MAINTENANCE AND PRESERVATION OF CULTURES 103 



ones by placing a pad of cotton in the base; then the small vial is inserted 

 and in turn is covered with a wad of shredded asbestos which is pressed 

 rather firmly against its top. The asbestos plug protects the cotton from 

 scorching and the inner vial from excessive heat while the upper part of 

 the large vial is drawn out to a thin neck. This step is carried out by 

 heating the vial, about 4 cm from its open end, in a gas-air torch set to 

 give a narrow pointed flame, rotating it so as to soften and constrict 

 uniformly, until an isthmus about 5 cm long and 2-3 mm in outside 

 diameter can be drawn out. Great care must be taken that the bore of 

 the isthmus is not closed in this process. The open end of the vial, which 

 still has its original diameter, serves for attachment to a manifold for the 

 final exhaustion and sealing. 



A six-outlet manifold is convenient for this purpose, and for expeditious 

 progress if many vials are to be sealed, two such manifolds, each provided 

 with a shutoff valve, are connected by a Y tube to a manometer and then 

 to a vacuum pump. The connection between vial and manifold outlet is 

 made through a snug-fitting rubber cork, which must be kept well lubri- 

 cated with stopcock grease. The vials remain on the manifold under as 

 high a vacuum as the apparatus will pull (it should be at least 100 to 

 150 /x, and 50 is preferable) for about 10 min; then each is separated in 

 succession by burning off the neck in a micro bunsen burner. Care must 

 be taken not to overheat the rounded end of the vial back of the tip; 

 otherwise it may collapse because of the vacuum within and thus form a 

 precarious seal. 



Subsequently, usually within a day or two and again for about 10 days, 

 before the preparations are filed away as reliable, the vials are tested with 

 a high-frequenc}^ vacuum tester. A glow within the vial when the tip of 

 the apparatus is brought near it is indicative of a suitable vacuum, but 

 only momentary exposure to the discharge is advisable, as microscopic 

 perforations of the glass may result, with loss of vacuum. 



If the survival capacity of the organism under lyophilization is not 

 known, a check for viability should be made after one month and again 

 after six. If viability is demonstrated at these intervals, it may be 

 assumed that the preparation will remain viable for the average survival 

 period of the species. This may vary from 2 to 10 or more years. The 

 over-all viability gradually declines, the rate depending largely on the 

 number of viable cells in the original preparation. 



Most lyophilized preparations survive well if stored at room tempera- 

 ture, but some species of Hemophilus^ Lactobacillus, Neisseria, Pasteurella, 

 and doubtless others are short lived unless refrigerated. 



For reconstituting the culture the tip of the vial above the asbestos wad 

 is heated moderately in a gas flame, then a drop of water is placed on it. 

 The resultant cracking breaks the vacuum, and the inner vial, protected 

 by its cotton plug from contamination, is withdrawn. Reviving the 



