The hay infusion method

The hay infusion is a time-honored method for securing bacteria for study. Pour hot water on a handful of hay, and filter the fluid through blotting paper. Place the fluid in a glass dish, and cover with a piece of glass to keep out the dust. When the fluid begins to appear turbid, bacteria will be abundant. The active movements are easily observed in a mount from the turbid water. As the bacteria pass into the resting condition, they form a scum on the surface of the water. Usually, the first to appear is a somewhat rod-shaped form, the Bacterium termo of the older texts. Spirillum and Coccus forms often appear later.

Fine preparations may be obtained by inoculating a mouse with Anthrax, and then cutting paraffin sections of favorable organs. For making mounts of a dangerous form like Anthrax, secure properly fixed material from a bacteriologist. Stain in gentian-violet or crystal-violet. The following schedule gives good results with Anthrax and many other bacteria:

1. Gentian-violet, 5 minutes. 2. Rinse in water a few seconds. 3. Gram's solution (iodine 1 g., potassium iodide 2 g., water 300 c.c.) until the color is almost or quite black; this will generally require 1 or 2 minutes. 4. 95 per cent alcohol until the color has nearly disappeared. 5. Rinse in water and examine. If the bacteria are well stained, a counter-stain may be added. 6. Light green or erythrosin, 5 seconds; or Bismarck brown, 5 or 10 seconds. 7. 95 and 100 per cent alcohol, dehydrating as rapidly as possible. Not more than 5 or 10 seconds can usually be allowed. 8. Xylol, 1 to 5 minutes. 9. Balsam.

After the rinsing in water of stage 5, the preparation may be dehydrated rapidly in 95 per cent and 100 per cent alcohol, and then stained for 5 or 10 seconds in orange dissolved in clove oil. From the clove oil, transfer to xylol and mount in balsam.

With sections not more than 5( thick, excellent results can be obtained by staining in iron-alum haematoxylin.

The following rapid method gives fairly good results:

1. Place on a clean cover a drop of water containing the bacteria and dry completely in a flame or on a hot plate. 2. Stain 2 to 5 minutes in gentianviolet or methyl violet. 3. Rinse quickly in water. 4. Dip into 95 per cent alcohol to reduce the stain. 5. Remove most of the alcohol by touching a corner of the cover with filter paper and then dry completely by passing through a flame. 6. Mount in balsam.

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