Long-Term Preservation by Freeze-Drying of Pathogenic Bacteria of the Czechoslovak National Collection of Type Cultures

Abstract

On the basis of long-term experience with the preservation of the pathogenic bacteria of the Czechoslovak National Collection of Type Cultures, it is concluded that the suspending medium is a factor of primary importance in the preservation of these organisms in the freeze-dried state. The primary index of suspending medium effectiveness is the level of protection it affords against the adverse effects of low (subzero) temperatures, dehydration, long-term storage, and rehydration. A number of simple and compound suspending media were studied for their protective effects on cultures from over 50 genera of predominantly pathogenic bacteria. The results indicate that not only each bacterial genus and species but also frequently every strain of a given serological type must be looked upon as a distinct biological entity, the preservation of the viability, activity, and desirable characters of which must be tested in different protective media in preliminary experiments. Heat testing (at 75 to 100 C) of freeze-dried cultures kept under high vacuum is recommended as a good indicator of adequate protection provided by a suspending medium. With the freeze-drying technique used, the best results for the largest number of species were obtained with a mixture of calf serum or defibrinated sheep blood and lactose solution (to a final concentration of 5 to 10%) and Annear's peptone-containing medium. Individual suspending media were evaluated in relation to individual strains, and recommendations are made as to the preferred medium on the basis of control tests performed at 5-, lo-, and 15-year intervals.Twenty-five years have elapsed since the establishment of the Czechoslovak National Collection of Type Cultures (CNCTC) at the Institute of Epidemiology and Microbiology (in 197 1, the Institute of Epidemiology and Microbiology became a part of the newly established Institute of Hygiene and Epidemiology under the name Center of Epidemiology and Microbiology). At present, the CNCTC contains over 7,000 strains of bacteria, viruses, and fungi, the majority of which have been successfully preserved by the relatively modern method of freeze-drying, first employed at the CNCTC in 1948. The freeze-drying technique has made important headway both in Czechoslovakia and abroad (16), the continuing refinement of this technique having proceeded along the lines of improvements in the freeze-drying apparatus (19) and in the suspending media used for freeze-drying ( 1 , 4 , 6 , 7, 9, 10, 12, 20, 2 1).A number of laboratories have recently summarized their long experience with freezedrying as applied t o the preservation of microorganisms (3,5,9,10,17) and by so doing have provided valuable information for culture collections and other laboratories interested in microorganism preservation. We present our experience on the preservation of the pathogenic bacteria maintained and stored at the CNCTC and of various recently isolated bacteria maintained at the National Reference Laboratories of the Institute of Hygiene and Epidemiology (22). MAT...

Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperatures

“…In cattle with a weakened pulmonary defence, inhalation of the bacterium results in rapid colonization of the lungs and development of an economically important disease called shipping fever pneumonia (Rehmtulla and Thomson 1981). Lyophilization and freezing have been used for the preservation of this species as well as other members of the family Pasteurellaceae (Šourek 1974; Gibson and Khoury 1986; Aulet de Saab et al. 2001).…”

Simple bacterial preservation medium and its application to proficiency testing in water bacteriology

“…Sourek (17) suggested that every bacterial strain of a given serological type had different viability characteristics. This conclusion was supported by the results of Gray and Lowe and by our own investigation.…”

“…The evaluation of the ability of a bacteriological preservation medium to maintain stable cul- tures depends, to a large extent, on the methods used to recover and enumerate the cells after storage (5,15,17). Our inability to duplicate the results of Gray and Lowe, using nutrient with boric acid, may be related to our use of a pour plate, rather than a most-probable-number, technique for enumeration.…”

“…Unfortunately, the bacterial preservation techniques useful in the qualitative proficiency testing programs of clinical bacteriology, such as Stuart transport medium (3), Bact-Chek disks (7), and lyophilization (9), are not suitable for quantitative investigations in water bacteriology. Although many bacterial preservation methods will ensure that the organisms will remain viable, none are able to maintain a constant number of bacterial cells (2,6,15,17).…”

Attempt at gene reversion of a desired phenotype as a rescue against strain degeneration

“…Strain degeneration, which has been observed in a strain of Aspergillus versicolor producing versi-Colin, an antifungal antibiotic, is a fairly well established phenomenon. The maintenance of multiple cultures of an active strain has become a standard practice to meet the exigency (Raper & Alexander 1945;Buell & Weston 1947;Sourek 1974;Wellington & Williams 1977), although at times regeneration becomes an absolute need in the interest of a given research programme. Mutation techniques have been used successfully in the production of high, low or non-producer mutants or sometimes even producer mutants with an altered phenotype (Boronin 1970;Edwards et al 1974;Blumauerova et al 1976Blumauerova et al , 1978aLee & Ryu 1979;Simpson & Caten 1979;Takiguchi et al 1979…”