2. Connect up the inlet tube of the Bulloch's jar with the sterile gas filter, and this again with the delivery tube of the gasometer or gas generator. 3. Open both stop-cocks of the Bulloch's apparatus and pass the gas through until it has completely replaced the air in the bell jar as shown by the result of analyses of samples collected from the exit tube. 4. Incubate under optimum conditions as to temperature. 5. Examine the cultivations at intervals of twenty-four hours, until the completion of seven days. 6. Remove one tube from the interior of the apparatus each day. If no growth is visible, incubate the tube under optimum conditions as to temperature _and_ atmosphere, and in this way determine the length of exposure to the action of the gas necessary to kill the organisms under observation. 7. Control these results. ~II. Temperature.~-- (A) _Range._-- 1. Prepare a series of ten tube cultivations, in fluid media, of optimum reaction. 2. Arrange a series of incubators at fixed temperatures, varying 5° C. and including temperatures between 5° C. and 50° C. (In the absence of a sufficient number of incubators utilise the water-bath employed in testing the thermal death-point of vegetative forms.) 3. Incubate one tube cultivation of the organism aerobically or anaerobically, as may be necessary, in each incubator, and examine at half-hour intervals for from five to eighteen hours. 4. Note that temperature at which growth is first observed macroscopically (Optimum temperature). 5. Continue the incubation until the completion of seven days. Note the extremes of temperature at which growth takes place (Range of temperature). 6. Control these results--if considered necessary arranging the series of incubators to include each degree centigrade for five degrees beyond each of the extremes previously noted. (B) _Optimum._-- 1. Prepare a second series of ten tube cultivations under similar conditions as to reaction of medium. 2. Incubate in a series of incubators in which the temperature is regulated at intervals of 1° C. for five degrees on either side of optimum temperature observed in the previous experiment (A, step 4). 3. Observe again at half-hour intervals and note that temperature at which growth is first visible to the naked eye = Optimum temperature. (C) _Thermal Death-point (t. d. p.)_-- Moist--Vegetative Forms: The _t. d. p._ here is that ~temperature~ which with certainty kills a watery suspension of the organisms in question after an exposure of ~10 minutes~. [Illustration: FIG. 155.--Hearson's water-bath.] _Apparatus Required:_