Long-Term storage of Tularemia Agent strains

Keeping microorganisms alive, saving initial practically vital properties, is a major issue in microbiology. Objective of this research was to study preservation of the key biological properties of lyophilized cultures of 18 F. tularensis strains of different subspecies from the cultures collection of the «48 Central Research Institute» of the Ministry of Defense of the Russian Federation under varying conditions of preliminary preparation (including animalization through the guinea pigs’ body or without it). The microbe storage life at below-freezing temperatures ranged from 5 to 50 years. Materials and methods. The survival of microbes in the process of drying and subsequent storage was determined in accordance with the commonly-accepted methods; morphological, cultural, biochemical, antigenic, and pathogenic properties of F. tularensis strain cultures with different storage term and conditions for preparation to lyophilization were assessed and compared with datasheet specification. The results and conclusions. The research showed that under preliminary stabilization of F. tularensis strain properties using animalization, the storage in lyophilized state at below-freezing temperatures under vacuum for up to 50 years or less protected them from changes in phenotypic properties. At the same time, after lyophilization of cultures, which were repeatedly sub-cultured on dense nutrient media without initial signs of properties’ breach, phenomena of dissociation, the decrease in titers of cultures in the agglutination reaction from 2 to 4 times, and an increase in LD50 index by times were found.

1. Kudryavtseva T.Yu., Trankvilevsky D.V., Mokrievich A.N., Popov V.P., Morozova N.S., Zarochentsev M.V., Mazepa A.V., Okunev L.P., Kholin A.V, Kosilko S.A., Fedorov Yu.M., Khramov M.V., Dyatlov I.A. [Epizootic and Epidemic Situation on Tularemia in the Russian Federation in 2015 and Prognosis for 2016]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous

2. Mokrievich A.N., Timofeev V.S., Kudryavtseva T.Yu., Ulanova G.I., Karbysheva S.B., Mironova R.I., Vakhrameeva G.M., Gubareva T.I., Pavlov V.M., Dyatlov I.A. [Isolation of Central Asian Subspecies of Tularemia Agent in the Altai Territory]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections] 2013; 1:66-9. DOl: 10.21055/0370-1069-2013-1-66-69.

3. Whipp M.J., Davis J.M., Lum G., de Boer J., Zhou Y, Bearden S.W., Petersen J.M., Chu M.C., Hogg G. Characterization of a novicida-like subspecies of Francisella tularensis isolated in Australia. J. Med. Microbiol. 2003; 52:839-42. DOI: 10.1099/jmm.0.05245-0.

4. WHO Guidelines on Tularemia. Epidemic and Pandemic Alert and Response; 2007. 115 p.

5. Guidance for the operation of biological research centres (BRCs) Part 2. Microorgamsmsdomen 2001. [Электронный ресурс]. URL: http//www.oecd.org/sti/biotech/38l77417 (дата обращения 05.11.2015 г).

6. Kupletskaya M.B., Netrusov A.I. [Livability of lyophilized microorganisms after 50 years of storage]. Mikrobiologiya. 2011; 80(6):842-6.

7. Pokhilenko V.D., Baranov A.M., Detushev K.V [Long-term storage methods for collection cultures of microorganisms and development trends]. Izvestiya VUZov. 2009; 4:99-121.

8. Solov'ev E.A., Sayapina L.V., Osina N.A., Davydov D.S., Bondarev V.P. [Characteristics of Phenotypic and Genetic Properties of Francisella tularensis 15 NIIEG Vaccine Strain with an Extended Storage Period]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2015; 4:91-5. DOI: 10.21055/0370-1069-2015-4-91-95.

9. Gracheva I.V., Osin A.V. [Mechanisms of Damaging Bacteria during Lyophilization and Protective Activity of Shielding Media]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2016; 3:5-12. DOI: 10.21055/0370-1069-2016-3-5-12.

10. Metelkin A.I. [Tsenkovsky L.S. - the Founder of the National Microbiologists School, 1822-1897]. M.: “Medgiz”. 1950. P. 227-56.

11. Saltykov R.A., Motornaya V.P., Sirotyuk L.V. [25-year studies of the stability of tularemia vaccine strains’ properties]. Mikrobiologiya. 1972; 12:64-70.

12. Faibich M.M. [Stabilization of vaccinal preparations in the process of drying and storing]. Zhurnal Mikrobiologii Epidemiologii i Immunobiologii. 1968; 2:59-66.

13. Onishchenko G.G., Kutyrev V.V., editors. [Laboratory Diagnostics of Dangerous Infectious Diseases. Practice Guidelines]. M.: “Shiko” CJSC; 2013. 560 p.