One Solution Technique for Direct Microscopic Counting of Bacteria in Milk.

Abstract

A method has been devised for simplifying the direct microscopic method of Breed for counting bacteria in milk.

In the official Breed method, 0.01 ml. of milk is spread over an area of 1 square centimeter on a glass slide. When dry, the slide is immersed in xylene for at least one minute (to remove the fat from the milk); then dried, immersed in 90 per cent alcohol for one or more minutes (to fix the smear to the slide); dipped in Loeffler's methylene blue until the milk smear is overstained; washed in water; decolorized in alcohol; again washed in water; dried and examined under a standardized microscope.

The new method eliminates the use of two solutions, two Coplin jars and four separate operations, and substitutes a single solution which simultaneously dissolves out the fat; fixes the milk smear to the slide and stains the bacteria and leucocytes. Excellent contrast between bacteria and background is secured without recourse to decolorization. Any one of the following solutions may be used:

Dissolve methylene blue∗ in warm (70° C.) alcohol, adding the powdered stain very slowly. Then add the xylene and acetic acid. Filter. Keep in tightly stoppered bottle.

1. Milk smears are made on a glass slide in the usual manner. When dry, the slide is immersed in the above solution for ½ to 4 minutes.

2. Remove the slide from the solution and allow it to drain until the smear is thoroughly dry. In warm weather, this seldom requires longer than half a minute.

3. Wash thoroughly in water.

Add the alcohol to the tetrachlorethane in a flask and bring to a temperature not to exceed 70° C.; add to the powdered methylene blue.