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 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 dark or 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 45°C. 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 at 60°C for 10 min) and incubated. 

 Similarly, glass-stoppered bottles completely filled with a medium con- 

 sisting of 0.5 per cent yeast extract and 2 per cent of glucose or 10 per cent 

 of sucrose are inoculated with raw sewage. Shredded cabbage or other 

 plant tissue, pressed tightly and covered with water, or ground grain 

 mash can serve as enrichment media. Further types can be isolated by 

 streaking throat swabs on blood agar plates (described below) and 

 incubating at 37°C. When growth has occurred in the enrichment 

 media, plates of yeast-extract-glucose (or sucrose) agar containing 1 per 

 cent CaCOs (sterilized separately by dry heat) are streaked, and portions 

 of isolated colonies tested for catalase (lactic acid bacteria are catalase 

 negative) with 5 per cent H2O2 before streaking again to obtain pure 

 cultures. With certain types which show some sensitivity to atmospheric 

 oxygen in primary cultures, poured, seeded plates may be more successful 

 than streaked plates. Plates should be incubated at temperatures used 

 for the corresponding enrichment. It is apparent that many variations 

 of enrichment media may be employed and that the predominant occur- 

 rence of given lactic acid bacterial types depends upon the source used. 



The coliform group. Owing to the vast amount of work performed 

 with the Enter ohacteriaceae, highly specialized media have been developed 

 for their rapid isolation and identification. Enrichment media for the 



