Flagella Staining of Anaerobic Bacilli

Abstract

An improved technic for staining flagella of anaerobic bacilli is descr. This involves the use of the flagella stain descr. by Bailey, and careful attention to several details. Those considered of most importance are listed, as follows At least 2 rapid subcultures were made in broth containing peptone, meat infusion and glucose; the broth was centrifugalized and the cells washed in sterile dist. water before inoculation upon anaerobic agar slants. All dist. water used was sterilized by passing-through a sterile Mandler filter to remove precipitate; the temp, of the water ranged from 30[degree] to 37[degree] C, and the pH was approx. 6.8. The harvested cells were incubated at 37[degree] C 1/2 hr. before staining to obtain better distribution. A small drop of suspension was placed near one end of specially prepd. slides, allowed to flow slowly over the surface, and placed in the incubator, without heating, in a slanted position to dry. Freshly made and freshly filtered solns. were always used, and (he stains washed off with dist. water blown gently through a wash bottle. With the use of carbol fuchsin, careful experimentation was required as to the amt. of heat necessary to stain the flagella. The limits of time appeared to be between 1/2 and 1 min. The slides were air-dried and not blotted. In the writer''s hands, the following technique has proved of value for the staining of flagella of pathogenic bacteria where ordinary methods have failed. A 3-yr.-old stock culture of a pathogenic strain of Bacillus difficilis was used. A guinea pig was injd. subcut. with 2 cc. of a 48-hr. brain-broth culture, and the bacillus recovered at autopsy. With careful and appropriate microscopical and cultural methods the bacillus was found to correspond to the original descr. and pathogenicity for another guinea pig proven. These cells produced satisfactorily stained flagella; the control slides, using the stock culture, showed rods with few and faintly stained flagella.