102 MANUAL OF MICROBIOLOGICAL METHODS 



inside. To fill such small vials or reconstitute the culture requires a 

 capillary pipet or hypodermic syringe, and the vials, especially if hard 

 glass is used, are difficult to open and are subject to considerable risk of 

 contamination by inrush of air when the vacuum is broken. 



The procedure which has long been in use (15 years) at the American 

 Type Culture Collection eliminates most of these difficulties, and espe- 

 cially the hazard of contamination when the culture is opened, by using 

 a double-vial arrangement, one within the other, the inner one plugged 

 with cotton like an ordinary culture tube. 



Specifications for the vials have been given. The small vials are 

 cleaned, cotton-plugged, and autoclaved in advance. Prior to use they 

 are labeled with glass-marking ink. Filling is done with an ordinary 

 measuring pipet, 1 ml size, graduated in 0.1 ml to a basal line (not to the 

 tip, as it is undesirable to blow the last drop of fluid into the small vial). 

 For most cultures, the use of slightly over 1 ml of fluid to wash down the 

 slope will proA^ide a cell suspension of sufficient density so that 0.15- 

 0.2 ml dispensed in each vial yields satisfactory lyophilized preparations. 

 Thus from one growing culture five preserved ones can be obtained. The 

 last drop or two from the pipet is customarily placed on an agar slant 

 and spread evenly over the surface or transferred to other suitable media 

 as a check on the purity of the culture during these manipulations. 



After filling, the cotton plugs are trimmed just above the rim of the 

 vials, and the vials are quickly transferred to a freezing mixture of 

 roughly equal parts of finety crushed dry ice and alcohol. A convenient 

 receptacle for this is a large petri dish, covered with a coarse wire screen 

 through Avhich the vials will pass and which holds them upright. The 

 freezing mixture need not be more than about 1 cm deep. As soon as the 

 culture has solidified, the vials are transferred by means of long forceps 

 to a stout bottle having a ground-glass top, to which is connected a 

 U tube terminating in a moisture trap and having a side outlet near the 

 top which is connected to a vacuum pump. The vessel holding the vials 

 is enclosed in a suitable container which is packed with coarsely crushed 

 dry ice, serving as a cold jacket to keep the material frozen through the 

 initial stage of dehydration. This will ordinarily be within an hour or 

 two, and this time should not be greatly exceeded, as prolonged freezing 

 at this temperature is inimical to survival. Throughout the process of 

 dehydration the distal end of the U tube, with the moisture trap, is kept 

 in a thermos vessel surrounded with dry ice and alcohol, hence serving as 

 the cold element of the distilling apparatus. The moisture trap serves to 

 protect the oil in the vacuum pump from absorption of water. The pump 

 must run until desiccation is complete, which will ordinarily require about 

 6 hr, but overnight operation is a convenient way to ensure enough time, 

 and the sealing is completed the next morning. 



In the meantime a set of the large vials is prepared to receive the small 



