228 MANUAL OF MICROBIOLOGICAL METHODS 



cultures of true pathogens when serial passages are employed to regain 

 the virulence of a degraded type. 



Although no longer regarded as the sine qua non standard of patho- 

 genicity, it is well to conclude this section with a brief recapitulation of 

 Koch's postulates. Fulfillment of the postulates is often difficult, even 

 with accepted pathogens, as with the gonococcus. In certain instances, 

 such as synergistic infection, they are not applicable. Nevertheless, the 

 basic intent of Koch is valid and directed against premature or unwar- 

 ranted assumptions of pathogenicity. The postulates are as follows: 



(1) The organism must always be present where the disease occurs, 



(2) the organism must be obtained in pure culture from the infected 

 tissue, (3) this culture must cause the disease when injected into a favor- 

 able region or tissue of a normal susceptible animal, and (4) the organism 

 must be recovered from the latter. 



PREPARATION OF INOCULA 



It is always desirable to prepare bacterial inocula in a consistent and 

 standardized manner so that experiments can be readily duplicated. 

 Commonly used diluents for the preparation of suspensions are any of 

 several nutrient broths, physiological saline solution, distilled water, or 

 an isotonic buffer solution. (Methods followed in preparing tissue sus- 

 pensions will be found in Chap. XI.) Suspensions may be obtained from 

 washings or scrapings of growth on solid media or from fluid cultures. 

 Old cultures are generally less pathogenic than young growths. In each 

 case the largest cell aggregates are removed by light centrifugation, and 

 when necessary, the final suspension is standardized for density with a 

 colorimeter or densitometer. 



As a check of purity approximately 0.1 ml of the final inoculum should 

 be cultured on a suitable agar medium. Bacterial counts of the inoculum 

 can be performed either turbidimetrically or by culture ; the latter is often 

 the preferable procedure, as it counts viable bacteria only. When the 

 toxicity of a preparation is being determined, the cells or cell debris are 

 usually removed by centrifugation or filtration. It should be borne in 

 mind that certain toxins, e.g., the murine toxin of Pasteurella pestis, may 

 be inactivated by filtration, and this procedure is generally not recom- 

 mended for toxin preparation. Sterility tests of toxins are also desirable 

 to ensure that the death of an experimental animal is not due to a con- 

 taminated preparation. 



The storage of bacterial inocula for periods of a few days is best done at 

 4-8°C, whereas for longer periods freezing, at —20 or — 70°C, is often 

 resorted to. However, considerable killing may occur with even a single 

 freeze and thaw, and this method is not recommended for the storage of 



