An Improved Staining Method for Demonstrating Bacterial Capsules, with Particular Reference to Pasteurella

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Abstract
The amt. of culture that can be picked up by a fine, straight platinum wire from a colony or an agar slant is suspended in a loopful of physiol. saline containing 0.5-1% phenol and 10% blood serum, and spread into a thin film on a clean polished slide. To fix the dried film, the slide is dipped rapidly into methyl alcohol, drained, and flamed to burn off the excess alcohol. The prepn. can then be stained for 30 sec. to 1 min. with any of the common bacterial stains, and washed with water. No particular advantage was found in washing the slide with 20% CuSO4 soln., as in Hiss''s method. With crystal violet (prepd. as in Hucker''s modification of the Gram stain) the organisms stain dark blue to black, whereas the background takes a light blue or purple stain and the capsule appears as a distinct colorless halo around the organism. The capsules can also be demonstrated in a prepn. stained by the Gram method, using dilute carbol fuchsin as a counterstain, if the film is prepd. and fixed as described above. With cultures of P. mastiditis, it was found that the organisms from the iridescent peacock-blue (smooth) colonies were 90-100% encapsulated, whereas those from the translucent grey to opaque (rough) colonies showed little or no encapsulation. Capsules have also been demonstrated by this method in smooth cultures of Pasteurella septica, Salmonella pullorum, S. suipestifer, Escherichia coli, Eberthella typhosa, Shigella gallinarum, and Brucella abortus.

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