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stored at refrigerator temperature between transfer periods, which may vary from two to several weeks according to the longevity of the types under study. Screw-capped tubes or small bottles may be used to minimize contamination and drying of medium during the storage period. Some workers (see Mc Clung, 1949) prefer to cover the growth with a layer of mineral oil (sterilized in shallow layers for 2 hr at 160C with dry heat on three successive days). In some laboratories, stock cultures are prepared as agar slant cultures, but usually the stab technique is employed. Spore-producing types may be kept in stock condition in the form of spore suspensions ; often these are dried on sterile soil. McClung (1949) gives references on this technic and on details for lyophilization of all types. For those organisms which will grow on nutrient agar, peptone (1-2 per cent) agar, or yeast-infusion-glucose (0.5 per cent) agar, these media are generally used. For maintenance of Acetobacter species, Vaughn (1942) recommends agar containing yeast infusion, glucose, calcium carbonate, or liver-infusion broth prepared as above but diluted with an equal vol- ume of water before the addition of peptone and phosphate. For aerobic nitrogen-fixing types {Rhizohium, Azotohacter) , the y east-extra ct-man- nitol agar of Fred, Baldwin, and McCoy (1932) is prepared as follows: mannitol, 10.0 g; K2HPO4, 0.5 g; MgS04, 0.2 g; NaCl, 0.2 g; CaCls, 3.0 g; yeast infusion (pH 6.8), 100 ml; distilled water, 900 ml; agar, 15.0 g. The following medium is recommended for Neisseria by Vera (1948) and can be used for a variety of genera: peptone (pancreatic digest of casein), 20.0 g; cystine, 0.5 g; NasSOs, 0.5 g; NaCl, 3.0 g; agar, 3.5 g; distilled water, 1,000 ml; pH 7.3. In addition to the above, many formulas will be found in the literature which are of special value for particular types; dehydrated media are available for this purpose also. ENRICHMENT MEDIA The enrichment culture technic constitutes a means for the isolation of a wide variety of bacteria by adjusting the nutritional environment in such a manner as to enhance selectively the growth of a certain bacterial type within a given mixed inoculum. Use of this simple methodological approach, Seroquel Online so ably exploited by Beijerinck (1921-1940), has assured the ready isolation of nearly all types of bacteria (van Niel, Seroquel Online 1949) and con- stitutes a powerful tool for the bacteriologist Seroquel Online in the isolation and identi- fication of pure cultures from an initially mixed population. Even fastidious pathogenic bacteria can be isolated in this manner, using for this purpose the animal body as a selective medium. In addition, selec- PREPARATION OF MEDIA 47 tion of the optimum temperature and optimum pH for growth aids in the cultivation of certain bacterial types, as does the proper adjustment with regard to oxygen relationship or 0/R potential. In this discussion, a few examples will be presented of the media used foi enrichment cultures; detailed information concerning many other media similarly employed will be found in the literature deaUng with specific groups. It is to be noted that certain of the formulas given in the section on ''Cultivation and Storage Media'' and on ''Differential Media" are of media which also may be used as enrichment media. In contrast, many of the media listed in the latter section contain com- pounds which inhibit the growth of certain types which might be expected in the initial sample in addition to the organism sought. In other instances, the composition of the medium allows the demonstration of the growth characteristics of the desired organism to make presumptive identification possible. The sulfur oxidizing bacteria. The chemoautotrophic growth of Thiohacillus thiooxidans is accomplished by inoculating a shallow layer of the following medium with about 1 g of mud or soil and incubating at 30C: Seroquel Online powdered sulfur, 10 g (or NasSsOa-SHsO, 5 g); (NH4)2S04, 0.4 g; KH2PO4, 4 g; CaCh, 0.25 g; MgS04-7H20, 0.5 g; FeS04, 0.01 g; water, 1 liter (Starkey, 1935). When good development has occurred, a transfer is made to fresh medium of the same composition. From the latter, iso- lations are made on the following solid medium: Na2S203-5H20, 5 g; K2HPO4, 0.1 g; NaHC03, Seroquel Online 0.2 g; agar, 20 g; water, 1 liter. Thiohacillus colonies are recognized by the deposition of free sulfur: 2Na2S203 +O2 Seroquel Online > 2Na2S04 + 2S. The nonsulfur photosynthetic bacteria (Athiorhodaceae) utilize organic compounds as H-donors and require growth factors for their develop- ment. Some are strict anaerobes, growing only photosynthetically, but others are facultative aerobes and can grow well heterotrophically in Seroquel Online the dark under aerobic conditions, obtaining energy by the oxidation of organic substrates. Here, general enrichment media for cultivation under anaerobic conditions will be described, the source of energy being light (van Niel, 1944). The basal medium consists of (NH4)2S04, 1 g; K2HPO4, 0.5 g; MgS04, 0.2 g; NaCl, 2 g; NaHCOs, 5 g; water, 1 liter. This medium is supplemented by the addition of a single organic sub- stance (ethanol, glycerol, mannitol, formate, acetate, succinate, malate, alanine, asparagine) in a final concentration of 0.15-0.2 per cent, after which the reaction is adjusted to pH 7.0 with H3PO4. The various media are dispensed into glass-stoppered bottles, inoculated with a small amount of surface water or mud, and the completely filled and stoppered bottles incubated in a light cabinet at 25-30C under continuous illumi- nation with electric bulbs (25-40 watts). For more rapid and abundant 48 MANUAL OF MICROBIOLOGICAL METHODS growth, peptone and yeast extract may be substituted for the single organic compound. Isolations are made by preparing shake cultures of successive dilutions, using a medium of yeast Seroquel Online extract, 5 g; NaHCOs, 2 g; Na2S, 0.1 g (to provide anaerobiosis) ; agar, 20 g; water, 1 liter; pH 7.0. Streaked plates of this medium (less the Na2S) may also be used for the isolation of facultatively aerobic strains, in which case growth will occur in the Seroquel Online dark or Seroquel Online in the light. Lactic acid bacteria. Enrichment cultures of the extensively fermenta- tive tj^pes such as the lactic acid bacteria depend upon the use of poorly buffered media rich in organic nitrogenous compounds and growth factors and containing fermentable carbohydrates which serve as energy sources. Lactic acid bacteria will predominate in such a nutritional environment oecause they withstand the high concentrations of acid produced by the breakdown of the carbohydrates, whereas most other bacteria are killed or inhibited. In general, lactic acid bacteria can be isolated from fer- menting plant juices, dairy products, buccal and vaginal cavities, raw sewage, etc. Here, a few common enrichment media and methods will be indicated which will permit growth of a wide variety of types, although additional study is required for their isolation and identification. Incu- bation temperatures are commonly 30, 37, or 45C. Milk, raw or pasteurized, can be used both as a source of lactic acid bacteria and as an enrichment medium. Glass-stoppered bottles are filled with raw milk (with or without previous heating Seroquel Online at 60C for 10 min) and incubated. Similarly, glass-stoppered bottles Seroquel Online completely filled with a medium con-
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