32 LABORATORY EXERCISES IN BACTERIOLOGY. 



drawn, and the temperature allowed to fall to somewhat less than 100 C., and the pres- 

 sure to normal, before the cover is removed and the sterilized materials taken from the 

 interior. Should one attempt to open the cover without having allowed equalization of 

 pressure to have taken place, fluids in the interior, as culture media in flasks or tubes, 

 are liable to bubble violently, wet the stoppers, or the escaping gases may force them 

 from the necks of the flasks or tubes ; and the operator, if careless, is liable to accidental 

 scalding from the rush of steam from the apparatus. One such exposure is easily equal 

 to the three exposures in the ordinary method of operation, but it should be recalled that 

 the high temperature may be unsuited to some materials. 



Exercise 10. Repeat exercise 8 in duplicate, employing, instead of dry 

 heat, for one set of tubes the ordinary fractional sterilization in steam with- 

 out pressure, and for a second set sterilization in the autoclave, tempera- 

 ture 120 C. and a pressure of thirty pounds. In the first set allow one in- 

 fected tube to be exposed to the full process, one to but two exposures, one to 

 a single exposure of twenty minutes, one to a single exposure of one hour. In 

 the second set let one tube be exposed to the full twenty minutes' heating, 

 one to steam for fifteen minutes, one ten minutes, and one five minutes. 

 Place all tubes in the incubator and note results at the close of twenty-four, 

 forty-eight, and seventy-two hours. The tube in each case with least expo- 

 sure showing no occurrence of growth marks the lethal exposure of the given 

 germ to the mode of procedure employed. 



In determination of the thermal death-point of a given microorganism, material de- 

 void of spores is selected (this determined by microscopic examination or by the success 

 of inoculations from cultures which have been exposed to a temperature of 80 C. for ten 

 minutes) . This material may be either diffused in a sterile fluid in tubes or smeared dry 

 on sterile glass slides; each specimen is then placed in one of a series of ovens the tem- 

 perature of which ranges several degrees apart through a number of degrees, as 50, 52, 

 54, 56, 58, and 60 C. At intervals inoculations are made from each specimen. The 

 shortest exposure to the lowest degree of heat after which such inoculations fail of 

 growth represents the temperature lethal to the bacterium. In the record note should 

 be made of whether the organism was in moist or dry condition, as well as the degree of 

 temperature and the duration of exposure. 



Exercise 1 1. In order to observe the relative value of steaming without 

 pressure and under pressure, some infected material, as a fresh surgical 

 dressing from some suppurating wound or bits of sterile threads dipped in 

 a bouillon culture of some known organism, as the colon bacillus, is care- 

 fully wrapped in a known number of thicknesses of a towel. One such 

 preparation is to be placed in the autoclave and subjected to the usual 

 steam exposure ; the other is placed in the ordinary steam sterilizer for the 

 same period. As soon as removed, inoculations are to be made from each 

 of these preparations to fresh tubes of nutrient medium, incubated, and the 

 results noted as usual at the close of twenty-four, forty -eight, and seventy- 



