‘Spiromed’: a rapid and inexpensive Spiroplasma isolation technique

Abstract

An isolation technique for Spiroplasma species (class: Mollicutes) was developed which did not require the labour and cost intensive process of sample filtration. Isolation of spiroplasmas depended upon the ability of these organisms to resist high concentrations of a variety of antibiotics during 45 min prior to subculture in spiroplasma growth medium SP4 enriched with penicillin, sodium hypochlorite and mycostatin. Some 1450 insect samples were treated without contamination of culture media by nondesirable bacteria or fungi, while 96 water samples were associated with an acceptable contamination rate of 6%. The latter figure could probably have been improved upon had the concentration of penicillin in the final culture medium been increased. Eleven Spiroplasma strains or species grew successfully using this isolation technique. Utilisation of 96-well plastic titration plates meant that large numbers of arthropods could be processed rapidly. Non significant difference was observed in the isolation rate achieved using ‘Spiromed’ and the classical approach of filtration and culture in SP4 medium.