170 MANUAL OF MICROBIOLOGICAL METHODS 



GROWTH MEASUREMENTS 



In routine tests for taxonomic purposes, semiquantitative measure- 

 ments of growth are usually deemed satisfactory. However, for research 

 problems in the physiological and biochemical aspects of bacteriology, 

 quantitative methods are generally necessary. Either the rate or extent 

 of growth is used for quantitative purposes, and the extent of growth 

 generally represents the most accurate expression, particularly w^hen 

 related to small experimental differences. Microbiological assay methods 

 for the determination of amino acids, vitamins, and other growth factors, 

 as well as antibiotics, are usually dependent upon growth measurements 

 either directly or indirectly. Investigations into the nutritional require- 

 ments of bacteria, although often satisfied by qualitative methods, are 

 more definitively answered by quantitative growth determinations. 

 Within the area of nutritional studies is included the elucidation of bio- 

 chemical pathways by means of biochemical mutants. Various mutants 

 must be defined on the basis of nutritional requirements before becoming 

 useful in such a study. 



Several methods for the quantitative estimation of growth are described 

 in detail below. Since most investigators publish results in terms of the 

 milligrams of N or milligrams of cells (dry weight) used, the method 

 employed for measuring is generally related by means of a standard curve 

 to one of these unitages. For example, using a given suspension of bac- 

 terial cells, one may determine turbidity, dry weight, and total Kjeldahl 

 nitrogen and then construct curves of turbidity as a function of dry 

 weight and of nitrogen. With subsequent suspensions, only the turbidity 

 needs to be determined, and the dry weight may then be read from the 

 previously constructed curve. However, it should be pointed out that 

 the margin of error will vary with cells harvested at different stages of 

 growth. In addition, the turbidity-dry-weight relationships determined 

 for one species do not necessarily apply to another species or under differ- 

 ent conditions of growth. 



Quantitative Methods 



Cell counts. Indirect method. Dispense sterile saline solution (0.85 

 per cent; w/v) in 9.0-ml amounts in sterile, cotton-plugged test tubes. 

 Serially dilute 1.0 ml of a culture or suspension of the organism in the 

 saline to give 1:10, 1:100, 1:1,000, etc., dilutions, respectively, of the 

 culture. 



Dispense 10- to 15-ml quantities of melted nutrient agar in sterile 

 plugged tubes, and hold at 40-45°C. To each agar tube, add 1.0 ml of a 

 suitable dilution of bacterial suspension, mix well, pour into a sterile petri 

 dish; allow to harden, and incubate. The incubated agar plate should 

 contain 30-300 colonies for accuracy in counting, and all dilutions should 



