Outlines of dairy bacteriology, 10th edition / A concise manual for the use of students in dairying

by H. L. Russell; E. G. Hastings

All kinds of materials may be sterilized by treatment with steam under pressure. An exposure for a few moments at 250° F., a temperature attained with 15 pounds steam pressure, will destroy all kinds of bacteria and their spores. This method of sterilization is used in the canning of meats and vegetables and in the preparation of evaporated milk. To avoid contamination of the media after sterilization, the flasks and tubes are, after being filled, stoppered with plugs of cotton-wool, which effectually filter out all bacteria and mold spores from the air, and yet allow the air to pass freely in and out of the containers.

Methods of determining the number of bacteria. The method of determining the number and kinds of bacteria in any substance can be illustrated by the process as applied to milk. For this purpose the method of procedure is as follows: Sterile gelatin in glass tubes is melted and then cooled until it is barely warm. To this melted gelatin a definite quantity of milk is added. The medium is gently shaken, so as to thoroughly mix the milk and gelatine, and the mixture then poured into a sterile, flat, glass dish, and quickly covered, where it is allowed to cool until the gelatin hardens. After the culture plate has been left for twenty-four to thirty-six hours at the proper temperature, tiny spots will begin to appear on the surface, or in the depth of the culture-medium. These spots are called colonies, and are composed of an almost infinite number of individual cells, the result of the continued growth of a single organism that was in the drop of milk and which was firmly held in place when the gelatin solidified. The number of these colonies represents approximately the number of living bacteria that were present in the amount of milk added to the tube of gelatin. If the plate is not too thickly sown with the bacteria, the colonies will continue to grow and increase in size, and as they do, minute differences will begin to appear. These differences may be in the color, the contour, and the texture of the colony, or the manner in which it acts toward gelatin.

Fig. 4.—Plate Culture.
Each of the dots is a colony that has been formed by the growth of an organism embedded in the solid culture-medium. By counting the colonies, the number of living bacteria in the amount of milk added to the culture is determined.

In order to make sure that the number of colonies is not so numerous as to prevent counting and further study of their characteristics, a series of plate cultures is usually made in which varying amounts of milk are added to the tubes of gelatine. This is attained by adding a definite amount of the milk or other substance to be examined to a measured amount of sterile water, e.g., one cubic centimeter of milk to ninety-nine cubic centimeters of water. One cubic centimeter of this mixture may be used for the inoculation of the plate culture. This dilution may be carried on to any desired extent; in the examination of many dairy products, it is necessary to use very minute quantities of material, often only one one-millionth of a cubic centimeter.

To study further the peculiarities of the different bacteria, small portions of the individual colonies are transferred to tubes of sterile culture-media. In order to do this the colony is touched with a piece of platinum wire; the minute amount of growth that adheres to the wire is sufficient to seed the tube of fresh culture-medium. The inoculating needle must always be sterilized before use by passing it through a gas flame.

A culture thus obtained is called a pure culture since it contains but a single kind of an organism, as the colony is the result of the growth of a single cell. These cultures then serve as a basis for continued study, and must be planted and grown upon the different kinds of media that are obtainable. In this way the slightest variations in the growth of different forms are detected, and the peculiar characteristics are determined, so that the student is able to recognize this form when he meets it again.

Fig. 5.—Different Kinds of Bacteria Growing in Gelatin.
A, meager growth, no liquefaction or surface growth; B, profuse surface growth, radiating filaments from the growth below the surface; C, a rapid liquefying form; D, a gas producer that grows equally well in the presence or absence of air; E, form that grows only in the absence of air, an anaerob.