54 MUSHROOM GROWING AND SPAWN MAKING. 



pure-culture methods necessitates to a considerable extent a knowl- 

 edge of the bacteria and molds which are everywhere i)resent in the 

 air and which are especially abundant wherever there are dusty or 

 damp, moldy conditions. The principle of making pure cultures is 

 briefly this : The materials, or media, and all the vessels employed 

 must be sterilized, which implies being heated at a temperature suffi- 

 cient to kill all germs present in the vessels or materials used. If 

 the vessels used are test tubes or other pieces of glassware with small 

 mouths, they should, previous to sterilization, be plugged with cotton 

 batting. This cotton batting prevents, when carefully manipulated, 

 the entrance of germs from the air, and therefore keeps the vessel 

 or medium in a pure or sterile condition. If such a vessel is opened, 

 this should be done in a room free from currents of air or falling 

 dust particles ; and, while open, tubes and other apparatus should be 

 held in a more or less horizontal position, so that they will be less 

 liable to contamination. It follows, of course, that the cotton plug, 

 if removed, shoidd not come in contact with any unsterilized sub- 

 stances. If, now, a small quantity of the growing mycelium of a 

 mushroom from a pure culture is transferred to such a sterilized 

 tube, using for this transfer sterile needles, or scalpels, there will be 

 little danger fi'oui foreign organisms, and the piece of mycelium 

 inserted will therefore grow as a ])ure cidtui-e free from all other 

 fimgi or bacteria. 



77ie thsve-cvlfiirc ixefhod. — In making jjure cultures of musln-ooins, 

 large test tubes or wide-mouthed bottles nuiy be used. These should 

 be carefully cleaned, and, if possible, a sterilization should be given 

 by means of dry heat as a preliminary precaution. In this event the 

 tubes are i)lugged with cotton i)lugs and placed in a dry oven made 

 for the purpose. They are heated to a temperature of about 150° C, 

 and this temperature should be maintained for nearly an hour. 

 Ordinarily, however, in rough work it is not essential to employ this 

 preliminary sterilization. In either case the tubes are next partially 

 filled (about two-thirds) with the manure, or half-decayed leaves, 

 upon which it is desired to grow the virgin spawn. A plug is 

 inserted in each tube, and the tubes are then sterilized in a steam 

 boiler or under pressure. If sterilized under steam pressure, as in an 

 autoclave, it is necessary to use about 15 pounds pressure and to allow 

 the tubes to remain at this pressure for from fifteen mimites to half 

 an hour. If the sterilization must be effected in a boiler or in an ojien 

 Avater bath, it can only be done at 100° C, of course; and it is then 

 desirable to boil the tubes for at least one hour on each of two or three 

 successive days. 



With the tubes thoroughly sterile, the next step is to make the 

 cultures or inoculations. By the tissue-culture method .it is implied 



