MAINTENANCE AND PRESERVATION OF CULTURES 101 



of cells and perhaps interfering with dehydration. The following speci- 

 fications have given consistent satisfaction. 



Warm 200 ml of distilled water, and add 40 g of powdered skim milk. 

 Stir thoroughly, preferably with an electric mixer. Filter through 

 cheesecloth and tube, 5-6 ml per tube. Autoclave exactly 13 min at 

 115°C (approximately 11 lb). Ample space for the circulation of heat 

 around the tubes must be provided to ensure sterilization at this exposure. 



Vaspar, a mixture of equal parts of 45° mp paraffin and white petro- 

 latum, makes an excellent seal for cultures of anaerobes and for liquid 

 cultures that are transported or mailed. Sometimes a layer of melted 

 agar is added above the culture, for example of Lactobacillus in milk, to 

 accomplish the same purpose. This effects a tighter seal, since wax may 

 shrink away from the glass and permit leakage of fluid, but a wax seal has 

 the advantage, on the other hand, that a pipet can be pushed through it 

 without disturbing the seal or clogging, thus permitting easy transfer of 

 the culture. 



White mineral oil or lipuid petrolatum, of the medicinal grade, is com- 

 monly used to seal cultures of various kinds of microorganisms, on agar 

 slants or stabs and also broth cultures, against desiccation and oxidation, 

 thus preserving them for months or sometimes several years beyond the 

 survival of unsealed cultures. Both vaspar and mineral oil can be steril- 

 ized by autoclaving at 15 lb pressure for 60 min and then driving off any 

 entrapped moisture by heating in a drying oven at 110°C for about 1 hr. 



DORMANT CONSERVATION 



Lyophilization. The essentials of the freeze-drying method of pre- 

 serving cultures may be stated as follows: (1) Obtain as dense as possible 

 a suspension of cells of the organism to be preserved by washing down a 

 culture that is grown to the proper stage of maturity with a suitable fluid, 

 commonly serum, milk, or 3 per cent lactose; (2) transfer 0.1-0.2 ml of 

 the suspension to vials; (3) quick-freeze these in a mixture of dry ice and 

 95 per cent alcohol; (4) evacuate while frozen until completely desiccated; 

 (5) hermetically seal the vials while evacuated. There are many varia- 

 tions of the method, and some elaborate types of equipment have been 

 devised to accommodate a large number of vials at one time and to con- 

 trol environmental factors. 



In commercial establishments and some institutions where lyophilized 

 preparations are produced in great quantities, it is customary to use glass 

 tubing, cut in short lengths and sealed at one end, or very small culture 

 tubes, which are handled en bloc through the freezing, dehydrating, and 

 sealing processes, by attaching them to a multioutlet manifold. The 

 tubes or vials may be as small as 3-4 mm in outside diameter, 1.5-2 mm 



