172 MANUAL OF MICROBIOLOGICAL METHODS 



According to Wilson and Knight (1949) the bacterial suspension should 

 contain about 10^-10^ cells per ml for accurate counting. This cell con- 

 centration results in 3-12 cells per square of the counting chamber. A 

 total count of about 400 cells per slide results in a final count correct to 

 within 10 per cent. For best results, the direct count is made with dark- 

 field illumination. 



Results obtained with the counting chamber will always be higher than 

 those from the colony-count method, since those cells which are not 

 capable of reproduction are also counted. The direct-count method is, 

 however, more rapid. 



Cell volume. This technic involves the hematocrit principle of blood- 

 cell determination. Unlike blood cells, however, bacterial cells occupy a 

 smaller proportion of the volume of the suspending medium, and larger 

 samples must be employed. A tube similar to the Hopkins vaccine tube, 

 which holds 5- to 10-ml samples containing 0-0.05 ml of cell volume, is 

 satisfactory. Using the Hopkins tube, the cell volume may be estimated 

 directly, and if desired, the number of cells may be estimated by calcula- 

 tion from the known cell size. Schmidt and Fischer (1930) centrifuged 

 cell suspensions in capillary tubes and then measured the height of the 

 columns of sedimented bacteria. 



A number of disadvantages accompany this method. Unless standard 

 conditions are rigidly followed, the final cell volume observed will be 

 variable, since the cells can be packed loosely or tightly, depending upon 

 the rate and time of centrifugation. The average volume per cell also 

 changes during the course of growth. Thus, the problem of relating 

 total cell volume to other growth measurements becomes complex. Any 

 variation in suspending medium will probably affect the water content of 

 the cells, thus causing variable results in the total volume observed. In 

 this method the differences observed are often small, thus allowing much 

 greater opportunity for error. 



Dry weight. Using distilled water, wash and resuspend the bacterial 

 cells. Add aliquots of varying volume to small tared watch glasses or 

 weighing bottles, and dry in an oven overnight at 85°C. After accurate 

 weighing on an analytical balance, the dry weight of the bacterial mass in 

 the original suspension is calculated. 



If salt or other solutions are used in place of distilled water, it is neces- 

 sary to determine the dry weight of the solution constituents. The 

 determination, as calculated from such results, will contain an error intro- 

 duced by the wet volume of the bacterial cells. On the other hand, the 

 use of water for washing and resuspending the cells tends to extract salts 

 or other soluble compounds from the cells, resulting in a final weight 

 which is lower than the true weight. 



Since the turbidity of a cell suspension is more easily and rapidly 



