Enrichment and isolation of aerobic chemoheterotrophic spirilla from mud and sand samples.

Abstract

Pure culture isolation of aerobic chemoheterotrophic spirilla from mud and sand samples collected from various freshwater and seawater areas in Japan was accomplished by applying the following three methods in the sequence shown: (i) boiled shellfish infusion method for enrichment of spirilla; (ii) glass capillary method for selection of spirilla; and (iii) ordinary streak plate method for pure culture isolation of spirilla. The aerobic spirilla grew well in all enrichment cultures in the 20 experiments performed. Crude cultures containing abundant spirilla were obtained from all enrichment cultures by use of glass capillaries. Pure culture isolation of spirilla from the crude cultures was reliably achieved by the ordinary streak plate method.The putrid infusion of crushed raw shellfish was appropriate for the enrichment of freshwater as well as marine spirilla, and many strains were isolated by direct inoculation onto plates of nutrient agar or nutrient agar using seawater (1,2). Effective enrichment cultures of water spirilla were also obtained by adding the infusion of shellfish prepared by boiling to mud samples collected from freshwater or seawater areas (2), and glass capillaries were also used for the selection of spirilla in that study at the suggestion of Watanabe. Subsequently, the present author found that aerobic chemoheterotrophic spirilla could easily be isolated from mud or sand samples by combining the following three methods in the sequence presented: (i) boiled shellfish infusion method for enrichment of spirilla; (ii) glass capillary method for selection of spirilla; and (iii) ordinary streak plate method for pure culture isolation of spirilla. Then, the present study was carried out to ascertain whether effective enrichment cultures are acquired by chance or the methods are reliable for the enrichment and isolation of aerobic chemoheterotrophic spirilla. This paper deals with the details of the methods and presents the results obtained by applying the methods to mud and sand samples collected from various locations in Japan. 395