Metronidazole Strip Method for Differentiating Single Colonies of Obligate from Facultative Anaerobes on Primary Agar Plates

Metronidazole is active in vitro against obligate anaerobes such as Bacteroides, Fusobacterium, Veillonella, Peptococcus and Peptostreptococcus, but has no activity against facultative anaerobes. However it is generally less active against non-sporing Gram-positive bacilli such as Actinomyces and Propionibacterium, it is also less active against some species of Peptostreptococcus, but less sensitive strains are usually not truly anaerobic. Metronidazole may, therefore, be a useful tool in differentiation of obligate anaerobic bacteria.And so paper strips each containing 5μg of metronidazole were prepared to differentiate single colonies of obligate from facultative anaerobes on primary plate cultures of mixed bacterial suspensions containing obligate and facultative anaerobes. This suspension was inoculated onto a GAM agar plate to obtain single colonies and a metronidazole strip was placed at right angles to the well of inoculum, so that it extended into the area where single colonies would be expected.After 48 hours of anaerobic incubation, in the area uniformly inoculated, a zone of inhibition around the metronidazole strip with intrazonal growth revealed the presence of an obligate anaerobe together with a facultative anaerobe. The area where this phenomenon is recognized is called “sparser zone”. Complete inhibition of growth around the metronidazole strip indicated that obligate anaerobes only were present. In the area where single colonies were obtained, colonies of facultative anaerobes were recognized even right up to the edge of the strip and colonies of obligate anaerobes were recognized at a distance of 5 to 15 mm at least from the edge of the strip. Therefore it seems to be able to differentiate single colonies of obligate from facultative anaerobes without examining the plate which was incubated aerobically.Facultative anaerobes have little influence on the metronidazole strip method. Although metronidazole was inactivated by certain facultative anaerobes such as Streptococcus faecalis, their presence in mixed culture with obligate anaerobes did not inhibit the activity of metronidazole. This was because of the more rapid bactericidal effect of metronidazole before any significant inactivation by the facultative anaerobes. Therefore the metronidazole strip method is able to detect obligate anaerobes on primary plate cultures of clinical specimens containing 102 to 103 times as many facultative anaerobes as obligate anaerobes so that it is difficult to obtain single colonies of the obligate anaerobes. In this case multiple anaerobic selective media are recommended for the isolation of the obligate anaerobes of all types.