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Indian Journal of Medical Microbiology, Vol. 26, No. 2, April-June, 2008, pp. 198-199 Correspondence Preservation of Vibrio cholerae by suspension in normal saline Chitnis S, Chitnis V, Nanda H, Chitnis DS Department of Immunology, Choithram Hospital and Research Centre Date of Submission: 13-Aug-2007 Code Number: mb08059 Dear editor, Sporadic cases and small outbreaks of cholera continue to occur in Asian countries. Epidemiological and microbiological studies demand preservation of the isolated strains in the region. Vibrio cholerae can be preserved by lyophilisation and at ultra low temperature [1],[2] however, this facility may not exist in most laboratories. Members of Enterobacteriaceae family can be well preserved on egg media or by stab method [3] but not V. cholerae (personal observation). Survival of V. cholerae for more than a month is not possible even on nutrient agar slants stored at 4°C. Our accidental observation that V. cholerae suspended in normal saline pH 8 and kept at 4°C retained viability for more than six months prompted us to study preservation of suspension of V. cholerae in normal saline with pH 8 solution stored at 4°C. The pH of the normal saline is adjusted to 8 because vibrios prefer alkaline pH and are susceptible to acid pH. Classical V.cholerae 569 B Inaba procured from Haffkine institute, Mumbai, India was received as lyophilized culture. Nine isolates of V.cholerae El Tor Ogawa isolated during 2003-2006 from cases of cholera in Indore city and preserved in 15% glycerol containing nutrient broth (Hi Media, India) at −70°C were included in the study. The nutrient agar (Hi Media, India) slants were inoculated with cultures and incubated overnight at 37°C. The growth on nutrient agar slant was harvested in 5 ml of the normal saline pH 8 and the opacity adjusted to 0.5 at 635 nm using colorimeter (ERBA Japan). The suspensions were aliquoted in 9 mL amount in 10 mL injection vials in triplicate. The vials were capped with rubber bungs, sealed with aluminium caps and stored in domestic refrigerator. The temperature was recorded every morning and evening round the year and was in the range of 4-8°C. The initial mean count for standard strain of V. cholerae 569B Inaba and other clinical isolates of V. cholerae were 0.8-1.5 x 10 9 CFU/mL in normal saline. The suspension of standard strain of V. cholerae 569B Inaba and isolates number 4 and 8 (randomly selected) were subjected to viable counts [1] on the day one and subsequently once a month for 12 consecutive months. One hundred micro litre of the ten fold dilutions were plated on nutrient agar plates in duplicate and the plates showing colonies 50-200 were used for the viable count estimation. The remaining culture suspensions were subjected to viable count on day one and then after 12 months. The viability study results for the standard strain 569B and isolate number 4 and 7 were similar [Figure]. The colony morphology remained typical after revival from normal saline and the subculture at the end of one year did not show any change in staining and biochemical characters. The antigenic properties with regards to agglutinability with the antisera remained the same. The mean counts of surviving bacteria among the nine isolates of V. cholerae were 0.8-1.8 x 10 4 CFU/mL in normal saline. Thus, consistent survival of V. cholerae was seen in normal saline with pH 8. The suspensions in normal saline showed almost four log fall in 1 month of storage. However, subsequently there was no marked fall in the viability during an observation period of 12 months. Initial fall in viable count from 10 9 to 10 5 CFU/mL at the end of 1 month is possibly due to accumulation of lethal metabolites and/or depletion of nutrients. [4] However, the subsequent loss in viability in normal saline was minimal till 12 months. The survival of vibrios could be due to very low metabolic activity without autolysis in the suspending fluid. The storage in domestic refrigerator is practical for any average microbiological laboratory in the economically developing countries that do not have freezers or lyophilizing equipment for preservation and the simple method will prove valuable for preservation of V. cholerae. Acknowledgement We are thankful to Choithram Hospital and Research Centre for providing facilities during the research work. We also acknowledge the Council of Scientific and Industrial Research, India for the award of Senior Research Fellowship to Dr. Sheetal chitnis to carry out the research work.References
Copyright 2008 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb08059f1.jpg] |
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