12 STERILIZATION BY MOIST HEAT 



temperature and pressure must be maintained for 20 minutes, reckoning from the moment 

 when it reaches 115 C. (e) When the necessary time has elapsed, remove the source of 

 heat and allow the autoclave to cool until the pressure reaches zero on the manometer, 

 then open the steam-tap (not shown in the illustration), and raise the cover. 



Note. The autoclave may also be used for sterilization at 100 0. The technique is 

 the same as in the preceding case, except that the valve D is never raised. Under these 

 conditions steam will issue in a powerful jet during the whole operation. The tem- 

 perature must be maintained for 30 minutes after reaching 100 C. 



Sterilization can also be effected at any temperature between 100 and 115 C. by 

 suitably altering the position of the knobbed handle of the valve ; the further the handle 

 is fronTthe vertical the less will the temperature rise above 100 C. 



Method of controlling the temperatures at which sterilization was effected. 



In laboratories where the sterilization of apparatus, etc., is entrusted to laboratory 

 assistants, it is convenient to have a method of controlling the temperature at which 

 sterilization was effected. This may be done by placing a maximum thermometer 

 or, more conveniently, a fragment of fusible alloy or some chemical compound 

 of suitable melting point (110-120 C.) alongside the apparatus in the autoclave. 

 If a powder be used it may be mixed, as suggested by Demandre, with a trace of 

 some dye, and sealed up in a small glass ampoule. The small amount of dye used 

 is not visible in the powder, but when the latter melts the dye diffuses through 

 it, and on cooling forms a coloured bead. 



The following substances are suitable for the purpose, the temperature in brackets 

 indicating the melting point: benzonaphthol (110C.); antipyrine and sulphur 

 (113 C.); resorcin (119 C.) ; benzoic acid (121 C.). 



For coloured beads the following formulae may be employed : 



Melting at 110 C. Benzonaphthol, - 100 grams. 



Safranin, - O'Ol gram. 



Melting at 121 C. Benzoic acid, - 100 grams. 



Brilliant green, - O'Ol gram. 



3. Sterilization by discontinuous heating at low 

 temperatures. 



Some substances used as culture media, being rich in albumin, cannot be 

 heated to boiling point without marked alteration and to some extent 

 destruction of their properties. Serum is a case in point. 



Pasteur showed that such media can be better sterilized by heating them 

 at a low temperature (55-60 C.) for a long time than at a high tempera- 

 ture for a short time. This prolonged heating at a low temperature con- 

 stitutes Pasteurization. In practice however it is found that to be effectual, 

 pasteurization must be combined with the method of discontinuous heating- 

 devised by Tyndall (p. 7). 



Technique. Distribute the medium into a series of sterile flasks with 

 long necks (fig. 35, p. 46), each flask being about three-fourths filled, and 

 seal the mouths in a blow-pipe. [Flasks and test-tubes covered with india- 

 rubber caps (p. 29) over the wool plugs can be used equally well.] 



Place the flasks in a water bath fitted with a thermometer, slowly raise 

 the temperature and regulate the gas flame so that it remains constant at 

 56-57 C. for an hour, then turn out the gas, but leave the flasks in the 

 bath until they are quite cool. 



The flasks must be heated in the same way daily for a week before the 

 contents can be regarded as sterile ; and even then they ought to be incu- 

 bated at 37 C. for two or three days, and any flask in which a growth appears 

 must, of course, be rejected. 



Water baths. 



Conducted in the manner described, this method of sterilization is tedious, 

 and it is difficult to avoid exceeding a temperature of 58 C., with the result 

 that the albumin coagulates, rendering the medium useless for the purpose 



