The preservation of bacterial cultures under paraffin oil.

The preservation of a large collection of bacterial cultures is often a task of major proportions, especially if one wishes to maintain weakly viable and/or fastidious species. Variation in physiological or in morphological characteristics is frequently an undesirable consequence when stock cultures are held for some time, unless special methods are used in the preservation process. The method of Lumibre and Chevrotier, described in 1914, appeared to avoid the variation mentioned above and to allow for considerable longevity of the culture. These authors were successful in preserving strains of gonococci for several months under sterile paraffin oil. Morton and Pulaski (1938) reviewed the literature on the application of the method by other investigators and reported its advantages when preserving stock strains of normal and variant types of pathogenic and nonpathogenic bacteria. Forty-four strains of bacteria, mostly pathogens, from 22 genera were included in their study. The value of the method was recorded by Morton and Pulaski, as follows: (a) It greatly reduces the frequency of contamination, especially with molds, thus permitting cultures to be maintained with greater success in surroundings which are not conducive to precise bacteriological work. (b) No preliminary treatment of the cultures is necessary. (c) Practically all the organisms tested live longer under oil than in the control tubes. (d) Changes in cultural and biochemical characteristics-other than the sometimes-prolonged lag phase of growth on subculturing-have not been observed. (e) The cultures are available at all times for transplantation without interfering with the preservation of the stock cultures. (f) The method is applicable to single colonies or mass cultures. (g) It is especially advantageous in working with unstable variants, where occasional transferring to fresh media or growth in mass culture results in a change in the developmental stage of the strain. (h) No seals, such as rubber caps, waxes, cements, etc., are needed for the culture tubes. (i) No special apparatus is required, such as a centrifuge, desiccator or vacuum pump. Since 1938, the literature records that Sterzi (1939) preserved fragments of syphilomata (Truffis strain) for 40 days under a mixture of horse serum and paraffin oil. Typical staining characteristics and the form of the treponemata persisted throughout this period. Hac (1940) used the methods of Morton and Pulaski to preserve 30 strains of gonococci. All strains survived two transfers made at 12-month intervals. This study is especially interesting since the cultures were grown on five different media before applying the paraffin oil. The strains that remained viable longest were grown on Huntoon's agar. Simmons (1942) preserved streptococci of animal origin for 12 months, some Group A streptococci for 30 months, staphylococci for 24 months, and Corynebacterium diphtheriae for 19 months by layering 24-hour agar-slant cultures with heavy paraffin oil. Groups B, C, and G streptococci survived 15 to 19 months when preserved by this method. Neisseria meningitidis grown on liver-agar stabs survived for 12 months even when held at 37 C. Sherf (1943) demonstrated the practicability of the method by maintaining the very fastidious plant pathogen Corynebacterium sepedonicum for 18 months under oil without loss of pathogenicity. Similar results were obtained with Peudomonas medwaginis and Xanthomonas phaseoli. Certain plant-pathogenic fungi remained viable under oil immersion for 6 months and produced normal spore forms and mycelium when removed. These were Fusarium eumartii, F. oxysporum, F. avenaceum, F. lycopersici, and an Alternaria species. Wernham (1946) had similar success with cultures of Diplodia zeae, Gibberella zeae, Cochliobolus heterostrophus, and Nigrospora oryzae. Norris (1944) used the methods of Sherf and successfully preserved cultures of other pathogenic fungi and bacteria for 6 months, even though storage was at room temperature. His cultures were strains of Pleospora herbarum, Pleospora rehmiana, Pseudoplea trifolii, Colletotrichum trifolii, Ascochyta imperfecta, Vermicuaria species, Fusarium species, Macrosporium species, and Rhizobium from Medicago scutellata. He, too, concluded that the method was very effective and efficient since it saved much time, media, and materials, and eliminated the necessity of making frequent transfers of the cultures. Gordon and Smith (1947) held 43 genera of bacteria