The Drying and Preservation of Bacterial Cultures

Abstract

SUMMARY: Working details are given for keeping as desiccates a collection of some 1500 strains of bacteria. The efficacy of drying, the various methods of freezing, and the effect of storage were tested by viable counts, using a spinning bottle modification of the roll-tube method. The survival rate of bacteria suspended in broth or other protective colloids, and subjected to freezing at -78", varied with the species from about 100 yo with the resistant Staphylococcus aureus to about 10 yo with the sensitive Neisserz'a gonorrhoeae, and from about 100 to 1.5 yo or less, respectively, when the organisms were suspended in saline. The percentage of organisms which survive the freeze-drying process was found to vary with the species, from 100% to less than 1.0 yo when the organisms were suspended in broth and from 100 yo to no survivors when the organisms were suspended in saline. The storage loss of dried cultures was found to be a function of the storage temperature ; however, suitably dried cultures could be kept a t room temperature for very long periods. By far the most important factor influencing loss on storage was the presence of traces of moisture, and to ensure optimal survival the cultures must be as dry as possible. It appears that even with adequately dried cultures the presence of oxygen is deleterious.The survival rate of bacteria in a sample of dried culture after heating to 80' for 1 hr., determined by plating a suspension of the heated sample in broth, provides a simple measure of the capacity of the particular batch of the dried culture to remain viable on storage at ordinary temperatures.The drying of bacteria arouses most general interest as a solution of the utilitarian problem of avoiding the continuous subcultivation of strains and of maintaining them in any particular metabolic state. The essential requirements of a collection of dried cultures is that, with the routine employed, all strains should be viable as required, and not that particular batches of dried cultures should survive for very long periods. On this point, rather strangely in view of the spate of published methods for the preservation of bacteria, there is almost no information. We have maintained as desiccates a collection of some 1500 strains of different organisms, mainly pathogenic bacteria, for several years and have examined quantitatively the effect of freezing, drying, and storing dried bacteria under different conditions. METHODSRoutine for drying bacterial cultures. The method used is that of Swift (1937) with minor modification%, and the routine we adopted is as follows. Most aerobic organisms are grown for 24-48 hr. on agar slopes of suitable media, and the growth emulsified with about 2 ml. of nutrient broth. The broth, containing c. 3 g.N/l., is made by extracting fresh horse muscle with a papain digest of horse muscle containing c. 1.5 g.N/1.Most anaerobic organisms are grown in 250 ml. amounts of nutrient broth, containing 0.01 yo thiolacetic acid, incubated for 24-48 hr., and the culture

The Effect of Phenol, 2-Phenoxyethanol and Cetyltrimethylammonium Bromide on the Oxidation of various Substrates by Escherichia coli

“…coli NCTC 5934. It was subcultured in nutrient broth, and freeze-dried (Proom & Hemmons, 1949). Culture medium.…”

The Influence of Rehydration on the Viability of Dried Micro-Organisms

“…On the other hand, several other fluids have been used, and in amounts which have also varied rather widely. Broth, which was recommended by the Medical Research Council (1958), was used by Stamp (1947), Proom & Hemmons (1949), Rhodes (1950) and Annear (1956). The volume of broth used by these workers has varied from less than 5 to more than 100 times the volume of the water removed.…”

The Preservation of Lactobacilli by Freeze-drying

“…The data in Table 5 show that survival rate varied with species, as Proom & Hemmons (1949) and Rhodes & Fisher (1950) found with other organisms; strain differences were not so marked as in Record & Taylor's (1953) work with Freeze-drying of 1 actobacilli Escherichia coli. All the freshly isolated cultures gave high survival rates save for two in group V; however, most of the strains in this group dried relatively poorly.…”

“…The apparatus, illustrated in P1. 1, was similar to Swift's (1937) and also to that used by Proom & Hemmons (1949), the chief components being an efficient vacuum pump ('Speedivac', W. Edwards and Co. (London) Ltd.), a desiccator and a large lagged box. Vacuum in the system was determined by means of an Edwards's 'Vacustat', and dry air was introduced as required through a column of silica gel.…”