76 PRELIMINARY COLD STORAGE STUDIES. 



able liquid carrier for the organisms, generally physiological salt, 

 was then put into the flask and the whole was shaken with sufficient 

 vigor to agitate both the pieces of glass and the tissue. Since the 

 portions of the latter were not large, the combined action of the sharp 

 glass and the liquid served for their disintegration. The longer the 

 shaking, the more perfect was the diffusion of the particles. It could 

 not, however, be continued beyond a comparatively short period 

 of time, because of the multiplication of the organisms. With the 

 quantities of tissue above stated, ten minutes' shaking was selected 

 as a happy medium between an undesirable multiplication of the 

 organisms on the one hand and the retention of the organisms by the 

 tissue, and the consequent lowering of the numbers found, on the 

 other. 



Still it is quite possible that by the method above described not 

 all of the organisms are dislodged from the tissue. The fact that 

 all of the experiments were conducted in exactly the same way, how- 

 ever, would render the results reported strictly comparable. 



The suspension of organisms, prepared according to this method, 

 was transferred by accurately graduated pipettes in portions of 1 cc, 

 or more, to petri dishes and the desired nutrient material added. 

 Such a procedure sufficed for the detection of aerobes and facultative 

 organisms. For the anaerobes the double-plate method of Wright 

 was adopted. This consists in the use of the cover of the petri dish as 

 a holder for the medium and the organisms, and into it is set the 

 lower part of the dish. If the dishes are flat, the cover is of fair 

 depth, and the inner dish is lowered into the outer one in a slanting- 

 fashion, air bubbles will ordinarily be expelled, and there will be an 

 air-free layer of nutrient media between two flat glass surfaces, en- 

 abling one to study or count readily the colonies which develop. 



Since these plates are easily contaminated after sterilization, 

 even in the short time required for cooling and in the transference 

 during manipulation, a special metal container was obtained, in 

 which they were sterilized and kept until the moment of inoculation. 



There is generally a small space between the inner and the outer 

 dish. This permits a drying out of the media, even after short 

 periods of incubation, and makes the plates quite impracticable for 

 longer periods. Therefore, the space between the two dishes was 

 filled with a low melting paraffin, which had the additional advantage 

 of excluding both air and dust. 



The number of colonies developing on plates so prepared permits 

 the enumeration of both facultative and anaerobic organisms, but 

 for the separation of these two classes it would be necessary to trans- 

 plant each colony or type of colony, submitting it to appropriate 

 conditions to determine its relation to oxygen. An attempt was 



