Culture Media

CULTURE MEDIA, artificial media of various compositions intended for the cultivation of microbes and protozoa under laboratory conditions. They were first introduced for the isolation of individual species of bacteria by R. Koch in 1881, which created the foundation for all modern microbiology and contributed to its rapid flourishing. The significance of culture media is enormous: thanks to their use, it is possible not only to isolate a microbe in pure form but also to constantly maintain it in culture in the laboratory, which facilitates its comprehensive study. Every microbe studied to date is capable of living and multiplying under artificial conditions on appropriate culture media. The reaction of the culture media is of great importance. The growth and multiplication of microbes are possible at a certain acidity (resp. alkalinity) of the medium, the limits of which are quite narrow for the majority of microbial species. This reaction of the culture media is determined by colorimetric, electrometric, or titration methods. Litmus and phenolphthalein, which were widely used previously as indicators, are currently being displaced by other, more sensitive indicators associated with the method of determining the concentration of hydrogen ions in the medium. To establish the reaction of a culture medium using litmus, it is tested on litmus paper, blue or red, then 10 cm³ of the medium is measured into a test tube, and if the reaction of the medium according to litmus was acidic, then enough 1/10th normal solution of sodium carbonate is added to the test tube from a burette so that a drop of the liquid applied to red paper causes only a slight turning to blue, i.e., so that the reaction is neutral. The amount of soda solution used for alkalizing 10 cm³ of the medium is calculated for the total amount of the medium and added to the latter; the medium must then be boiled and its reaction checked once more. With some practice, one can establish the reaction of the medium by carefully adding a 10% soda solution or a normal solution of NaOH to it. When using phenolphthalein as an indicator, the culture medium cannot be alkalized with a soda solution, because phenolphthalein is decolorized under the influence of CO₂ released during neutralization (phenolphthalein is used in a 0.5% solution in 50% alcohol). In a weakly alkaline medium (pH=8), phenolphthalein causes a pale pink coloration of the medium; with further alkalization, it turns red; at a more acidic reaction, phenolphthalein is colorless. Titration is performed with a 1/10th normal solution... [The text continues with instructions on filtration using a sterilized funnel, clarification with egg white, sterilization methods such as autoclaving at 120°C for 20-30 minutes for broth and agar, fractional sterilization for gelatin and carbohydrate media, and the storage of media. It further details the history of dehydrated media, starting with Doerr (1909), and lists various commercial preparations like those from Merck, as well as specific recipes for broth, agar, and gelatin powders. Finally, it outlines the preparation of general protein-based media, including meat infusion and peptone solutions, and the use of Hottinger's solution.]

Preparation method: 1 kg of coarsely chopped meat is placed in 1.5 liters of boiling water, brought to a boil again, the meat is removed and passed through a meat grinder. After cooling, the meat broth is poured into a two-liter bottle, 1.5 g of soda (Natr. carbon.), 3-5 g of pancreatin (Pancreatinum siccum), and 15-20 g of chloroform are added (pancreatin can be replaced with glycerinated pancreas). The contents of the bottle are shaken well, and the minced meat is added to it. The bottle is closed with a stopper and shaken well, holding the stopper so that the chloroform vapors do not push it out. It is left at room temperature for 5 days for digestion (at 37° for 2 days), until the contents of the bottle turn into a transparent, saturated yellow liquid with a fine sediment at the bottom; filtration, dispensing, sterilization at 120° for 20 minutes. 1. Meat media. 4) Meat-peptone agar, see Agar-agar. Agar media are used for growing bacteria on a surface and by stab culture (see Microorganisms, cultivation of microbes) in the depth of the agar (anaerobes). For the first purpose, test tubes with freshly poured agar are placed at an acute angle to the surface for the agar to solidify in an inclined position ("slant" agar). For stab culture, so-called "straight" agar is used, which has solidified in the straight position of the test tube as a column. 5) Glycerin agar, 6) Glycerin broth, see below for special media for tubercle bacilli, No. 1 and No. 2. 7) Meat-peptone broth (see Broth): 1 liter of meat water, 10 g of peptone, 5 g of NaCl. 8) Buffered broth: instead of 5 g of NaCl per 1 liter of meat water, 2-3 g of commercial sodium phosphate is added. 9) Liebig broth (from Liebig meat extract): 10 g of meat extract, 10 g of peptone, 5 g of NaCl, 1,000 cm³ of distilled water. The media are usually somewhat darker than those prepared with meat water and are somewhat worse for bacterial growth. Some extracts may contain resistant spores and a peptonizing enzyme, which interferes with the coagulation of gelatin prepared on such an extract. 10) Maggi nutrient broth: 1,000 cm³ of distilled water, 10 g of Maggi meat powder, 10 g of peptone, 3 g of NaCl. Boiled for 1 hour in a Koch apparatus. The rest is as usual. 11) Martin's broth, which was previously used almost exclusively for the preparation of diphtheria toxin, is now also used for the cultivation of a whole range of microbes. Preparation method: a) Peptone solution: from washed with tap water and finely chopped pig stomachs (not less than 5), the following mixture is prepared for self-digestion: for 200 g of slurry, 10 g of pure HCl and 1,000 cm³ of water at 50°. It is left in a water bath (or in a thermostat) at 50° for 12-24 hours (at this temperature, pepsin digests tissues better, converting them into peptone). Bacteria do not develop in an acidic environment. Then, heating in an autoclave for 1/2 hour at 100° (to destroy the pepsin), filtration through a sieve covered with absorbent cotton or through cloth. Before mixing with meat water, the peptone solution is heated to 80° and neutralized by adding 10% sodium hydroxide until red litmus paper turns slightly blue (about 30 cm³ per 1 liter). b) Meat water: 500 g of chopped fat-free meat (veal) in 1 liter of water is infused for 20-24 hours (at 37°). Then the temperature is brought to 50°, the liquid is passed through cloth, squeezed out, and 5 g of NaCl per 1 liter is added. Mix 500 g of peptone solution with 500 g of meat water; heat to 70° (in clay or porcelain ware) in flowing steam; after heating, pass through a towel, adjust to a neutral reaction by adding 7 cm³ of a normal NaOH solution per 1 liter of liquid. Sterilization in an autoclave for 20 minutes at 120°, filtration through paper, dispensing, sterilization at 100° for 3 days for 20 minutes each. 12) Hottinger broth: the basic Hottinger solution is diluted with distilled water 3.5-10 times (depending on the type of microbe), 0.7% NaCl and 0.1% K₂HPO₄ are added; boil, adjust the reaction, boil again, filter through a paper filter, check the reaction, dispense into test tubes, and sterilize in an autoclave (120° - 20 minutes). 13) Nutrient gelatin, see Gelatin. 14) Peptone water: 1% peptone solution (2% for anaerobes) or a basic peptone solution diluted 1:10 with distilled water. 15) Peptone water with carbohydrates (Hiss medium) is used to study the fermentation of carbohydrates by bacteria. Composition: 1% peptone solution + 0.4-1% of one or another carbohydrate [arabinose, xylose, rhamnose (pentose group); d-glucose, levulose, galactose (hexose group); sucrose, lactose, maltose, raffinose (disaccharides); inulin, dextrin (polysaccharides); glycerin, erythritol, adonitol, mannitol, dulcitol, sorbitol, inositol (polyhydric alcohols)] and litmus tincture until the medium is clearly blue. Fractional sterilization in flowing steam for 3 days in a row for 15 minutes each. 2. Media with body fluids. 16) Ascites agar is prepared like serum agar (see below). 17) Ascites broth (ascitic broth): sterilely collected ascitic fluid is added to sterile slightly alkaline broth dispensed into test tubes in a ratio of 1:2. Control for sterility (the liquid can be pre-sterilized by heating in a water bath at 56° for 3 hours or filtered through a candle). 18) Blood agar, see Blood-blood as a nutrient medium. 19) Thalmann's blood agar: to 5-10 cm³ of slightly alkaline agar, melted and cooled to 45°, 5 drops of human blood are added; mix by gentle shaking. 20) Broth with blood: to the finished broth, sterile defibrinated blood (sheep, rabbit, etc.) is added in an amount of 5-10%. Control for sterility. 21) Coagulated blood serum: sterilely collected blood of a horse, cattle, human, or rabbit is allowed to settle, the serum is sterilely aspirated from the clot, dispensed into test tubes, and coagulated in an inclined position in a special Koch apparatus at 75-80°. Check for sterility in a thermostat at 37° for 24-48 hours. Human serum remaining after the Wassermann reaction and removed sterilely from the clots can be used; pre-heat for 1-2 hours at 58° in a water bath. 22) Serum broth (serum broth) is widely used for the cultivation of streptococci, pneumococci, etc. 23) Serum agar (serum agar) is prepared by adding, while observing all rules of asepsis, 1/3-1/4 volume of sterile serum to finished slightly alkaline melted and cooled to 45° sterile agar, dispensed into test tubes; slant; control for sterility. 24) Milk, 25) litmus milk, see Milk-milk as a nutrient medium. 26) Bile, see Bile-bile as a nutrient medium. Sterilely collected hydrocele, pleural, etc., fluids can also be used as protein fluids. 3. Egg media, see Eggs-eggs as a nutrient medium, and below-special media for tubercle bacilli. B. Media of plant origin. 1. Potato media, see Potato. 2. Yeast media are used for the cultivation of fungi and demanding bacteria (instead of serum broth); recently they have been successfully used by some institutes of the USSR for the preparation of vaccines (intestinal-typhoid group) as a substitute for meat media, yielding a good output of microbes. 27) Broth with yeast extract (instead of serum broth for demanding bacteria): 100 g of brewer's yeast is stirred in 400 cm³ of distilled water, acidified to pH 4.5, and boiled on a fire through a wire gauze with constant stirring (10 minutes). After centrifugation, the transparent liquid is poured into small flasks and sterilized in flowing steam or in a water bath for no more than 10 minutes. This extract is added to meat broth in an amount of 5-10%. 28) Yeast agar for fungi: brewer's yeast is sterilized for 1 hour in a Koch apparatus and allowed to settle; 2% agar is added to the clear filtrate, it is boiled for 1 hour in a Koch apparatus, poured into flasks and test tubes, and sterilized in an autoclave in the usual way. 29) Regenerated yeast agar for vaccine: 30 liters of liquid brewer's yeast, 60 liters of washed regenerated agar, 150 g of NaCl, 600 g of dry agar, boiled in an autoclave for 30 minutes at 1 atmosphere. 30) Tragacanth medium was developed in 1930 by laboratory assistant Zamkovaya (Moscow) as a partial substitute for the scarce agar-agar: 20 g of tragacanth + 500 cm³ of water is left for a day to swell, then filtered through gauze. For 1 liter of swollen tragacanth, 10 g of dry agar is taken, the rest is like ordinary meat-peptone agar. 31) Soy media-see Soy. C. Protein-free media (synthetic) were introduced into bacteriological technique back in the time of Pasteur, who noted that yeast and bacteria can use ammonia nitrogen. Cohn (F. Cohn) provided a further modification, the so-called

a "normal nutrient solution for bacteria" of the following composition: 0.1 g potassium phosphate, 0.1 g crystalline magnesium sulfate, 0.1 g tribasic calcium phosphate, 20 cm³ distilled water, and 0.2 g ammonium tartrate (the source of nitrogen is ammonium, and of carbon is tartaric acid). At the present time, there exists a number of synthetic media suitable for the cultivation of bacteria (see table).

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CULTURE MEDIA

Asparagine and ammonium aspartate are the main sources of nitrogen in them. Sterilization is performed with flowing steam in test tubes (3 days in a row for 15-25 minutes). Synthetic media are especially readily used for the cultivation of tubercle bacilli (see below). Galimard and Lacomme add urea or other amines (arginine, leucine, tyrosine, glycocoll) to a solution containing 0.5% NaCl, 0.05% magnesium sulfate, 0.2% calcium glycerophosphate, 1.5% glycerin, and potassium carbonate to a slightly alkaline reaction. Solid protein-free media were introduced into practice by Winogradsky and Sullivan. Raulin's protein-free medium: 1,500 cm³ water, 70 g cane sugar, 4 g tartaric acid, 0.4 g ammonium nitrate, 0.6 g ammonium phosphate, 0.6 g potassium carbonate, 0.4 g magnesium carbonate, 0.25 g ammonium sulfate, 0.07 g iron sulfate, 0.07 g zinc sulfate, 0.07 g potassium silicate, 0.07 g manganese carbonate.

Special culture media, or media for specific purposes, are used for the isolation and cultivation of a specific type of bacteria (though other bacteria may also grow well on them). Media on which only or almost exclusively a specific type of microbe develops are called elective. Many special media are used as differential-diagnostic media. I. Special media for the cultivation and differentiation of tubercle bacilli. 1) Glycerin agar: standard preparation of meat-peptone agar with 4-5% glycerin. 2) Glycerin broth: standard preparation of meat-peptone broth (1% peptone and 0.5% NaCl) with 3-5% pure glycerin; pH = 6.8-7.2 (used for mass production of tuberculin by inoculating a piece of the pellicle of a tubercle culture onto the surface). 3) Marmorek's glycerin broth: 0.5 kg of calf spleen is infused in 1 liter of water for 24 hours, boiled for 10 minutes over an open flame, filtered, 1% Chapoteaut peptone and 0.5% NaCl (or monobasic potassium phosphate) are added, filtered, a slightly alkaline reaction is established with a 10% soda solution, boiled for 25 minutes in an autoclave, filtered, 2-3% glycerin is added, poured into flasks, and sterilized in an autoclave at 115°. 4) Jochmann's glycerin broth: 5% of Heiden nutrient medium is added to 3% glycerin broth, sterilized in flowing steam. 5) Anzilotti's glycerin potato: potato wedges are boiled in a 6% glycerin solution (alkalized with a 1% sodium carbonate solution) for about 2 minutes. If the reaction changes during this process, the liquid is re-alkalized. The wedges are placed in Roux tubes, into the expansion of which, up to the constriction, 6% glycerin broth or 5-6% glycerin water is poured. Sterilization in an autoclave for 20 minutes at 120°. 6) Pavlovsky's glycerin potato (for preparation method, see Potato); no special alkalization is required. 7) Potato with bile (according to Calmette and Guérin) is prepared like glycerin potato. Potato slices are placed in sterile bovine bile with 5% glycerin and heated in a water bath at 75° for 3 hours, then transferred to Roux tubes, into which 5% glycerinized sterile bile is poured up to the constriction. Sterilization for 30 minutes at 120°. On this medium, the growth of tubercle bacilli (typ. bovin.) is abundant and moist. 8) Esmarch's potato: round slices of potato (peeled and washed), 1 cm thick and 4-5 cm in diameter, are placed in double glass dishes. Sterilization in flowing steam. 9) Waudremer's potato broth: 500.0 g of peeled potato is boiled in 1 liter of water, filtered. A neutral reaction is established (with soda) using litmus. Sterilization for 1/2 hour at 120°; kept for 24 hours in an incubator, decanted, and sterilized again (used mainly for studying non-acid-fast strains of tubercle bacilli). 10) Tsekhnovitser's potato broth: same as the previous one, with the addition of 4-5% glycerin. 11) Yurevich's potato broth: grated potato is covered with a double amount of water, left for a day in the cold, shaken, and squeezed through cloth. It is allowed to settle for half an hour and decanted; an equal amount of meat water, 1/2% peptone, and 1/2% NaCl are added to the decanted liquid, boiled for 1 hour, and filtered. 3% glycerin is added, an alkaline reaction is established, it is sterilized (1/4-1/2 hour) in an autoclave, filtered, and sterilized again. 12) Kumbari medium and 13) Despeignes medium - see Tubercle bacilli. 14) Ficker's brain media: an equal volume of distilled water is added to finely ground fresh brain, boiled with constant stirring for 1/4 hour, passed through cloth, squeezed firmly, and sterilized for 2 hours under pressure. When serum or 3% glycerin is added to the brain mash (coagulation at the appropriate temperature in tubes), a solid serum-brain medium is obtained; when an equal part of 2.5% agar and 3% glycerin is added to the brain mash, a brain agar is obtained (the brain mash should be acidic; do not neutralize). 15) Ivanov and Sweany's cream medium for differentiating typ. hum. and bovin. bacilli - see Tubercle bacilli. 16) Löwenstein medium with Congo red for isolating tubercle bacilli from blood - see Tubercle bacilli. 17) Petragnani medium: potato-milk-egg medium with the addition of Malachite green; good results for the isolation of tubercle bacilli (difference in colonies of different types of tubercle bacilli). 18) Besredka's egg medium: a liquid medium consisting of eggs and water, alkalized with a 1% sodium carbonate solution (see Tubercle bacilli). 19) Dorset's egg medium (for egg preparation, see Eggs, eggs as a culture medium): the contents of the eggs are blown entirely into a jar (with beads), sterile water is added in an amount of 10% relative to the weight of the eggs, and it is shaken until a completely homogeneous emulsion is obtained (carefully, so that foam does not form); it is filtered through a gauze funnel (aseptically) and poured into tubes. Coagulation at 70° for 2 hours, in a slanted position; sterility control in an incubator (3 days); store under rubber caps. The medium is especially suitable for isolating bovine-type tubercle bacilli; the human type grows worse on it. 20) Lubenau's egg medium: same preparation method (as Dorset medium), only yolks are taken, and instead of water, an equal volume of 5% glycerin broth with a neutral reaction is added (for 100 cm³ of broth - 5-6 eggs); the medium is poured into tubes. Coagulation in a slanted position at 85-90° (place a dish with water in the apparatus); the human-type tubercle bacillus grows well. 21) Petroff's egg medium (all preparation is sterile): 250 g of fresh veal or beef, passed through a meat grinder, is covered with an equal amount of 5% glycerin water, infused overnight in an icebox, and filtered through sterile gauze; mix 2 volumes of the egg white and yolk mixture thoroughly, filter through sterile gauze, mix with 1 volume (per 400 cm³ - 200 cm³) of meat water, and add 1 cm³ of a 1% alcoholic solution (95%) of gentian violet per 100 cm³ of this medium. Mix well and pour into tubes. Coagulation in a slanted position; first day at 85° for 30 minutes; second day at 75° for 30 minutes; third day at 75° for 30 minutes. The lilac color of the medium advantageously highlights the growing colonies; it is used for isolating tubercle bacilli from tuberculous material. For subcultures, the yellow Petroff medium (of the same composition, without dye) is used. 22) Hohn's egg medium: fresh eggs (cleaned as in other methods) are released entirely into a jar with beads, shaken (without foam), and poured into a graduated cylinder; 1/3 volume of 5% glycerin broth (pH 6.2-6.4) is added, the mixture is shaken, filtered through gauze, and poured into sterile tubes. Coagulation in a slanted position, heating first over a high flame to 80°, then bringing it to 87° over a low flame and holding it at this temperature for 30 minutes. After cooling, add 0.8 cm³ of sterile acidic broth without glycerin (pH 6.2-6.4) to each tube, put on caps (seal with paraffin); check in an incubator for 48 hours. Store in an icebox. The medium is widely used for isolating tubercle bacilli from material. 23) Hohn's hematin medium - a modification of the previous medium: 1/3 acidic 5% glycerin broth and 3% Hb solution are added to the eggs. Coagulation as for Hohn's medium. Typ. bovin. bacilli grow well. 24) Coagulated blood serum according to R. Koch, 25) coagulated serum with 3% glycerin according to Nocard and Roux, preparation method - see General media, No. 21. Protein-free media for cultivating tubercle bacilli are used with success. 26) Sauton's medium (see Bacteria - Calmette-Guérin bacillus) gives a good yield of tubercle bacilli, greater than on glycerin broth; it is specifically used by Calmette for cultivating BCG. 27) Long's medium, 28) Model's medium (see).

Tubercle bacilli) are used for the preparation of protein-free tuberculin. 29) Löwenstein medium: 6 g of ammonium phosphate, 40 g of glycerol, 1,000 cm³ of water. 30) Massol and Breton medium: 1 l of distilled water, 1 g of sodium carbonate, 0.04 g of iron sulfate, 0.05 g of magnesium sulfate, 1 g of potassium phosphate, 8.5 g of NaCl, 10 g of glucose, 2 g of asparagine. 31) Lockemann medium, see General media table. 32) Heyden-Hesse medium: 5 g of Heyden nutrose is dissolved in 50 cm³ of water, then 5 g of NaCl, 30 cm³ of glycerol, 10-20 g of agar, 5 cm³ of 0.1 N soda solution and 950 cm³ of water are added. Boil for 15 minutes, filter, sterilize at 115°, and pour into Petri dishes. II. Special media for diphtheria bacilli: 1) Meat-peptone broth, pH = 7.3-7.6. 2) Löffler serum: classic medium for diphtheria bacilli (elective): 3 parts serum (calf or sheep, horse is also acceptable) + 1 part neutralized broth with 1% glucose; coagulation of the medium at 90-95°; sterilization for 3 consecutive days at 80° for 1 hour. 3) Coagulated blood serum (horse, human, ox) - see above General media, No. 21. On both media, diphtheria bacilli grow rapidly (8-12 hours at 37°), whereas the growth of saprophytes lags significantly behind. 4) Drigalski and Bierast medium: to 600 cm³ of bovine serum, 174 cm³ of broth with 1% glucose and 26 cm³ of pure bovine bile are added, filtered, poured into Petri dishes and coagulated at 90-95°, then sterilized for three consecutive days for 1 hour at 80°. Rapid growth of diphtheria bacilli upon the first inoculation. 5) de Costa, Troisier, and Dauvergne medium: 100 cm³ of horse serum, 10 cm³ of sterilized 30% glucose solution, 30 drops of concentrated sterile litmus tincture, 3 cm³ of sterile sulfuric acid solution (10 g per 1000); coagulated in Petri dishes with a slow increase in temperature to 80° (1 hr. 15 min.). On this medium, colonies of diphtheria bacilli are red, those of diphtheroids are grayish-white. 6) Rothe medium; based on the same principle: 90 parts of serum broth (4 parts ox serum + 1 part neutralized broth) are mixed with 10 parts of litmus tincture containing 10% sugar (glucose or levulose), coagulated as usual in Petri dishes. 7) Thiel medium: 1.0 g of peptone, 1 g of nutrose, 1 g of glucose, 0.5 g of NaCl, 5 cm³ of litmus tincture, 100 cm³ of water; when diphtheria bacilli grow on the medium, fermentation of glucose occurs (unlike with pseudodiphtheria bacilli), resulting in reddening and coagulation of the medium. Tellurium media are based on the ability of diphtheria bacilli to reduce salts of telluric acid, as a result of which colonies of diphtheria bacilli are colored black and gray-black. 8) Bifalco tellurium medium: a) egg white is diluted in 3 parts of water with the addition of 0.5 cm³ of NaOH solution (1:10) and sterilized for 15 minutes at 115°; if a liquid medium is desired, it is sterilized at 50-55° in a water bath for 3 consecutive days for 30 minutes; b) Mix 75 cm³ of the egg white solution, 25 cm³ of yolk, and 2 cm³ of 1% sterile solution of Kal. tellurosi. Heat for 2 consecutive days for 30 minutes at 50-55°, and on the third day, coagulate. 9) Conradi and Troch tellurium agar: 1% sugar (glucose) broth [10 g of meat extract, 5 g of NaCl, 20 g of Witte peptone, and 6 g of Calc. bimalici (dimalate)] is mixed with fresh ox serum in a ratio of 1:3; to 100 cm³ of the mixture, 2 cm³ of 1% sodium tellurite solution is added; it is poured into Petri dishes and coagulated at 85°. Colonies of diphtheria bacilli are black due to the reduction of tellurium. 10) Pergola tellurium medium: 50 cm³ of serum, 50 cm³ of 0.8% NaCl solution, 2 cm³ of 1% potassium tellurite solution, 1 yolk; coagulation in Petri dishes at 85-90°. 11) Mandelbaum and Heinemann glycerol blood agar with 5% glycerol; the surface is smeared with sterile human blood; hemolysis - diphtheria bacilli. 12) Engering medium with sodium oleate for the differentiation of diphtheria bacilli; the latter do not grow on this medium, while diphtheroids show good growth. To 20 cm³ of molten agar, 0.4 cm³ of 10% sodium oleate solution is added with vigorous shaking and poured into Petri dishes. 13) Media for obtaining diphtheria toxin: a) meat cleaned of fat and tendons is passed through a meat grinder; 1 kg is covered with 2 l of water, yeast is added, and the mixture is placed in a warm place overnight. The meat sugar ferments and decomposes. The infusion is filtered through cloth, 2% Witte or Chapoteaut peptone and 5.0 g of NaCl or an equal part of peptone solution prepared by the Martin method from pig stomachs is added to it. The medium is sterilized for 45 minutes at 120°, filtered, neutralized with litmus (pH=8.2-8.4); in addition, 7 cm³ of normal soda solution is added per 1 l, sterilized for 20 minutes at 120°; filtration, pouring into flat flasks in a thin layer (3 cm). b) 1 l of veal meat water, 20 g of peptone, 3 g of NaCl, 2 g of Natr. phosph., several pieces of marble or one spoonful of chalk, 1 g of cane sugar (glucose). III. Special media for the enteric-typhoid group have a common basic principle: the ability of Bacterium coli to decompose certain carbohydrates, especially lactose, in contrast to the typhoid bacillus and other pathogenic microbes of this group, which is indicated by a change in the color of the medium containing one or another color indicator (color media); in addition, various substances are used that act to inhibit accompanying bacteria, mainly Bact. coli. The pH limits for the typhoid bacillus = 6.2-7.6; opt. pH = 6.8-7.2. 1) Conradi-Drigalski medium - see Drigalski-Conradi medium, a) Modification: 100 cm³ of meat-peptone agar (2-3%), 1.5 g of milk sugar, 5 cm³ of litmus tincture, 1 cm³ of 1‰ solution of Krystallviolett B. Höchst; b) To 2 l of meat water, 20 g of Witte peptone, 10 g of NaCl, 20 g of nutrose, 60 g of agar, 300 cm³ of litmus tincture, 30 g of lactose, 20 cm³ of 1‰ aqueous solution of Krystallviolett. The typhoid bacillus, paratyphoid bacilli, and dysentery bacilli produce colorless or bluish colonies, while the colon bacillus produces red ones. 2) Endo medium: 1,000 cm³ of meat-peptone (3%) agar, pH 7.5; 15.0 g of milk sugar (chemically pure), 5 cm³ of saturated alcoholic fuchsin solution, 25 cm³ of 10% aqueous solution of Natr. sulfurosi (sodium sulfite). The medium must be kept in the dark. Colonies of typhoid bacillus, paratyphoid, and dysentery are colorless after 24 hours, pink after 48 hours; colonies of Bact. coli are pink in the center after 15 hours, red after 24 hours. Endo medium, along with the previous ones, is the most commonly used for the typhoid-enteric group. 3) Gaehtgens caffeine-fuchsin agar - Endo agar with the addition of 0.33% chemically pure caffeine. The growth of the colon bacillus is inhibited, that of the typhoid bacillus is not. 4) Lentz and Tietz malachite green agar (for typhoid and paratyphoid); inhibits the growth of the colon bacillus and other alkali producers; typhoid and paratyphoid bacilli grow on the 1st day in the form of small colonies, later (2-3 days) - good growth. Neutral 2-3% agar with 2% peptone (pH 7-7.2) + aqueous solution (1:60) of Malachitgrün (Höchst) (1 cm³ per 100 cm³). The amount of dye is determined empirically for the given agar. 5) Padlewski agar: to 3% meat-peptone agar (2% peptone) of weakly alkaline reaction, 1% chemically pure milk sugar and 3% bovine bile (boiled and filtered) are added. Sterilization at 100° for 2 days for 30 minutes. Separate solutions: a) 1% aqueous solution of crystalline chemically pure malachite green (cryst. chem. pur. Höchst); b) 10% aqueous solution of Natr. sulfurosi. To 100 cm³ of agar (molten and cooled to 60-70°), 0.5 cm³ of solution (a), 0.75-1.0 cm³ of solution (b), and 0.5 cm³ of sterilized bile are added.

Bile favors the growth (enrichment) of the typhoid bacillus; the latter, paratyphoid and dysentery bacilli, produce first colorless, then golden colonies, Bact. coli produce green colonies (decomposition of milk sugar with the formation of acid). 6) Bitter's China-blau-Malachitgrun-Agar contains 1% Witte peptone, 0.5% NaCl, 2% milk sugar, 12% China-blau Hochst solution (8 drops per 100 cm³), and malachite green, crystalline, chemically pure (2.5 cm³ of a 0.1% solution per 100). Typhoid and paratyphoid produce colorless transparent colonies, Bact. coli blue, Proteus and Pyocyaneus also colorless, but less transparent. 7) Liebermann and Acel's Congo red agar: to 100 cm³ of 3% weakly alkaline meat-peptone agar (1% peptone, 1% nutrose, 0.5% NaCl) add 1.5 g of chemically pure lactose and 30 cm³ of a 1% aqueous solution of Congo red. Colonies of typhoid, paratyphoid, dysentery, and cholera bacilli are red, Bact. coli are black. 8) Schmitz's medium—serum Congo red agar, a medium of the same composition as the previous one; serum agar is prepared from serum and a decoction of blood clots (from a slaughterhouse). Caffeine can be added, which inhibits the growth of Bact. coli. On this medium, colonies of typhoid, paratyphoid, and dysentery (Shiga and Flexner) are dry, flat, dark red; Bact. coli are dark blue on a red background. A very good medium. 9) Leffler-Schuster's medium: Malachitgrun-Reinblau-Safranin-Agar: to 100 cm³ of agar add 3 cm³ of bovine bile, 1 cm³ of a 0.2% aqueous solution of safranin (rein; Grubler), 3 cm³ of a 1% aqueous solution of Reinblau doppelt konzentr. (Hochst), 3-4 cm³ of a 0.2% aqueous solution of malachite green (Malachitgrun-Chlorzink-Doppelsalz; Hochst). The medium is lilac-blue; typhoid bacilli form blue colonies, thin, irregular, with a metallic sheen; paratyphoid B the same; paratyphoid A forms round, transparent colonies, bluish, without a metallic sheen; the Gaertner group and coli form pink and red colonies. 10) Gassner's medium with Wasserblau and Metachromgelb [to 200 cm³ of agar add 17.5 cm³ of a 10% Wasserblau solution (with 10 g of lactose) and 12.5 cm³ of a 2% aqueous solution of Metachromgelb]. The medium inhibits the growth of spore-forming bacilli and cocci; the colon bacillus forms dark blue colonies, typhoid and dysentery bacilli form yellow ones. 11) Muller's medium: an elective medium for typhoid bacilli, contains iron-potassium tartrate, phenol, lactose, potassium ferrocyanide, and soda. Typhoid bacilli form orange colonies on a brown background of the medium, Bact. coli blue; the medium gives more indicative results than Endo and Conradi-Drigalski media. 12) Brilliant green agar (Brilliantgrun-agar): 1 liter of neutral meat-peptone agar, 8 cm³ of brilliant green solution 1:100, 13.2 cm³ of picric acid solution. Fresh medium inhibits the growth of Bact. coli. 13) Ficker's caffeine broth (for the accumulation of typhoid bacilli): 100 cm³ of meat broth (3% peptone) + 0.6 g of pure caffeine, 0.7 cm³ of crystal violet solution (1:1000). The following media are differential for the typh.-paratyph.-coli-dysenter. group, i.e., they serve mainly or exclusively for this purpose. 14) Petruschky's litmus milk whey (Lackmusmolke Petruschky) (see Milk, milk as a culture medium)—one of the basic media for the differentiation of typhoid, paratyphoid, coli, and dysentery bacilli. 15) Seitz's artificial litmus milk whey—a valuable substitute for Petruschky's whey: 20 g of chemically pure lactose, 0.4 g of glucose, 0.5 g of dibasic sodium phosphate, 1 g of ammonium phosphate, 2 g of sodium citrate (tribasic), 5 g of NaCl, 0.05 g of dry Witte peptone, 0.25 g of azolitmin (Kahlbaum), 1000 cm³ of distilled water. 16) Litmus milk—see Milk, milk as a culture medium. 17) Barzikov's medium I: dissolve 1% nutrose, 1% glucose, 0.5% NaCl in water and add 10% litmus tincture (Kahlbaum). Bact. coli and paratyphoid produce reddening, coagulation, and gas formation; Bact. typh. and dysenter. produce reddening and turbidity; Bac. faecalis alcaligenes produces blue coloration. To better capture gas, a float is lowered into the test tubes. 18) Barzikov's medium II—of the same composition, but with lactose instead of glucose; Bact. coli produces the same change as in I; typh., paratyph., and dysentery bacilli do not change the medium. 19) Derr's mannitol-nutrose-litmus solution for the differentiation of dysentery group bacilli. 20) Hetsch's maltose-nutrose-litmus solution, the same. 21) Hiss's media with various carbohydrates and litmus tincture (or another indicator)—see General media. 22) Russell's double sugar agar: 100 g of nutrient agar, 0.1 g of glucose, 1.0 g of lactose, litmus tincture; pH 7.3-7.4; for the differentiation of bacteria of the group: a) Bact. coli decomposes both sugars with acid and gas; b) the paratyphoid and enteritis group decomposes glucose with acid and gas; does not decompose lactose; c) typhoid and dysentery decompose glucose without gas, do not decompose lactose; d) the alkali-forming group does not decompose either lactose or glucose; the color does not change. 23) Besson, Ranque, and Senez's neutral red broth: 1 liter of meat water of weakly alkaline reaction, 4.0 g of glucose, and 3 cm³ of a 1% aqueous solution of neutral red; the medium is poured into test tubes with floats (Durham, Asch). 24) Omeliansky's medium: ordinary broth + 1/5% sodium formate (check the reaction) and 1/2% saturated aqueous solution of neutral red. Poured into test tubes with floats. Sterilization in flowing steam. For the change in the medium under the influence of the growth of bacteria of the group, see Rothberger's medium. 25) Rothberger's medium—agar with grape sugar and neutral red: meat-peptone agar (0.75%) + 0.3% grape sugar and 1% aqueous saturated (cold-filtered) neutral red; Bact. coli produces green fluorescence, gas, and decolorization of the medium (yellow); typhoid and dysentery produce a crimson coloration of the medium; paratyphoid A produces acid and a little gas, the medium is crimson, slight fluorescence; paratyphoid B produces fluorescence, gas, the medium is orange; alkali-formers do not change the medium. 26) Stern's glycerin-fuchsin broth for differentiation in the paratyphoid B group (Bac. Schottmulleri and Bac. enteritidis Gaertneri—reddening after 3 days as an exception; B. Breslau—reddening; Bact. suipestifer—does not change). 27) Bitter-Weigmann and Habs's serum with rhamnose for the differentiation of bacteria of the paratyphoid group; indicator—methyl red; B. Breslau—medium is red (cleaves more acids from rhamnose); B. Schottmulleri—medium is yellow; the same for B. enteritidis Gaertneri and B. suipestifer. 28) Rhamnose-Endo-agar for the same purpose (rhamnose, syn. isodulcitol). 29) Drigalski's litmus milk agar for the differentiation of typhoid, paratyphoid, dysentery, Bac. faecalis alcaligenes—blue growth, coli—red. IV. Media for cholera vibrios (opt. pH 7.0-7.4): 1) 1% peptone water. 2) Dieudonne's blood agar: 30 parts alkaline blood, 70 parts agar; contains a large amount of alkaline albuminates; as a result, favorable conditions are created for the growth of cholera vibrios and the inhibition of the multiplication of other intestinal bacteria. Bac. pyocyaneus and some cocci develop well; they are easily distinguished by the appearance of the colonies. 3) Moldovan's medium: the same components, but in a ratio of 1:4 (10+40). 4) Pilon's agar: 30 parts alkaline blood (soda) and 70 parts neutral agar. 5) Kabeshima's medium: agar with Pfeiffer's hemoglobin extract and sodium carbonate. 6) Esch's hemoglobin agar: 15 cm³ of normal potassium hydroxide solution, 15 cm³ of distilled water, 5 g of hemoglobin, 170 cm³ of neutral 3% agar. 7) Krumwiede's alkaline medium: eggs, crystalline sodium carbonate, meat-peptone agar. 8) Teruuchi and Hida's medium: a) casein-trypsin-peptone 4-5% water, an excellent medium for cholera vibrios; b) agar with 4-5% casein-trypsin-peptone, alkaline reaction; only cholera vibrios grow. 9) Aronson's medium (H. Aronson): with fuchsin and cane sugar, strongly alkaline reaction (Natr. carbon.), as a result of which other bacteria (coli) almost do not grow; cane sugar favors the growth of cholera vibrios. 10) Hesse's medium with malachite green and cane sugar. 11) Kodama and Takeda's medium: peptone water with starch (potato); proposed for the differentiation of vibrios, gives insufficiently clear results. 12) Ottolenghi's bile medium: to 100 cm³ of fresh bovine bile (filtered through paper) add 3 cm³ of the following solution: 10 g of sodium carbonate, 0.1 g of potassium nitrate (saltpeter), 100 cm³ of distilled water. Sterilization in an autoclave for 20 min at 115°.

Enrichment method for the first inoculation, very good results. 13) Kemal-Moukthar mineral medium: 0.8 g sodium phosphate, 0.4 g asparagine, 0.6 g ammonium lactate, 0.5 g NaCl, 100 cm³ distilled water; upon the first inoculation from faeces, the cholera vibrio quickly (5-6 hours) produces an almost pure culture; other intestinal microbes lag significantly in growth. 14) Medium for indole by Bujwid—see Cholera. V. Culture media for Pfeiffer's bacilli. Pfeiffer's bacilli grow best on media with blood and on vitamin media; pH range 6.2-7.6; opt. 7.0; opt. t° 37°. Growth after 18-24 hours in the form of small colonies, "dew drops"; on Leventhal's medium, the colonies are slightly turbid. 1) Agar with 5% defibrinated blood in Petri dishes (2 days to "mature"). 2) Leventhal's agar (with cooked blood): 2% meat-peptone agar, pH 7.3-7.5, melted and cooled to 60°, mixed in flasks (slowly) with 5% blood (sterile defibrinated human, horse, etc.). The medium is boiled on a burner with an asbestos mat (or in a Koch apparatus) for no more than 5 minutes at 1 L and 8-10 minutes at 2 L. The color of the agar darkens. When it begins to boil, remove and repeat the procedure once more (do not overheat). Aseptically filter at 60° through cotton wool or aspirate the transparent agar with a pipette after settling at 60°. Pour into test tubes as a column. Before use, slant (immerse in boiling water for 1-2 min.). Stored for 2-3 weeks. 3) Pfeiffer's blood agar—a little fresh, sterile blood is dropped or smeared over the surface of ready-made slanted agar (1-1.5%; pH 7.3-7.5) (control in a thermostat for 24 hours). 4) Legroux's vitamin agar: ordinary agar with 5-10% blood extract (blood diluted 4 times with physiological saline, heated for 15 minutes at 80°, sterilely filtered, and passed through a candle). 5) Chocolate agar according to Voges: add 15% horse or rabbit blood to boiling agar, mix, and immediately remove from the heat. When the agar cools to 50-60°, thoroughly stir the dark sediment, pour into dishes or test tubes (slant). Avery's medium: agar with sodium oleate (inhibits the growth of streptococci and pneumococci): a) vitamin 2% agar (pH 7.0-7.2); b) 2% aqueous solution of sodium oleate (sterilized in an autoclave); c) defibrinated human or rabbit blood (aspirate the serum and add physiological saline or broth to the original volume); d) to 94 cm³ of agar (90°) add 5 cm³ of 2% sodium oleate solution and 1 cm³ of erythrocyte suspension. VI. For whooping cough bacilli (Bordet-Gengou). They grow on blood media in the form of very small whitish colonies. 1) Ordinary blood agar. 2) Bordet-Gengou potato blood agar: a) 100 g of sliced potatoes are boiled in 200 cm³ of glycerin water (4%) at 120° for 30 min.; the liquid is filtered and squeezed through cloth; b) 5 g of agar is dissolved in 150 cm³ of 0.6% NaCl solution. To 150 cm³ of agar (b), add 50 cm³ of liquid (a), pour into test tubes in 2-3 cm³ portions, and sterilize in an autoclave at 120°. Before inoculation (the day before), add 1.5-3 cm³ of defibrinated human or rabbit blood to each test tube of agar (melted and cooled to 50°). Mix carefully, cool in a slanted position, put on rubber caps, and place for a day at 37° for verification. Growth after 24-48 hours (hemolysis). VII. Media for meningococci; opt. t° 37°, do not grow below 22°; strictly under aerobic conditions. For the first generations, media with blood or other protein liquid are necessary. From 2-3 generations, they grow on ordinary media. 1) Ascitic broth: 2-3 parts broth + 1 part sterile ascitic fluid containing not less than 5 g of albumin per 1 L; turbidity and formation of a small delicate sediment at the bottom. By the end of 3 days, a delicate, fragile film. 2) Ascitic agar or serum agar: melted and cooled agar + 1/3 volume of ascitic fluid or serum (3+1). 3) Ascitic agar or serum agar of Zeissler and Riedel: the same + 2% glucose to the agar. 4) Blood-ascites-maltose-agar—Esch's medium: 60 cm³ peptone agar, 20 cm³ sterile defibrinated sheep blood, 10 cm³ ascitic fluid, 1 g maltose dissolved in 3 cm³ broth. Growth is rapid. 5) Vitamin agar: 500 g fresh beef heart, 15 g peptone, 5 g NaCl, one egg, and 500 cm³ tap water. Heating at a temperature that preserves vitamins, on an open flame or water bath with constant stirring, until the color of the mass becomes brown (68-70°); the liquid is strained through a thick sieve or mesh (not through canvas, cotton, or paper, as this retains the vitamins). Separately, dissolve 15 g of agar in 500 cm³ of tap water, heat to 70°, and add to the first mixture. Sterilization in an autoclave at one atmosphere for 30 minutes, then leave until the next day. After removing the solidified mass from the pot, cut off the lower turbid part with the sediment. Dissolve the transparent part, pour into test tubes, and sterilize in an autoclave. 6) Agar with human blood (Schottmüller) and with grape sugar (Zeissler and Riedel): meat-peptone agar (cooled to 45°) + 20% sterilely collected human blood + 2% grape sugar. Differential medium for distinguishing from Micrococcus catarrhalis and Micrococcus pharyngitidis; colonies of all types are round, slightly elevated, gray-violet in color; the consistency of meningococci resembles thick mashed potatoes, while others' colonies can be removed from the medium entirely. 7) Ordinary 10% blood agar. 8) Kedrovsky's hemoglobin agar; human placenta passed through a meat grinder is infused for a day with 3-4 volumes of water, filtered through a candle. The filtrate is mixed with sterile 2% agar. 9) Kutscher's placental agar: maceration of 500 g of fresh placentas in 1 L of water; add: 2.5% agar, 0.5% NaCl, 1% glucose, 2% nutrose, 2% Chapoteaut peptone. To 3 parts of this agar, slightly alkalized and sterilized, add 1 volume of inactivated (at 60°) bovine serum. 10) Beily's media: a) hormone agar: dissolve 15 g of agar (thoroughly washed) in 1 L of water, cool to 50-60°, add 500 g of moderately ground beef heart or spleen. Bring to a boil and cook on low heat for 15-20 minutes. Filter again through a coarse sieve. Add 10 g of peptone and 5 g of NaCl, previously dissolved in a small amount of distilled water. Boil for 5 min. Adjust pH=7.4-7.5 (sodium carbonate). Let it settle for a few minutes (in a conical vessel), drain the transparent liquid from the sediment, pour into test tubes, sterilize fractionally or in an autoclave for 20 minutes at 115° (sterilize large bottles 2 times). b) Hormone gelatin: 10 g of gelatin (good quality) dissolved in 1 L of water; otherwise, follow the previous method. c) Blood hormone agar: ordinary agar + 5% fresh defibrinated human blood. Good medium for preserving cultures; on slanted solidified agar, growth at 37° for a day, sealing with liquid paraffin, storage in a slanted position in diffused light at room t°. Viability is maintained for 3-12 months. 11) Wassermann's medium (see below media for gonococci). 12) Brain medium: 1 part water + 2 parts brain rubbed through a sieve or strainer. Sterilization at 100° for 3 days in a row. 13) Sacquepee and Delater's medium. In an Erlenmeyer flask, mix the whites of 2 eggs, add triple the amount of distilled water and 0.5 (per 100) of 10% sodium hydroxide solution. Sterilization in an autoclave for 15 min. at 115°. Add 1 part of this liquid to 5 parts of sterile neutral agar (55°) and pour into dishes. 14) MM medium (Salimbeni and d'Herelle): 1 L of Martin peptone (6-8-hour); alkalization, precipitation by heating in an autoclave for 15 min. at 120°. Filtration through paper filter, addition of 2 g glucose, pouring, and sterilization in an autoclave for 15 min. at 112-115°. 15) Ungermann's medium—see media for protozoa, No. 5. 16) Buchman's serum, modification of Loeffler's serum: 75 parts bovine serum, 25 parts broth, 1% dextrose with the addition of neutral red (0.5 : 10,000); growth in the form of red colonies. VIII. Media for gonococci; opt. growth 36-37°. Range 25-40°. Reaction of the medium is slightly alkaline (pH 7.3); pH limits 6.0-8.3. 1) Blood agar (1 part defibrinated blood + 2 parts nutrient agar). 2) Ascitic agar according to Lingelsheim for the differentiation of various cocci.

To 100 cm³ of ascitic agar, add 15 cm³ of a sterile litmus tincture solution and 1 g of dextrose, maltose, or levulose (dissolve the sugar in 10 cm³ of distilled water and boil for 1/2 hour in a Koch apparatus; after cooling, add to the ascitic agar) and pour into Petri dishes (gonococcus colonies are colorless). 3) Ascitic broth (1 part + 3 parts). 4) Ikoma's citric acid ascitic agar, ascitic agar with 0.4% citric acid. 5) Kiefer's peptone-glycerin-ascitic agar: nutrient agar (3 1/2% agar, 0.5% NaCl, 1% peptone, 2% glycerin) + ascitic fluid aa (mix with agar cooled to 40°). 6) Wassermann's medium: 15 cm³ of pig serum (or serum of another animal), 30-50 cm³ of water, 3 cm³ of glycerin, 0.8 g of nutrose (casein sodium phosphate); boil for 15 minutes while stirring thoroughly. Pour into test tubes and sterilize in flowing steam. Before use, mix aa with 2% sterile agar, melted and cooled to 50-60°. A very good medium. 7) Leboeuf's medium: 4 l of liver broth, 400 cm³ of egg white solution, 20 g of potato starch (check the neutrality of the reaction), 20 g of agar per 1 l; sterilization for 35 minutes in an autoclave at 115°. A good medium for preparing vaccines. 8) Nastyukov's medium: to meat-peptone agar, melted and cooled to 45°, add 1/3 of egg yolks, collected sterilely and mixed with three volumes of sterilized water. 9) Nicolle's medium for vaccine preparation: 100 cm³ of meat broth, 0.4 g of urea, 2 g of pure glucose, 0.05 g of ammonium phosphate, 1 g of sea salt, 1.5 g of agar. To the finished medium, add 0.5 cm³ of rabbit serum per 5 cm³ of medium. 10) Sabouraud and Noire's medium: agar with milk whey, glucose, and urea; boil 1 l of fresh milk for 5 minutes; precipitate the casein by adding 2 cm³ of HCl, pass through a sieve covered with hydrophilic cotton. Add 1/2 volume of water, neutralize with a 10% soda solution. Sterilize in an autoclave for 10 minutes at 120°; filter; add 1% peptone, 1% glucose or sucrose, 0.3% urea, 1.6% agar, dissolve in an autoclave, filter through paper. Pour into test tubes, sterilize for 10 minutes at 110°. 11) Coagulated human serum. IX. Special media for streptococci; opt. t° 24-38°; pH range 5.5-8.0; opt. pH 6.2-7.0. 1) Blood agar (10% sheep blood). 2) Lactose litmus agar (colonies are blue). 3) 1% sugar broth. 4) Marmorek's serum broth: a) 1 part freshly prepared alkaline (approx. 5 cm³ of caustic soda per 1 l) broth + 2 parts fresh human serum; b) 2 parts broth + 1 part ascitic fluid; c) 1 part Martin's broth + 1 part human serum (inactivated for 30 minutes at 58°). 5) Weissenbach's enrichment medium: peptone water with glucose on egg white, alkaline; 100 cm³ of water, 4 g of Chapoteaut peptone, 0.5 g of NaCl; 0.2 g of glucose, 100 cm³ of egg white (alkalized with soda and diluted 3 times with distilled water). 6) Aldershoff's broth for obtaining scarlet fever toxin; 300 g of meat, 500 cm³ of distilled water, 500 cm³ of 0.8% soda solution, 5 cm³ of chloroform, 2 g of chemically pure pancreatin (Merck), 80 cm³ of hydrochloric acid (normal). For detailed preparation, see Streptococci. For differential media for various types of streptococci (haemol., anhaemolyt., viridans, Enterococc, Stf. mucosus, etc.), see the corresponding sections. 7) Bile-blood agar: a) 80 cm³ of 3% nutrient agar (melted and cooled to 50°); b) 10 cm³ of bovine bile (sterilization for 2 days in a row for 20 minutes in a Koch apparatus); c) 10 cm³ of defibrinated sheep blood. Before mixing, the bile is heated to 45-50° (Petri dishes). 8) Bieling's heated blood agar: 3% nutrient agar (1% peptone), heated to boiling + 15% defibrinated blood (sheep or horse), pour into dishes. 9) Kovacs' sugar-blood agar: 2% sugar agar + defibrinated sheep blood 1:5 (Petri dishes). 10) Rymovich's medium: ordinary agar (pH 7.6) + 1/3 volume of defibrinated and stroma-free laked pigeon blood; the difference between the scarlet fever streptococcus and other hemolytic streptococci is that it grows on Rymovich's media as white colonies without condensation. 11) Spanier's medium: 1 part serum, 2 parts distilled water, 0.2% esculin, 8% litmus tincture. Upon acid formation, reddening and protein precipitation occur. 12) Harrison and Wanderbeck's esculin medium: 1.5 g of peptone, 0.5 g of sodium taurocholate, 0.1 g of esculin, 0.05 g of iron citrate per 100 cm³ of water. Upon the breakdown of esculin, the medium turns black. Enterococcus breaks down esculin, is resistant to bile, does not hemolyze, and its growth is not inhibited by bile. 13) Kurt Meyer's esculin broth: ordinary broth + 0.2% esculin. Differentiation from enterococcus. X. Media for pneumococci. pH range 7.0-8.3; opt. pH 7.8. 1) Peptone agar with blood (rabbit or human). 2) Bieling's blood agar: 20 cm³ of distilled water, 40 cm³ of defibrinated horse blood, 60 cm³ of agar; pH 7.5. 3) Agar T. (Truche): same composition as medium T (see below) + 20 g of agar per 1 l. After dissolving the agar, alkalize to a slightly alkaline reaction. Without filtering, pour into test tubes, sterilize for 20 minutes at 110°. 4) Neufeld's serum broth of alkaline reaction (5-10% serum). The medium is good for maintaining virulence. 5) Weichselbaum's serum agar: 1 part serum + 2 parts agar. 6) Salimbeni and D'Herelle's medium: maceration of pig stomachs (see Martin's broth above) for 7-8 hours at 50°. At 55°, add 10 cm³ of HCl per 1 l and, while shaking, 300 g of slurry from stomachs (passed through a meat grinder). Keep for 7-8 hours at 50° and then raise the temperature to 80-90° to destroy pepsin and stop digestion. Alkalize and sterilize at 120°. Filter through a moistened paper filter (Chardin) and add 2 g of glucose. Pour, sterilize at 112-115°. A good medium for preserving pneumococci. 7) Truche's liquid medium: 4 g of Chapoteaut peptone, 0.5 g of NaCl, 0.2 g of glucose, 100 cm³ of distilled water, dissolve at 80°; slightly alkalize (faint pink coloring with phenolphthalein). Boil for 5 minutes, filter, pour, sterilize for 1/4 hour at 110°. For initial cultures, it is good to add 1/3 ascitic fluid. To preserve the virulence of pneumococci, add 2/3 gelatin (15%) dissolved in physiological saline and alkalized (semi-liquid medium). 8) Medium N. C. T. (Nicolle, Cotoni, Truche): in 1 l of liquid Truche medium, heated in a water bath, dissolve 150 g of gelatin at 55°, add egg white, alkalize, heat for 1/2 hour at 110°. Before use, the medium is dissolved in a water bath. The culture is inoculated 1 part to 2 parts of medium. Store in an icebox (viability and virulence are maintained for several months). 9) Huhtoon's medium, L. A. P.: 0.5% lactose, 0.2% amnoid, 0.1% peptone, 0.25% NaCl, 0.5% dibasic potassium phosphate, 0.03% monobasic potassium phosphate; the mixture is dissolved by boiling in tap water. Lactose is dissolved in an amount of 10-15% in distilled water, sterilized, and added to the general mixture. 10) Ungermann's serum (see media for spirochetes) for maintaining the longevity of cultures. 11) Optochin agar: a) 0.1 g of optochin hydrochloride dissolved by heating in 10 cm³ of distilled water (decomposes quickly); b) 1 cm³ of optochin solution (1%) is added to 150 cm³ of 2 1/2% agar; c) 60 cm³ of horse blood is mixed with 90 cm³ of sterile distilled water, kept for 1 hour at 60°. Optochin agar is poured into the blood mixture. Mix carefully. The growth of pneumococci on this medium is inhibited, streptococci are not. 12) Hiss's serum-inulin broth: 2 parts nutrient broth + 1 part serum; to 100 parts of this mixture, add 6 parts of ordinary litmus solution, in which 1 part of inulin has been dissolved by heating (1+5). Differentiation from pneumococci (acid). 13) Hiss's medium: 1 part bovine serum, 2 parts distilled water; to this, add 1% litmus tincture (5%); the mixture is heated to 100°, filtered, 1% inulin is added. Fractional sterilization at 100°. Differential medium—pneumococcus decomposes inulin (the medium turns red), streptococcus does not change it (exception: sometimes green streptococcus). 14) Salomon's medium for differentiation with Str. mucosus and pneumococcus (streptococcus decomposes soluble starch, sucrose, lactose with the formation of acid).

To 10 cm³ of nutrient agar (3%), melted and cooled to 58°, add 1.5 cm³ of a 10% solution of soluble starch (in litmus tincture) and then 5 cm³ of fresh ascitic fluid (inactivated by heating in a water bath for 30 minutes). Pour into Petri dishes. XI. Media for plague bacteria: opt. t° 30-35°; growth is possible at 5° (a distinction from other pathogenic bacteria); pH limits 5.6-7.5; opt. pH 6.5-7. Growth on ordinary media. 1) Agar with salt: ordinary meat-peptone agar + NaCl (2.5-5 per 100) - rapid appearance of involution forms. Differential medium for rodent pseudotuberculosis bacilli. 2) Himmelfarb medium (see Plague) for differentiation from plague bacilli. 3) Nikanorov medium: 100 cm³ of Martin's broth; 2 g of agar in powder, 0.01 g of cresol red. The broth is filtered through a candle. The mixture is boiled briefly, poured into sterile test tubes, and cooled in a slanted position. The color of the medium is yellow-orange (pH 7). It turns red after 24 hours during the growth of pseudotuberculosis bacilli (alkalinization); plague bacilli show almost no change after 24 hours. XII. Media for the tularemia pathogen. 1) Francis medium: meat-peptone agar + 5% horse serum, 1% glucose, 0.1 g cystine; or agar + 1% glucose + 5% rabbit blood; pH = 7.3; opt. growth 37°, colonies after 2-3 days in the form of dew drops, mucous, poorly emulsifiable in physiological saline. 2) Mac Coy and Chapin medium: egg (4 parts) and 1 part water or milk or 3 parts egg yolk and 2 parts physiological saline are mixed; the mixture is coagulated in a Koch apparatus, as usual. XIII. Media for anaerobes - see Microorganisms, cultivation of microbes. Growth under anaerobic conditions, opt. 37°, pH 6.0-7.6 for B. perfringens, pH 5.5-8.3, opt. 7.0 for B. tetani; it is preferable to use a solution of peptone prepared by the Martin method as the basis for the media. 1) Zeissler sugar-blood agar: weakly alkaline agar (by litmus) with 2-3% agar and 2% glucose is melted and cooled to 45° (either in large test tubes or poured into a measuring cylinder); about 20% fresh defibrinated blood is added to it (12-15 cm³ per 60 cm³), mixed, and poured into Petri dishes. Before inoculation, the dishes are kept at room temperature for 2 days. 2) Blood agar - see Blood, blood as a culture medium. 3) Neutral red broth (1%). 4) Broth with bile (1:5). 5) Kitt-Tarozzi liver broth: a) 2-3 pieces of fresh liver weighing 1-3 g (guinea pig, rabbit, calf) are placed in broth (pH 7.4-7.6) poured into test tubes (8 cm³), sterilized in an autoclave for 20 minutes at 110°; b) three times the amount of nutrient broth is added to the liver cut into pieces and boiled in a Koch apparatus for 30 minutes. The broth is filtered, the pieces are washed on a sieve with tap water and distributed into test tubes (3-4 pieces per tube + 7-8 cm³ of broth). Sterilization for 30 minutes at 110°. 6) Broth with pieces of meat: fresh ground meat is distributed into test tubes (about 3-4 g), covered with 8 cm³ of broth (pH 7.4-7.6), and sterilized in an autoclave for 30 minutes at 110°. 7) Broth with pieces of boiled egg white - see Microorganisms, cultivation of microbes (Tarozzi, Noguchi). 8) Kitt blood broth: weakly alkaline meat-peptone broth with the addition of fresh sterile blood of humans, sheep, etc. (3 cm³ per test tube). 9) Wurcker "fermented" broth: broth (750 cm³) in a flask with finely chopped liver (250 g), sterilized in an autoclave, is inoculated with a pure culture of Bac. putrificus Bienstock and placed in an incubator for 14 days; then it is sterilized under pressure, filtered through an asbestos filter, mixed equally with weakly alkaline broth, poured into test tubes, and sterilized. 10) Brain mash (Hibler): fresh brain, cleaned of membranes, is passed through a meat grinder; add 1 part tap water (neutral reaction) to 2 parts brain, pass through a hair sieve; boil for 2 hours in a Koch apparatus. Pour into test tubes (10 cm³), sterilize in an autoclave for 2 hours at 110°. On this medium, as well as on liver broth, anaerobes grow without special measures to prevent air access; brain tissue has a reducing effect. The medium has differential significance, as anaerobes that release H₂S and alkali "blacken" the medium (formation of iron sulfide), and those that release acid color it pink. The medium is suitable for preserving cultures. 11) Milk with pieces of liver according to Ruppert and Rottgardt: pieces of fresh liver or kidney (guinea pig) of 3-4 g are placed in test tubes with 8 cm³ of cow's milk, sterilized in an autoclave for 60 minutes at 110°. 12) Litmus milk - see Milk, milk as a culture medium. 13) Peptone water 2%. 14) Medium with cotton wool according to Wrublewski - see Microorganisms, cultivation of microbes. 15) Noguchi-Smith medium: ascitic broth with sterile pieces of fresh kidney. A kidney is sterilely removed from a bled rabbit, sterilely cut into 8 parts, washed with physiological saline, distributed into sterile test tubes, and 8-10 cm³ of sterile ascitic broth is added. Sterility control for 5-6 days at 37°. After inoculation, it is covered with sterile vaseline oil (1-2 cm thick). 16) Medium with cysteine according to Frei and Riedmüller: ordinary broth from the heart or meat of cattle or horses + 1% peptone, 0.5% NaCl, and 2.5% agar; pH 7.4. To the melted agar (70°), add 1% of 0.015% cysteine hydrochloride; 10% NaOH solution until pH = 7.4. Sterilization for 1/2-3/4 hour in flowing steam. Growth is possible under aerobic conditions. 17) Wilson and Blair medium: 100 cm³ of 3% sugar (glucose) agar, 10 cm³ of a 20% solution of sodium sulfite freshly prepared in distilled water, and 1 cm³ of an 8% solution of ferric chloride. Due to the reduction of sodium sulfite by some anaerobes (Bac. fallax, B. perfringens, septic vibrio, Bac. Chauvoei, B. sporogenes), the sodium sulfide combines with the ferric chloride to form a black precipitate of iron sulfide. Carbohydrate media (for studying fermentation). 18) Litmus broth with carbohydrates: nutrient broth (pH 7.2) + 7.5% litmus tincture and 1% carbohydrates (glycerin, mannitol, isodulcitol, glucose, galactose, levulose, sucrose, lactose, maltose, inulin, salicin). 19) Carbohydrates in 2% peptone water are prepared as stock solutions: 30% sucrose, 30% glucose, 17% lactose, 30% maltose, 15% mannitol, 30% levulose (do not sterilize); 30% galactose, 14% raffinose, 30% glycerin, 30% dulcitol, 30% arabinose, 15% xylose, 30% dextrin, 15% rhamnose (isodulcitol), dissolve by heating (on a wire gauze), filter through paper, pour into ampoules, seal, sterilize for 15 minutes at 110°. Add 5 drops of the stock solution to each test tube with 2% peptone water before inoculation. The result of fermentation is determined by adding 2-3 drops of litmus tincture to each test tube after growth. XIV. Media for pathogenic fungi. 1) Sabouraud medium for isolation (Milieu d'epreuve de Sabouraud): 1,000 cm³ water, 18 g agar, 40 g maltose (brute de Chanut), 10 g peptone (granulee de Chassaing). Separately boil the agar in water for 1/2 hour and separately dissolve the peptone and maltose in water. Mix, place in an autoclave, heat to 120°, remove from the autoclave, filter hot through a Chardin filter, and pour. Sterilize as indicated above. 2) Sabouraud medium for preservation (Milieu de conservation de Sabouraud): 1.8% agar, 1% peptone, 1,000 cm³ water. Preparation as the previous one. 3) Sabouraud medium for sporothrix: 10 g agar, 10 g Chassaing peptone, 40 g glucose, 1,000 cm³ water. Cultivation at room t°; opt. 22-30°. 4) Plaut medium for initial culture: 1-2 g peptone, 1 g glucose, 0.5 g glycerin, 2 g agar, 0.5 g NaCl, 100 cm³ water. Neutralization is not performed. 5) Grütz medium: 5 g Knoll peptone (dissolved in a few cm³ of water with slight heating), 10 g glucose, 5 g glycerin, 5 g NaCl, 1,000 cm³ of 1.8% agar. 6) Grütz medium for preservation: 30 g peptone, 1,000 cm³ of 1.8% agar. Cultivation of pathogenic fungi is carried out at room t°, without closing with rubber caps, in diffused light. XV. Media for protozoa - see Microorganisms, cultivation of protozoa. A. Spirochetes. Spirochetes grow under conditions of relative anaerobiosis. 1) Noguchi ascitic agar for Treponema pallidum, Spirochaeta obermeieri, Sp. gallinarum, Sp. icterogenes: 2 parts weakly alkaline 2% agar + 1 part ascitic or hydrocele fluid.

Dispense into test tubes in 15 cm³ portions, place a small piece of fresh rabbit organ (kidney, testicle) into each, and cover the top with a 3 cm layer of liquid paraffin (the ascitic fluid must not contain bile). 2) Aristovsky and Holtzer medium (for Treponema pallidum): rabbit serum (undiluted or diluted with physiological saline 1:2), inactivated for 1 hour at 60°, with a piece of fresh testicle or brain. (Instead of rabbit serum, one may take ascitic fluid or human serum.) 3) Medium with blood extract: a) grind horse blood clots through a sieve, add double the volume of physiological NaCl solution (8‰) while heating; b) heat to 75° for 15-20 minutes, filter through paper, then through a Chamberland L3 candle, add a little sterile rabbit serum (5%), sterilize fractionally for 3 days in a row at 56° for 30 minutes (medium for Sp. icterohaemorrhag., the spirochete of Weil's disease). 4) Noguchi medium (for various blood spirochetes): 80 cm³ of sterile physiological solution, 10 cm³ of rabbit serum, fresh and sterile, 10 cm³ of 2% meat-peptone agar (pH 7.2), melted and cooled to 95°, 0.5 cm³ of sterile Hb solution. It solidifies as a column. After inoculation, cover with a layer of vaseline oil. 5) Ungermann medium: sterile fresh serum (rabbit), diluted with a small amount of physiological saline or Ringer's solution, is dispensed into test tubes, heated for 1/2 hour at 58-60°, and sealed from air access with sterile paraffin (for cultivating spirochetes icterogenes, Obermeieri, Duttoni). 6) Shereshevsky medium (for Treponema pallidum): coagulated horse serum (in a water bath with a slow temperature rise to 70°) as a column. 7) Noguchi serum water (for Treponema pallidum): 1 part rabbit serum + 3 parts distilled water; a piece of kidney or testicle is added to the mixture. Cover with paraffin, as above. 8) Uhlenhuth serum water for spirochete icterogenes: mix sterile rabbit serum and tap water in a ratio of 1:30. After inoculation, cover with a layer of paraffin. 9) Chmamine horse serum for Treponema pallidum: to 200 cm³ of horse serum, add 1-1.5 g of sodium nucleinate while shaking the vessel. Pass CO₂ (from a Kipp apparatus) for 2-3 minutes—transparency of the medium is achieved. The serum is dispensed in a high layer in test tubes, heated for 3 days for 1 hour at 60° and the 4th day slowly to 70°. 10) Hata horse serum for Obermeier's spirochetes: fresh horse serum is dispensed into test tubes (15-17 mm wide) in 4 cm³ portions; 8 cm³ of physiological saline is added to each and mixed. Then the test tubes are kept in a water bath for 3 hours at 58°; the temperature is slowly raised to 70-71°, and held for 1/2 hour at 71°. A small piece of rabbit kidney is placed into the semi-solid serum. Inoculate into the depth. B. Amoebae. 1) Straw infusion and decoction. 2) Mouton gelatin: 900 cm³ of weakly alkaline water, 100 cm³ of ordinary nutrient broth, 20 g of gelatin. 3) Schardinger agar: 30 g of hay + 1 l of water, add 1-1.5 g of powdered lime hydrate; keep in a thermostat for 24-36 hours; filter, alkalize. Addition of 1-2.5% agar. Inoculate material into the condensation water. 4) Frosch agar: 0.5 g of agar, 90 cm³ of tap water, 10 cm³ of weakly alkaline nutrient broth. 5) Musgrave and Clegg agar: 20 g of agar, 1,000 cm³ of distilled water, 0.3-0.5 g of meat extract, 0.3-0.5 g of NaCl. Slightly alkalize with caustic soda. Pathogenic amoebae are cultivated on protein media. C. Flagellate protozoa. 1) MacNeal and Novy medium: 2 parts defibrinated rabbit blood and 1 part agar. Growth in condensation water. 2) NNN-agar (Nicolle)—a modification of the previous one: 900 cm³ of distilled water, 14 g of agar-agar, 6 g of NaCl. At a temperature of 43°, add defibrinated rabbit blood in a ratio of 1:2. 3) Laveran and Petit liquid medium: 2 g of Chapoteaut peptone, 6 g of NaCl, 900 cm³ of distilled water. This peptone water is mixed equally with defibrinated rabbit blood. 4) Noller blood agar: 25 g of agar, 20 g of glucose, 1,000 cm³ of weakly alkaline horse broth. Dispense into test tubes. Before adding blood to the melted agar, an equal or half volume of defibrinated horse blood is poured in, and the medium is left to solidify in a slanted position. Agar regeneration aims to refresh already used agar (for economic purposes). During regeneration, the following points are taken into account: 1) bacteria and their metabolic products must be removed; 2) consumed nutrients must be replenished; 3) specially added substances (indicators, etc.) must be removed. Regeneration of simple agar is performed as follows: the agar is sterilized, poured into pots, and left to solidify. After solidification, it is removed in one piece, the lower cloudy part is cut off, the remaining mass of agar is cut into pieces and washed in running water for 1 day or more. 1) To 60 l of washed agar, add 15 l of meat water, 15 l of liquid peptone, 600 g of dry agar, 150 g of NaCl. 2) To 1 l of regenerated agar, add 500 cm³ of distilled water, the mixture is melted on a fire. To 1 l of the initial medium, add 3 cm³ of 10% soda solution, boil for 1/2 hour for sterilization. Then add 20 g of animal charcoal per 1 l of medium and boil for another half hour; the medium clears due to the absorption of bacteria by the charcoal. Let cool to 50-60°, add 30 cm³ of serum (instead of egg white). Instead of peptone solutions and Liebig's extract, one can add 1/2 l of ordinary broth or Gottinger broth per 1 l of regenerated medium. 3) Agar regeneration according to the method of the Control Institute. Used agar is sterilized in an autoclave for 20 minutes at 125°, poured into a low wide pot, cooled, and cut into small pieces; the latter are placed on a sieve and put under a stream of running water for 18-24 hours. Then it is melted, checked, and the reaction is adjusted using litmus paper, the volume is measured, and 1/3 of this volume of neutral broth and 2.5% of dry agar-agar for the total amount are added. Before sterilization, add egg white (1-2 per liter) and place in an autoclave for 20 minutes at a temperature of 120° (mix the egg white in a small amount of distilled water before adding). Filter, dispense into test tubes, and sterilize at 120°.