic medium for sweet fermentations, are the following:

0.5M solution of glucose, mannitol, mesoinositol, sorbitol, rhamnose, sucrose, arabinose, adonitol, fructose, galactose, maltose, trehalose, xylose, or me -Iibiosis,

0.3M lactose solution,

0.25M solution of raffinose, or amygdalin,

0.15M solution of salicin, or dulcitol,

the method being applicable to any other sugar.

2. Environment for the search for acetoin

Enterobacteria acidify the culture medium by fermentation of the glucose it contains, and some of them are capable of correcting this acidity by transforming the pyruvate formed from glucose into acetylmethylcarbinol, a substance neutral from the point of view of pH. after:

2CH3 - CO - COOH - »CH3 - CH - C - CH3 4- 2C02

I II

OH O

Acetylmethylcarbinol can be detected by the Vosges-Proskauer reaction, which results in the appearance, 30 min after addition of a potassium hydroxide solution and a-naphthol, a redcurrant shade. A defect in this reaction lies in the fact that, with bacteria reputed to be incapable of producing acetoin, very late pale pink hues are obtained, this coloration being supposed to be due to the reagents themselves, but more probably to a very low production of acetoin.

Given the change in acidity which results from the transformation of pyruvate into acetylmethylcarbinol, it has been proposed to add a pH indicator which takes on a different shade depending on whether the bacteria produce or do not produce acetylmethylcarbinol. This procedure was applied in the form of a methyl red test, the latter turning red below pH 4.2 and yellow above pH 6.3. However, the responses thus obtained are not clear, since the average pH obtained with bacteria which do not produce acetoin is 4.70, and that obtained with bacteria capable of producing it is 5.50. This is why the shade produced with methyl red by the acetoin bacteria (-) is only very little different from that obtained with the acetoin bacteria (+). This results in the difficulty of using the methyl red test, to which is added the fact that the latter is not very soluble and that bacteria reduce it, thus making it insensitive to variations in pH, which prevents its introduction in advance in the identification broth.

It has been established that, if the medium contains, in addition to glucose, pyruvate or lactate, the average pH of the media seeded with acetoin bacteria (-) is 5.4, while that of the media seeded with acetoin bacteria (+) is 6.9, that is to say that the difference between these two means is approximately double what it was with a medium containing only glucose. Under these conditions, it is possible to use as a pH indicator the bromothymol blue which turns yellow at pH below 6, and green, then blue at pH above 6. This indicator is easily soluble and not transformed by bacteria. In addition, the results are immediately read, without the need to add developers after incubation, and the results are consistent between this new method and that using the Vosges-Proskauer reaction.

In this medium, for the search for acetoin, substances activating the production of acetoin are used, and a small quantity of creatine, arginine, or of the two components used simultaneously.

The medium for acetoin research has the following preferred composition:

10 g yeast extract

50 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2 100 mg bromothymol blue

20 mmol of creatine, or 10 mmol of arginine, or 20 mmol of creatine and 10 mmol of arginine

20 to 100 mmol of glucose

200 mmol of pyruvate or 100 mmol of lactate distilled water q.s.p. 1000 ml.

Bacteria other than enterobacteria are less active in producing acetoin and are unable to bring the pH back to near neutrality from the acid products formed by the fermentation of glucose. The method using the colored indicator is then inapplicable and acetoin must be revealed by the V osges-Proskauer reaction.

In this case, the medium for acetoin research has the following preferred composition:

10 g yeast extract

50 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2

20 mmol of creatine, or 10 mmol of arginine, or 20 mmol of creatine and 10 mmol of arginine

20 to 100 mmol of glucose

200 mmol of pyruvate or 100 mmol of lactate distilled water q.s.p. 1000 ml.

3. Medium for the search for amino acid oxidases

The usual techniques for searching for amino acids oxidases consist in demonstrating by iron perchloride the formation of α-ketonic acid from phenylalanine or tryp-tophane (test of phenylalanine deaminase or tryptophan deaminase) . The subject of the present invention is the development of various media allowing rapid and intense reactions (3 to 14 hours depending on the germ) which, sometimes, do not require the use of revealing reagents.

It has been shown that many amino acids can be transformed into a-ketonic acids, but that all these a-ketonic acids do not react with iron perchloride ("An im-proved ferrie chloride test for differentiating. Proteus - Providence Group from other enterobacteriacees ", J. Singer et al., 1954). On the other hand, the media used combine various amino acids in defined proportions, which has proved particularly advantageous for obtaining rapidly positive reactions. Thus, the combination of L-tryptophan, L-leucine and L-arginine, or the combination of L-tryptophan, L-methionine and L-arginine were

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shown to be particularly interesting and better than L-tryptophan or L-methionine or L-leucine or L-arginine alone.

The amino acid oxidase reaction can be accelerated by adding a moderate dose of various oxidants, such as nitrates (sodium or potassium nitrate), persulfates (ammonium persulfate), or chloramine T. L ' atmospheric oxygen can also clearly activate the reaction without causing the inhibitory effects observed with too high doses of the oxidants mentioned above. The effect promoting atmospheric oxygen can be further intensified by introducing peroxidase (horseradish peroxide) into the medium. In this case, the amount of α-ketonic acid produced by the bacteria having the amino acid oxidases from the mixture of L-tryptophan, L-leucine and L-arginine, is such that the medium takes on a buff-brown hue. which is easily distinguished from the colorless medium when it is inoculated with bacteria that do not have amino acid oxidases. In this case, it is therefore no longer necessary to use a reaction developer. The preferred compositions of the media corresponding to the above indications are the following:

A. Medium requiring the use of a solution of iron perchloride (20 mmol) in 2.5M hydrochloric acid as developer, the preferred composition of which is:

42 mmol of L-tryptophan 6 mmol of L-leucine 0.25 mmol of L-arginine

0.25 to 0.75 mmol of sodium or potassium nitrate or 0.25 to 0.50 mmol of ammonium persulfate or 2.5 mmol of chloramine T

2.25 g of yeast extract distilled water q.s.p. 1000 ml, or trihydroxymethylaminomethane buffer 50 mmol / hydrochloric acid q.s.p. pH 7.2, q.s.p. 1000 ml,

or piperazine dichloride buffer 50 mmol / soda q.s.p. pH 6.5, q.s.p. 1000 ml.

B. Medium making optional the revelation by the solution of 20 mmol of iron perchloride in 2.5M hydrochloric acid,

whose preferred composition is:

42 mmol of L-tryptophan 6 mmol of L-leucine 0.25 mmol of L-arginine 2.25 g of yeast extract 10 mg of horseradish peroxidase distilled water q.s.p. 1000 ml.

Some colored substances can behave as an oxidant in the reaction of amino acids oxidases. Their contribution to the reaction reduces them and, therefore, causes them to change color, or discolour them. Thus, dichlorophenolindophenol changes from dark blue (oxidized state) to a pale pink tint (reduced state), when the amino acids involved are oxidized to corresponding α-ketonic acids under the influence of bacterial amino acid oxidases. When bacteria do not have the enzyme, the medium remains dark blue. Some amino acids are more suitable for this reaction than others; this is why a mixture of L-tryptophan, L-methionine and L-arginine was chosen. The peptone or cell extract introduced as a growth substance into the medium must not give rise to non-specific reduction reactions for dichlorophenolindophenol; this is why a papain soy peptone is proposed. Finally, atmospheric oxygen and peroxidase improve the reaction and reinforce the difference between positive results (pale pink) and negative results (dark blue).

Therefore, this reaction principle makes revelation with iron perchloride completely unnecessary, and the results are read directly. The preferred composition of the medium is as follows:

C. 20 mmol of L-tryptophan 20 mmol of L-methionine 0.25 mmol of L-arginine

600 (dichlorophenolindophenol imol

5 g of papain soy peptone 10 mg of horseradish peroxidase distilled water q.s.p. 1000 ml.

5 4. Environment for research into indole production

Conventionally, the production of indole is studied in a medium containing tryptophan. After an overnight incubation, the indole possibly produced reacts with the Kowacks reagent, giving a redcurrant shade. Another method consists in introducing into the culture medium either a bacterium producing an indol-3-hydroxylase, such as Pseudomonas indoloxydans, or any other bacterium capable of transforming indole into indoxyl by hydroxylation, or the culture juice. concentrated and purified of a bacterium producing this hydroxylase in its medium. The indole produced by the indologene species is transformed under the influence of this specific hydrolase into indoxyl which spontaneously condenses into dark blue colored indigo. The use of this enzyme preparation is possible, since Pseudomonas indoloxydans does not produce enzymes active on tryptophan or its catabolites other than indole. Therefore, the use of a reaction developer such as Kowacks' reagent becomes unnecessary. The preferred compositions of the media corresponding to these two solutions are as follows:

at. Medium requiring the use of Kowacks reagent, consisting of:

25 30 mmol of L-tryptophan 2.5 g of yeast extract distilled water q.s.p. 1000 ml, or trihydroxymethylaminomethane buffer 50 mmol / hydrochloric acid q.s.p. pH 7.2, q.s.p. 1000 ml,

30 or piperazine dichloride buffer 50 mmol / soda q.s.p. pH 6.5, q.s.p. 1000 ml.

b. Medium rendering unnecessary the use of a developer consisting of: 30 mmol of L-tryptophan

2.5 g of yeast extract 35 25 ml of purified and concentrated culture juice of a bacterium producing in its medium indol-3-hydroxylase distilled water q.s.p. 1000 ml.

5. Medium for testing for ß-galactosidase and nitrate-40 reductase

The medium for the search for ß-galactosidase and for nitrate reductase is characterized by the combination of two substrates: orthonitropltenyl-ß-D-galactopyrannoside and sodium nitrate in solution in a trihydroxymethylaminomethane / hydrochloric acid buffer. , which is recognized experimentally favorable to the use of sugars. It is also known that, when the bacterium has ß-galactosidase, orthonitrophinyl-ß-D-galactopyrannoside is degraded to orthonitrophenol yellow in color. After observation of this first bacterial property, the so-nitrite optionally formed by bacterial reduction of the nitrate is sought using the a-naphthylamine in solution in acetic acid (second Griess reagent) which is added to the medium after incubation and thanks to orthonitroph6nyl-ß-D-galactopyrannoside or to gold-thonitrophenol contained in the medium according to one of the following reactions:

A) Nitrite + a-naphthylamine + orthonitrophenyl-P-D-galactopyrannoside-► azo red in color

B) Nitrite + a-naphthylamine -I- orthonitrophenol- »azo red in color.

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This way of operating avoids the use of the first Griess reagent consisting of sulfanilic acid in acetic solution.

The nitrite generally formed by all enterobacteria exerts an inhibitory effect on the fermentation of galactose released by bac-65 teria from orthonitrophenyl-PD-galactopyrannoside, which prevents the accumulation of fixed acids, thus strengthening the buffer and thereby ensuring better readability of the β-galactosidase detection reactions.

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The medium for the search for ß-galactosidase and for nitrate reductase has the following preferred composition:

3 mmol of orthonitrophenyl-p-D-galactopyrannoside 50 mmol of sodium nitrate 2.25 g of yeast extract

100 mmol of trihydroxymethylaminomethane / hydrochloric acid pH 7.2

distilled water q.s.p. 1000 ml.

6. Environment for urease testing

The use of this medium is based on the well-known principle that the bacterial transformation of urea into ammonium carbonate alkalinizes the medium and causes the color of a pH indicator to change.

Usually, conventional media contain a buffer allowing the initial pH to be maintained in an acidic zone compatible with bacterial life; the media are such that the bacteria inactive on the urea do not change the color of the indicator. Unlike these media, the medium according to the invention does not contain a buffer and uses glucose fermentation as a developer; the glucose, which is fermented by all enterobacteria, generates acid products in quantities low enough to be able to be largely neutralized by the ammonium carbonate produced by urolysis, so that the medium turns to alkaline, causing the color to change of the colored indicator. When the bacteria is inactive on urea, the medium remains acidic.

In addition, the fermentation of glucose very strongly activates urolysis, when the bacteria has a urease. Like other activator, the medium contains yeast extract. The medium contains an amount of urea between 26 and 44 mmol, so that the urolysis reaction is optimal. To avoid non-specific alkalinization - the result either of the alkaline transformation of the yeast extract, or of the pH reversion as a result of oxidative processes or of the transformation of the acids formed by fermentation of glucose into acetylmethylcarbinol - it is proposed:

1) to use a sufficient quantity of glucose so that the alkaline products formed from the yeast extract are neutralized, but sufficiently weak so that the change to alkali under the influence of urolysis is not prevented or delayed;

2) to introduce into the medium an enolase inhibitor making it possible to avoid the loss of acids by transformation of the acid products formed by fermentation of glucose into substances such as acetylmethylcarbinol; the inhibitor used is sodium fluoride at a concentration of between 1 and 2 mmol / l, the concentration at which it is inactive on urolysis.

This medium is clearly superior to conventional mediums, because it has been shown that ureolysis is very strongly activated by the presence of glucose in the medium, thus making it possible to obtain rapid results in 3 to 12 h depending on the bacteria studied.

This medium for urease research has the following preferred composition:

35 mmol of urea 16 to 20 mmol of glucose 1 to 2 mmol of sodium fluoride 2.5 g of yeast extract

1 ml of bromocresol purple solution at 16 g / l in ethanol or 100 mg of bromothymol blue 16 mmol of potassium chloride distilled water q.s.p. 1000 ml.

7. Medium for the search for lysine decarboxylase and ornithine decarboxylase

The media for the detection of lysine decarboxylase and ornithine decarboxylase are based on the well known principle according to which the decarboxylation of lysine or ornithine alkalinizes the culture medium and fires a pH indicator . When the bacteria is inactive on lysine or ornithine, the medium becomes acidic under the influence of the fermentation of the glucose it contains. To avoid rapid nonspecific alkalinizations, the result of the transformation of cellular extracts, peptones and the degradation of acid catabolites of glucose, it has been proposed to incubate under a layer of vaseline oil ("Simplified tests for some aminoacid decarboxylases and for the arginine dihydrolase system ", V. Möller," Acta Path. ", XXXVI, 2.1954;" Rapid identification of subgenera of the Enterobacter group ", C. Richard," Ann. Biol. Clin. ", 1970, 28, 186-189). Unfortunately, it has also been shown that the anaerobiosis produced by the use of oil inhibits the decarboxylases which are not very active in certain bacteria (C. Richard, "Ann. Biol. Clin.", 1970, 28, 185-189) and that non-specific alkalinizations are not avoided with other bacteria. On the other hand, the media according to the invention make it possible to avoid this defect while not using petroleum jelly oil. These environments have the following particularities:

1) they contain lysine or ornithine in an amount respectively between 36 and 48 mmol / l and 24 and 36 mmol / l, so that:

a) the reactions of lysine decarboxylase or ornithine decarboxylase are maximum,

b) the inhibition by excess of substrate observed with certain bacteria is avoided

2) obtaining an optimal lysine decarboxylase or ornithine decarboxylase reaction makes it possible to increase the amount of glucose in the media, respectively to 9 mmol / l and 8 mmol / l (instead of 5 mmol in the circles of Möller and Richard); in doing so, the acid products of the glucose fermentation will be formed in greater quantity, allowing the correct neutralization of the alkaline substances formed from the peptones or cellular extracts contained in the media;

3) they contain an enolase inhibitor which makes it possible to avoid the transformation of the acid products formed by fermentation of the glucose and, consequently, make it possible to avoid the non-specific alkaline reactions of the medium; the inhibitor used is sodium fluoride at a concentration of between 1 and 2 mmol / l at which it is inactive on lysine decarboxylase and ornithine decarboxylase;

4) the combination of a substrate (lysine or Ornithine) in optimal quantities with glucose and the enolase inhibitor in suitable quantities makes it possible to avoid the use of petrolatum oil.

The medium for detecting lysine decarboxylase has the following preferential composition:

40 mmol of lysine hydrochloride 9 mmol of D-glucose 1 to 2 mmol of sodium fluoride 2.5 g of yeast extract

1 ml of bromocresol purple solution at 16 g / l in ethanol or 100 mg of bromothymol blue distilled water q.s.p. 1000 ml.

The medium for detecting ornithine decarboxylase preferably has the following composition:

30 mmol of ornithine hydrochloride 8 mmol of glucose 1.5 g of yeast extract 1 to 2 mmol of sodium fluoride

1 ml of bromocresol purple solution at 16 g / l in ethanol or 100 mg of bromothymol blue distilled water q.s.p. 1000 ml.

8. Citrate media and for studying the use of malonate

The media for checking the use of citrate and malonate are characterized by a buffer consisting of a mixture of suitable proportions of sodium citrate and citric acid or of sodium malonate and malonic acid in order to obtain a pH of the medium close to 6, the buffer of the medium also constituting the substrate of the bacteria. When the bacteria uses citrate or malonate, the resulting decarboxylation gradually destroys the buffering capacity of the substrate, and as the initial pH of the medium is

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not far from the limit of the buffering capacity of the citrate / citric acid or malonate / malonic acid system, the pH of the medium quickly changes to alkaline, which is manifested by the turning to blue of the colored indicator. This way of operating avoids the use of additional chemical substances having the quality of a buffer and makes it possible to obtain, at identical molar concentrations of citrate or malonate, much faster responses, of the order of 12 to 16 h.

The preferred composition of the medium for verifying the use of citrate is as follows:

22 mmol of trisodium citrate 3 mmol of citric acid 100 mg of bromothymol blue 0.6 g of yeast extract distilled water q.s.p. 1000 ml.

The medium for verifying the use of the malonate preferably has the following composition:

22.5 mmol sodium malonate 2.5 mmol malonic acid 100 mg bromothymol blue 0.6 g yeast extract distilled water q.s.p. 1000 ml.

9. Medium for the search for arginine dihydrolase This medium is preferably composed of:

25 mmol of arginine base

20 mg phenol red

2.25 g of hydrochloric acid yeast extract q.s.p. pH 6.2

0.01M phosphate buffer pH 6.2, q.s.p. 1000 ml

(disodium phosphate, monopotassium phosphate).

10. Medium for the detection of gelatinase

The gelatinolysis of the gelatin of a photographic film is facilitated by the nature of the peptone of the culture medium and by the addition of mineral salts such as calcium chloride. The fastest producing peptone is the pancreatic gelatin peptone.

This medium for the detection of gelatinase has the following preferred composition:

5 to 10 g of pancreatic peptone of gelatin 2.5 to 10 g of yeast extract 150 mmol of sodium chloride 50 mmol of calcium chloride distilled water q.s.p. 1000 ml.

11. Medium for demonstrating the production of hydrogen sulfide

The production of hydrogen sulfide is usually done either from cysteine or from hyposulfite, and it is evidenced by the reaction with a heavy metal introduced into the medium which forms with it insoluble metal sulfide at higher pH at 5.5 (see Brisou et al.). Conventional media use hypo-sulfite in the presence of ferrous iron and ammonium sulfate associated with a large amount of peptone, the choice of which seems to have been guided by the sulfur content. The use of a tampon was not considered useful. However, depending on the nature of the peptone and on the bacteria studied, the medium is seen to acidify or alkalinize, so that the formation of insoluble metal sulfide can be disturbed. The medium which is the subject of the invention consists of a trihydroxymethylaminomethane buffer (pH 7 to 7.4) and of substances strongly accelerating the reaction, such as arginine, magnesium chloride and a mixture of peptone and cell extracts , such as a pancreatic lactalbumin peptone and yeast extract. The combination of these various promoting factors (buffer, arginine, magnesium chloride, mixture of peptone and cellular extracts) makes it possible to reduce the total amount of peptone and cellular extracts, and to obtain rapid and intense reactions, even with bacteria known to be lazy to produce hydrogen sulfide. The preferred composition of the medium for demonstrating the production of hydrogen sulfide is as follows:

15 to 30 g of pancreatic lactalbumin peptone 1.2 parts,

yeast extract 1 part

18 mmol of L-arginine

2 mmol of magnesium chloride

2.5 mmol sodium hyposulfite

1.5 mmol of ferrous iron and ammonium sulfate 0.15M trihydroxymethylaminomethane buffer pH 7.2, q.s.p.

1000 ml.

12. Basic medium for fermentation and aerobic use of sugars by Cocci Gram (+) in catalase clusters (+)

or catalase (-)

The basic medium for the study of fermentation or the aerobic use of sugars is used to dilute the concentrated aqueous solutions described above to 1/10. The medium in accordance with the invention contains a large amount of yeast extract (20 g / l approximately) in a trihydroxymethylaminomethane / hydrochloric acid buffer, which has been shown to be experimentally favorable for the use of sugars. This large quantity of yeast extract makes it possible to obtain the results of sugared fermentations quickly in 12 to 16 hours in an oven at 35 ° C.

This basic medium for sweet fermentations has the following preferred composition:

20 g yeast extract

1 ml of bromocresol purple solution at 16 g / l in ethanol or 100 mg of bromothymol blue 50 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2 distilled water q.s.p. 1000 ml.

13. Environment for phosphatase research

The medium usually used gave lazy reactions and required the use of a developer after incubation. The medium object of the present invention contains a substrate such as paranitrophenylphosphate, colorless, which, under the action of phosphatase, provides phosphate and paranitrophenol of canary yellow color. It is therefore not necessary to use a developer after incubation. The optimal phosphatase action pH is between 8 and 10 and, between these pHs, the substrate spontaneously lyses, causing a non-specific yellow color to appear. To avoid spontaneous lysis of the substrate, the pH of the medium is maintained in the vicinity of neutrality by a trihydroxymethylamino-methane / hydrochloric acid buffer. This medium also contains papain peptone from soybean protein in an amount such that the growth of demanding bacteria such as staphylococci is possible.

This medium for phosphatase research has the following preferred composition:

12 mmol of paranitrophenylphosphate 10 g of papain peptone from soybean protein 50 mmol of trihydroxymethylaminomethane / hydrochloric acid pH 7.2 distilled water q.s.p. 1000 ml.

14. Medium for searching for nitrate reductase which is preferably constituted by:

20 mmol of sodium nitrate 5 g of tryptic casein peptone distilled water q.s.p. 1000 ml.

15. Medium for detecting coagulase consisting of a mixture of human oxalated plasmas sterilized by filtration.

16. Concentrated modified Koser's solution, consisting of an aqueous solution of:

430 mmol / l sodium chloride s

10

15

20

25

30

35

40

45

50

55

60

65

636,376

10

4 mmol / l magnesium sulfate 18 mmol / l potassium chloride 43 mmol / l ammonium chloride.

17. Gallery broth consisting of:

200 ml concentrated modified Koser's solution

4 to 7 g of yeast extract distilled water q.s.p. 1000 ml.

18. Medium for testing for nitrate reductase containing low concentrations of nitrates, preferably consisting of:

0.01 (imol of ammonium heptamolybdate

5 mmol sodium nitrate

0.5 g pancreatic peptone gelatin

50 mmol of glycerol distilled water q.s.p. 1000 ml.

19. Medium for the search for nitrate reductase containing high concentrations of nitrates, the preferred composition of which is as follows:

0.01 jimol of ammonium heptamolybdate

500 mmol sodium nitrate

0.5 g pancreatic peptone gelatin

50 mmol glycerol distilled water q.s.p. 1000 ml.

The reduction of nitrates is sought by the presence of nitrite in the medium, using the two Griess reagents added after incubation, at the rate of one drop each to the two media. The use of two media, one containing a very small amount of nitrate which can be easily reduced by capable bacteria beyond the nitrite stage, the other containing a very high amount of nitrate whose total reduction beyond the nitrite stage is impossible, allows to draw conclusions about the metabolic possibilities of the bacteria (reduction in nitrites, reduction beyond the nitrite stage, unable to reduce nitrates).

5 20. Medium for studying the use of glucose by the oxidative or fermentative route, the preferred composition of which is as follows:

50 mmol of D-glucose

10 to 25 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2

distilled water q.s.p. 1000 ml.

The media for the use of citrate, for the detection of gelatinase and arginine dihydrolase are the same as those described for enterobacteria.

15 In the case of establishing the diagnosis of the genus and species of enterobacterium on a bacterial suspension in 2.5 ml of salt water containing 9 g / l of sodium chloride, the reactions for the production of acetoin and the use of citrate or malonate are decarboxylation reactions producing carbon dioxide. When the reaction medium is in contact with a sufficient volume of air, this carbon dioxide can escape from the medium, thus contributing rapidly to the loss of acidity, therefore to the more rapid shift of the pH indicator. When the cups are closed by the adhesive paper, the volume of air between the surface of the liquid and the adhesive paper is insufficient, the carbon dioxide formed diffuses poorly through the paper, and consequently, the partial pressure of gas carbon dioxide increasing above the broth, a certain amount of acidity is retained in the medium, delaying or preventing the turn of the pH indicator. It is for these reasons that the adhesive paper of the cups containing these 30 media is pierced with a hole with a diameter of less than 1 mm. After 3 to 18 h of incubation in an oven at 35 ° C., the results are read from the table below:

Positive responses Negative responses

1. Fermentation of sugars (glucose, melibiose, rhamnose, or any other sugar)

yellow blue or green

2. Testing for colorless yellow ß-galactosidase

3. Reduction of nitrates to nitrites: add a drop of a-naphthylamine solution (second reagent from Griess) to the cup for the detection of ß-galactosidase

colorless redcurrant

4. Production of blue-green to intense blue-yellow acetoin

5. Use of citrate or intense blue green malonate

6. Presence of green or blue yellow urease, lysine decarboxylase or ornithine decarboxylase

7. Search for amino acid oxidases:

a) with medium requiring the addition of a drop of iron perchloride b) with medium making optional revelation with a solution of perchloride, without addition of perchloride c) with medium with dichlorophenolindophenol red-brown to purple - lie-de- wine brown buff pale pink colorless or straw yellow colorless dark blue

8. Search for indole production:

a) medium requiring the addition of two drops of Kowacks reagent b) medium with indol-3-hydroxylase red ring precipitated dark blue

colorless or slightly greenish ring unchanged middle

9. Production of black precipitated hydrogen sulfide medium unchanged

10. Search for pink to red arginine dihydrolase unchanged medium

11. Detection of gelatinase strip of transparent photographic film and precipitate black strip unchanged

11

636,376

In the case of biotyping of Cocci Gram (+) in a catalase (+) or catalase (-) cluster on a bacterial suspension of 1 ml of gallery broth, the reading of the results, after incubation for 16 to 20 h in an oven at 35 ° C, is carried out as indicated in the table below:

Positive reactions

Negative reactions

1. Fermentation of glucose or mannitol, or aerobic use of maltose, lactose, arabinose and mannitol, or any other yellow, green or blue sugar

2. Acetoin production: add a drop of 7M potassium hydroxide and a drop of 0.4M ci-naphthol in the middle after 30 min at ordinary temperature, colorless red currant

3. Search for colorless canary yellow phosphatase

4. Search for coagulase cloudy environment clear environment

5. Research for nitrate reduction: add a drop of each of the red currant Griess reagents turning colorless brown

In the case of the identification of pseudomonadaceae, the results are read as for enterobacteria, or as for Cocci Gram (+) in the case of the search for reduction of nitrates.

Claims (10)

636,376 1 ml of bromocresol purple solution at 16 g / l in ethanol, or 100 mg of bromothymol blue 50 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2 distilled water q.s.p. 1000 ml. 41. Medium according to claim 6, characterized in that the medium for the search for phosphatase contains as substrate the paranitrophenylphosphate. 42. Medium according to claim 41, characterized in that the medium for the search for phosphatase contains a buffer keeping the medium in the vicinity of neutrality to avoid spontaneous hydrolysis of the substrate. 43. Medium according to claims 41 and 42, characterized in that the medium for the search for phosphatase has the following composition: 12 mmol of paranitrophenylphosphate 10 g of papain peptone from soybean protein 50 mmol of trihydroxymethylaminomethane / hydrochloric acid pH 7.2 distilled water q.s.p. 1000 ml. 44. Medium according to claim 6, characterized in that the medium for the search for nitrate reductase consists of: 20 mmol of sodium nitrate 5 g of tryptic casein peptone distilled water q.s.p. 1000 ml. 45. Medium according to claim 6, characterized in that the medium for the search for nitrate reductase containing low concentrations of nitrates consists of: 0.01 nmol of ammonium heptamolybdate 1.5 mmol of ferrous iron and ammonium sulfate 0.15M trihydroxymethylaminomethane buffer pH 7.2 q.s.p. 1000 ml. 40. Medium according to claim 6, characterized in that the basic medium for sweet fermentations has the following composition: 20 g yeast extract 1 ml of bromocresol purple solution at 16 g / l in ethanol or 100 mg of bromothymol blue distilled water q.s.p. 1000 ml. 35. Medium according to claim 6, characterized in that the medium for verifying the use of citrate consists of: 22 mmol of trisodium citrate 3 mmol of citric acid 100 mg of bromothymol blue 0.6 g of yeast extract distilled water q.s.p. 1000 ml. 36. Medium according to claim 6, characterized in that the medium for verifying the use of the malonate has the following composition: 22.5 mmol sodium malonate 2.5 mmol malonic acid 100 mg bromothymol blue 0.6 g yeast extract distilled water q.s.p. 1000 ml. 37. Medium according to claim 6, characterized in that the medium for the search for arginine dihydrolase consists of: 25 mmol of arginine base 20 mg of phenol red 2.25 g of yeast extract hydrochloric acid q.s.p. pH 6.2 0.01 M phosphate buffer pH 6.2 q.s.p. 1000 ml (disodium phosphate, monopotassium phosphate). 38. Medium according to claim 6, characterized in that the medium for the detection of gelatinase has the following composition: 1 ml of bromocresol purple solution at 16 g / l in ethanol or 100 mg of bromothymol blue distilled water q.s.p. 1000 ml. 31. Medium according to claim 6, characterized in that the medium for the search for ornithine decarboxylase contains a concentration of ornithine between 24 and 36 mmol / l. 32. Medium according to claim 31, characterized in that the medium for the search for ornithine decarboxylase contains a quantity of glucose making it possible to neutralize the unspecified alkalinity. 10 15 20 25 30 35 40 45 50 55 60 65 636,376 1 ml of bromocresol purple solution at 16 g / l in ethanol or 100 mg of bromothymol blue 16 mmol of potassium chloride distilled water q.s.p. 100 ml. 27. Medium according to claim 6, characterized in that the medium for the search for lysine decarboxylase contains a concentration of lysine between 36 and 48 mmol / l. 28. Medium according to claim 27, characterized in that the medium for the search for lysine decarboxylase contains an amount of glucose making it possible to neutralize the non-specific alkalinity produced from peptones or cellular extracts contained in the media. 29. Medium according to one of claims 27 or 28, characterized in that the medium for the search for lysine decarboxylase contains as an enolase inhibitor sodium fluoride at a concentration of 1 to 2 mmol / l. 30. Medium according to claims 27 to 29, characterized in that the medium for the search for lysine decarboxylase has the following composition: 40 mmol of lysine hydrochloride 9 mmol of D-glucose 1 to 2 mmol of sodium fluoride 2.5 g of yeast extract 1 to 2 mmol of sodium fluoride 1 ml of bromocresol purple solution at 16 g / 1 in ethanol or 100 mg of bromothymol blue 10 to 100 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2 distilled water q.s.p. 1000 ml. 1 mm in diameter, and incubate the microplate thus inoculated at 35 ° C. for a period of 3 to 18 h, and finally to read the results. 1. Method for identifying bacteria, characterized in that it consists in suspending in salt water with 9 g / 1 of sodium chloride, or in a gallery broth, a colony or fraction of colony of bacteria to study, then distribute a drop of about 50 ni of this suspension in 100 ni of identification broth previously distributed in the plate wells for microtitra tion, to achieve anaerobic conditions by adding oil of petroleum jelly or aeration by an adhesive paper pierced with a hole 2 mmol of magnesium chloride 2.5 mmol of sodium hyposulfite 2.5 g yeast extract 2.25 g of yeast extract distilled water q.s.p. 1000 ml, or trihydroxymethylaminomethane buffer 50 mmol / hydrochloric acid q.s.p. pH 7.2, q.s.p. 1000 ml or piperazine dichloride buffer 50 mmol / soda q.s.p. pH 6.5, q.s.p. 1000 ml 17. Medium according to claim 15, characterized in that the medium for the search for amino acids oxidases contains a peroxidase having an activating effect, and the composition of which is: 42 mmol of L-tryptophan 6 mmol of L-leucine 0.25 mmol of L-arginine 2.25 g of yeast extract 10 mg of horseradish peroxidase distilled water q.s.p. 1000 ml. 18. Medium according to claim 15, characterized in that the medium for the detection of amino acid oxidases comprises as an oxidation-reduction indicator of dichlorophenolindophenol, as a peptone not giving a non-specific oxidation-reduction reaction the papain peptone from soybean and , as reaction accelerator, a Peroxidase, this medium consisting of: 20 mmol of L-tryptophan 20 mmol of L-methionine 0.25 mmol L-arginine 600 nmol of dichlorophenolindophenol 2.5 g yeast extract 1 to 2 mmol of sodium fluoride 2. Identification method according to claim 1, characterized in that, to obtain the diagnosis of the genus and sometimes of species of enterobacterium, the bacterial colonies obtained on isolation medium are used in relation to reagents chosen from media for the detection of glucose fermentation, melibiose, rhamnose, medium for the production of acetoin, medium for the search for amino acid oxidases and medium for the detection of indole production, medium for research ß-galactosidase and nitrate reductase, media for the detection of urease, lysine decarboxylase, and ornithine decarboxylase, citrate medium, and medium for the detection of the production of hydrofluoric acid. 2 3 mmol of orthonitrophenyl-p-D-galactopyrannoside 50 mmol of sodium nitrate 2.25 g of yeast extract 100 mmol of trihydroxymethylaminomethane / hydrochloric acid pH 7.2 distilled water q.s.p. 1000 ml. 23. Medium according to claim 6, characterized in that the medium for the search for urease combines glucose and urea as substrates. 24. Medium according to claim 23, characterized in that the medium for the detection of urease contains between 26 and 44 mmol / l of urea and between 16 and 20 mmol / l of glucose. 25. Medium according to claim 24, characterized in that the medium for the detection of urease contains as an enolase inhibitor sodium fluoride in an amount between 1 and 2 mmol. 26. Medium according to claim 25, characterized in that the medium for the search for urease consists of: 35 mmol of urea 16 to 20 mmol of glucose 3 636,376 10 g yeast extract 50 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2 20 mmol of creatine, or 10 mmol of arginine, or 20 mmol of creatine and 10 mmol of arginine 20 to 100 mmol of glucose 200 mmol of pyruvate or 100 mmol of lactate distilled water q.s.p. 1000 ml. 15. Medium according to claim 6, characterized in that it consists of a medium for the search for amino acids oxidases containing as oxidants sodium or potassium nitrate, ammonium persulfate or chloramine T, and a buffer trihydroxymethylaminomethane pH 7.2, or a piperazine dichloride / sodium hydroxide buffer pH 6.5 as well as a mixture of L-tryptophan, L-leucine, L-arginine. 16. Medium according to claim 15, characterized in that this medium has the following composition: 42 mmol of L-tryptophan 6 mmol of L-leucine 0.25 mmol of L-arginine 0.25 to 0.75 mmol of sodium or potassium nitrate or 0.25 to 0.50 mmol of ammonium persulfate or 2.5 mmol of chloramine T 3. Identification method according to claim 2, characterized in that the diagnosis is completed by the use of the medium for the research of arginine dihydrolase, of the medium for the study of the use of malonate, and / or other media for sweet fermentations, as well as media for the detection of gelatinase. 4 than produced from peptones or cellular extracts contained in the media. 33. Medium according to one of claims 31 or 32, characterized in that the medium for the search for ornithine decarboxylase contains as an enolase inhibitor sodium fluoride in a concentration of between 1 and 2 mmol / l . 34. Medium according to claims 31 to 33, characterized in that the medium for the search for ornithine decarboxylase consists of: 30 mmol of ornithine hydrochloride 8 mmol of glucose 1.5 g of yeast extract 1 to 2 mmol of sodium fluoride 4. Identification method according to claim 1, characterized in that it implements, with a view to biotyping Cocci Gram (+) into a catalase (+) or catalase (-) cluster, according to the classification of Baird Parker, a colony of bacteria taken from selective media or from blood agar or ordinary agar, in relation to reagents chosen from a medium for the fermentation of glucose and mannitol, media for the aerobic use of lactose, maltose, arabinose and mannitol, a medium for the detection of acetoin without a pH indicator, and mediums for the search for phosphatase and for the detection of coagulase, as well as a modified concentrated Koser solution, and a gallery broth. 5 mmol sodium nitrate 0.5 g pancreatic peptone gelatin 50 mmol glycerol distilled water q.s.p. 1000 ml. 46. Medium according to claim 6, characterized in that the medium for the search for nitrate reductase containing high concentrations of nitrates has the following composition: 0.01} imol of ammonium heptamolybdate 500 mmol sodium nitrate 0.5 g pancreatic peptone gelatin 50 mmol of glycerol distilled water q.s.p. 1000 ml. 47. Medium according to claim 6, characterized in that the medium for studying the use of glucose oxidatively consists of: 50 mmol of D-glucose 5 to 10 g of pancreatic peptone of gelatin 2.5 to 10 g of yeast extract 150 mmol of sodium chloride 50 mmol of calcium chloride distilled water q.s.p. 1000 ml. 39. Medium according to claim 6, characterized in that the medium for checking the production of hydrofluoric acid has the following composition: 15 to 30 g of pancreatic lactalbumin peptone: 1.2 parts; yeast extract: 1 part 18 mmol of L-arginine 5 g papain soy peptone 10 mg horseradish peroxidase distilled water q.s.p. 1000 ml. 19. Medium according to claim 6, characterized in that the medium for researching the production of indole uses a Kowacks reagent, and has the following composition: 30 mmol of L-triptophan 2.5 g of yeast extract distilled water q.s.p. 1000 ml or trihydroxymethylaminomethane buffer 50 mmol / hydrochloric acid q.s.p. pH 7.2, q.s.p. 1000 ml or piperazine dichloride buffer 50 mmol / soda q.s.p. pH 6.5, q.s.p. 1000 ml. 20. Medium according to claim 6, characterized in that the medium for researching the production of indole contains a concentrated and partially purified preparation of an indol-3-hydroxylase obtained from a culture juice of a bacterium <Pseudomonoas indoloxydans), this medium consisting of: 30 mmol of L-tryptophan 2.5 g of yeast extract 25 ml of purified and concentrated culture juice of a bacterium producing in its medium indol-3-hydroxylase distilled water q.s.p. 1000 ml. 21. Medium according to claim 6, characterized in that, in the medium for the search for ß-galactosidase and nitrate reductase allowing the search for nitrites by addition of the second Griess reagent (alphanaphthylamine in acetic solution), l he coupling agent consists of the substrate for β-galactosidase or the orthonitrophenol formed by the bacteria, both contained in the medium. 22. Medium according to claim 6, characterized in that the medium for the search for ß-galactosidase and for nitrate reductase consists of: 5 10 15 20 25 30 35 40 45 50 55 60 65 5. Identification method according to claim 1, characterized in that it implements, for the identification of pseudomono-adaceae (Gram bacilli (-) oxidase (+) or Gram bacilli (-) oxidase and nitrate (-) cultivating on selective lactose media or on ordinary agar) the following media: medium for researching the use of glucose aerobically, medium for researching the use of glucose by fermentation, medium for researching reduction of nitrate at low and high concentration of nitrate, medium for verifying the use of citrate, medium for detecting gelatinase, and medium for researching arginine dihydrolase. 6. Culture medium for the implementation of the identification method according to claim 1, characterized by the presence of enolase inhibitor in those intended for the study of sugar fermentations and in those intended for the demonstration negative reactions of urease, LDC and ODC by the fermentation of glucose, by the association of glucose and pyruvate or lactate in that for the research of acetoin, by the association of an oxidant or peroxidase with a mixture of amino acids in that intended for the research of the amino acid oxidase, by the presence of indole-3-hydroxylase in that for the research of indole, by the association of orthonitrophenyl-P-galactopyrannoside and sodium nitrate in that for the detection of ß-galactosidase and nitrate reductase, by the citric acid / sodium citrate mixture, adjusted to pH 5.9 with acid citric, contained in the medium for the use of citrate, by the mixture of malonic acid as / sodium malonate adjusted to pH 5.9 with malonic acid, contained in the medium for the use of malonate, by the mixture buffered to pH 7.2 with trihydroxymethylaminomethane, pancreatic peptone, lactalbumin yeast extract, aginine, magnesium chloride, sodium hyposulfite and iron and ammonium sulfate in that intended for the research of the production of hydrogen sulfide, by the mixture of pancreatic peptone of gelatin and of calcium chloride in that intended for the search for gelatinase, by the mixture of glucose and urea in that for the research for urease, by the presence of lysine in that for the research for lysine-decarboxylase, by the presence of ornithine in that for the search for ornithine decarboxylase, by the presence of arginine in that for the search for arginine-di-hydrolase, by the presence of p-nitrophenyl phosphate in that for phosphatase research, by mixing nitrates ate and peptone in that for the research of the nitrate-reductase, and by the mixture of glucose, trihydroxymethylaminométhane and hydrochloric acid in that for the study of the use of glucose by oxidative way. 7. Medium according to claim 6, characterized in that it contains, as an enolase inhibitor, sodium fluoride at a dose of between 1 and 2 mmol / 1, preventing pH reversals following blockage of catabolism fixed acids formed from sugar, but not preventing the transformation of the studied sugar. 8. Medium according to claim 7, characterized in that it consists of: 9. Medium according to claim 6, characterized in that it consists of concentrated aqueous solutions of diluted sugars before their use in the basic medium for sweet fermentations, namely: 0.5M solution of glucose, mannitol, mesoinositol, sorbitol, rhamnose, sucrose, arabinose, adonitol, fructose, galactose, maltose, trehalose, xylose, or melibiose , 0.3M lactose solution, 0.25M solution of raflinose, or amygdalin, 0.15M solution of sailicin, or dulcitol. 10. Medium according to claim 6, characterized in that it consists of a medium for the search for acetoin combining two substrates, namely glucose and one of its degradation products, pyruvate or lactate. 11. Medium according to claim 10, characterized in that the medium for the search for acetoin contains bromothymol blue as an indicator of the state of acidity of the media after incubation. 12. Medium according to one of claims 10 or 11, characterized in that the medium for the search for acetoin contains as activating substances for the production of acetoin an extract of yeast, creatine or arginine. 13. Medium according to claims 10 to 12, characterized in that the medium for the search for acetoin consists of: 10 g yeast extract 50 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2 100 mg bromothymol blue 20 mmol of creatine, or 10 mmol of arginine, or 20 mmol of creatine and 10 mmol of arginine 20 to 100 mmol of glucose 200 mmol of pyruvate or 100 mmol of lactate distilled water q.s.p. 1000 ml. 14. Medium according to claims 10 and 12, characterized in that the medium for the search for acetoin has the following composition: 10 to 25 mmol of trihydroxymethylaminomethane / hydrochloric acid q.s.p. pH 7.2 distilled water q.s.p. 1000 ml.