Abstract
The following method of flagella and capsule staining is offered as rapid, simple, and dependable. The method has been especially designed for staining Bacillus proteus, Bacillus subtilis, and the various members of the colon-typhoid group.
The procedure is as follows: 1. Make a thin smear of a 15-24 hour agar growth of the bacteria in a loopful of water on a clean slide. Air-dry. Do not heat. 2. Cover with mordant (5% tannic acid, 3 parts; 10% ferric chloride, 1 part) for 2 minutes. 3. Put 7 drops of mordant in a watch crystal or other small receptacle and add one drop of Ziehl-Neelson carbol fuchsin stain. Mix. Add one drop of concentrated hydrochloric acid. Mix. Add one drop of formaldehyde. Mix. 4. Pour off mordant from slide and cover smear with the mixture prepared in paragraph 3. Apply 7 minutes. 5. Wash in running water. 6. Cover with Ziehl-Neelson carbol fuchsin stain (Basic fuchsin, 10 gm.; 95% ethyl alcohol, 100 cc.; phenol, 5% water, 1000 cc.) and steam gently for one-half minute. 7. Wash in running water.
The following observations are in order. A. Remove a small portion of the youngest marginal growth from an agar culture. The medium must not be dry. Add to droplet of water upon a slide. Allow this to stand a few minutes. Then transfer a loopful to a second slide. Work with this second slide. B. The mordant will keep indefinitely and thus can be prepared in quantity. The mixture prepared in No. 3 should be used fresh. C. The action of the mordant usually requires 2 minutes. D. Steaming of the stain must not be too severe. Instead of using the recommended stain in NO. 6, the more powerful mixture of anilin gentian violet and carbol fuchsin may be preferred by some. E. Filtered mordant and stain yield better preparations than unfiltered materials.
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