Academic journal article Bulletin of the World Health Organization

Comparison of Different Culture Media and Storage Temperatures for the Long-Term Preservation of Streptococcus Pneumoniae in the Tropics

Academic journal article Bulletin of the World Health Organization

Comparison of Different Culture Media and Storage Temperatures for the Long-Term Preservation of Streptococcus Pneumoniae in the Tropics

Article excerpt

Bulletin of the World Health Organization, 2001, 79: 43-47.

Voir page 46 le resume en francais. En la pagina 47 figura un resumen en espanol.

Introduction

Pneumococcal infections cause substantial morbidity and mortality in infants and children in many developing countries (1, 2). Studies suggest that Streptococcus pneumoniae, together with Haemophilus influenzae, may be responsible for half of all pneumonias in children and 20-40 of all bacterial meningitis cases in the developing world, but little is known about their serotype distribution and age-specific disease rates (3, 4). Information on the regional distribution of pneumococcal serotypes is essential for the development and use of appropriate pneumococcal vaccines in developing countries (3). A multi-centre hospital surveillance study was therefore initiated in Vellore, Tamil Nadu, India in 1993 under the India Clinical Epidemiology Network (INDIACLEN), with support from the US Agency for International Development through the International Clinical Epidemiology Network (INCLEN). The objective was to describe the epidemiology of S. pneumoniae invasive infections in six hospitals located in widely different areas of India. Clinical samples and/or bacterial strains were sent from these centres to Vellore for confirmation of S. pneumoniae, including serotype and antibiotic susceptibility. All strains were preserved by lyophilization or freezing at -70 [degrees] C.

An essential component of this prospective surveillance study was the ability to preserve pneumococcal isolates reliably for comparatively long periods of time. S. pneumoniae, being a fragile organism, does not survive well in broth cultures in hot climates and can survive on culture plates for only two or three days. While freezing at -70 [degrees] C and lyophilization are recognized to be effective techniques, the high cost and difficulty in acquiring and maintaining equipment and test materials prevent their use in low-income regions of the world. We evaluated alternative low-cost techniques using locally available products against the standard techniques of -70 [degrees] C freezing and lyophilization. In order to identify suitable methods for the preservation of bacterial isolates for tests in developing countries, this study evaluated a variety of media and storage temperatures for the preservation of S. pneumoniae in a tropical setting.

Materials and methods

Bacterial strains

Cultures of S. pneumoniae, isolated from clinical samples in the Department of Clinical Microbiology, Christian Medical College and Hospital, Vellore, Tamil Nadu, India, were used for the study. We included capsular types 1, 5, 7, 19, and 23 -- representing the types commonly encountered in both India and the USA -- which were identified and serotyped by methods standardized in our laboratory (5). The isolates were subcultured on trypticase soy blood agar and then subjected to different methods of preservation as described below.

Freezing in glycerol-chocolate broth

Samples (0.5 ml) from an overnight glycerol-chocolate broth culture, supplemented with sterile glycerol (final concentration, approximately 8.7% v/v), were placed in freeze-resistant storage vials (Provial, Laxbro, Pune) and stored at-20 [degrees] C or-70 [degrees] C.

Freezing in skimmed milk

Samples (0.5 ml) of suspensions, made from harvesting overnight cultures on sheep blood agar slopes with 1.0 ml sterile skimmed milk, were dispensed in storage vials and stored at -20 [degrees] C or-70 [degrees] C.

Freezing in rabbit blood

Samples (0.5 ml) of culture suspension, obtained by harvesting overnight growth from sheep blood agar slopes with 1.0 ml sterile defibrinated rabbit blood, were placed in storage vials and stored at -20 [degrees] C or -70 [degrees] C.

Freezing in sheep blood

Samples (0.5 ml) of culture suspension obtained as described for rabbit blood, but harvested with 1. …

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