Pipette count method for obligate anaerobes.

The routine counting of obligate anaerobic bacteria is a serious problem in many laboratories. Reliable anaerobic chambers are either expensive or messy and cumbersome. Furthermore, the exposure of certain genera, notably Bacteroides and some spirochetes, to air may kill the organisms. The following technique does not require an anaerobic chamber, does not expose the organism to atmospheric oxygen, and has been used in our laboratory with a variety of genera including those mentioned above. All that is required is routine laboratory glassware, an inspection 10X binocular microscope, and a simple illuminator (Fig. 1) which can be constructed for a cost of less than three dollars. The organisms to be counted are dispersed into a screw-cap tube (13 by 125 mm) containing 9.0 or 9.9 ml of melted agar medium at 45 C. The sample is rotated with a number of glass beads or small marbles to eliminate clumping. Beads or marbles are added to fill the tubes as full as practicable to minimize incorporation of oxygen during shaking. Liquid dilutions are made to the desired end point with prewarmed 1.0-ml pipettes. The medium is drawn up into the pipette for a distance of 3 or 4 cm past the upper volumetric mark and held for a few seconds to allow the medium in the tip to harden. The pipette can then be put in horizontal position into a rack or canister and incubated without further manipulation. After 48 hr of incubation, oxygen will diffuse into the medium less than 1 cm from either end, and it is necessary to eliminate these areas from the count. For this reason, we prefer to use pipettes calibrated in 0.1 ml so that the lower 0.1 ml can be eliminated from the count and an equivalent volume length can be added to the upper portion of the pipette. Populations of 1,000 cells per pipette can be determined with the aid of the inspection microscope and counting chamber. When very high populations are involved and maximal accuracy is not needed, the calibration of the pipette will permit counting alternate tenths or other dilutions. INSPECTION MICROSCOPE