Of the environmental conditions influencing the growth of bacteria the following are the chief ones ordinarily determined:
A. Temperature.—The optimum temperature for growth is usually about the temperature of the natural environment and ordinarily one determines merely whether the organism grows at body temperature (37°) and at room temperature (20°) or not. For exact work the maximum, minimum and optimum temperature must be ascertained by growing in “incubators” with varying temperatures.
A bacteriological incubator is an apparatus for growing bacteria at a constant temperature. This may be any temperature within the limits for bacterial growth. If temperatures above that of an ordinary room are desired, some source of artificial heat is needed. Electricity, gas or oil may be used. A necessary adjunct is some device for maintaining the temperature constant, a “thermoregulator” or “thermostat.” For lower temperatures a cooling arrangement must be installed. For the great part of bacteriological work only two temperatures are used, 20° so-called “room temperature” (this applies to European “rooms” not to American) and 37° or body temperature. Incubators for 37° of almost any size and style desired may be secured from supply houses and need not be further described. Figs. 141 and 142 illustrate some of the types.
For use with large classes “incubator rooms” are to be preferred. The author has one such room for 37° work with 200 compartments for student use which did not cost over $60 to install.
The styles of incubators for lower temperatures, 20° and below, are not so numerous nor so satisfactory. The author has constructed a device which answers every purpose for a small class. The diagram, Fig. 143, explains it.
The thermal death-point is determined by exposing the organisms in thin tubes of broth at varying temperatures for ten-minute periods and then plating out to determine growth. The effect of heat may also be determined by exposing at a given temperature, e.g., 60°, for varying lengths of time and plating out.
B. Oxygen relations—whether the organism is aërobic, anaërobic, or facultative is determined by inoculation in gelatin or agar puncture or stab cultures and noting whether the most abundant growth is at the top, the bottom or all along the line of inoculation.
C. Reaction of the medium—acid, alkaline or neutral as influencing the rate and amount of growth.
D. The kind of medium on which the organism grows best.
E. The effect of injurious chemicals, as various disinfectants, on the growth.
F. Osmotic pressure conditions, though modifying decidedly the growth of bacteria, are not usually studied as aids in their recognition, nor are the effects of various forms of energy, such as light, electricity, x-rays, etc.
Among the “Physiological Activities” discussed in Chapters IX–XII those which, in addition to the staining reactions described, are of most use in the identification of non-pathogenic bacteria are the first ten listed below. For pathogenic bacteria the entire thirteen are needed.
1. Liquefaction of gelatin.
2. Digestion of blood serum.
3. Coagulation and digestion of milk.
4. Acid or gaseous fermentation in milk, or both.
5. Acid or gaseous fermentation of various carbohydrates in carbohydrate broth, or both.
6. Production of indol in “indol solution.”
7. Production of pigments on various media.
8. Reduction of nitrates to nitrites, ammonia, or free nitrogen.
9. Production of enzymes as illustrated in the above activities.
10. Appearance of growth on different culture media.
11. Production of free toxins as determined by injection of animals with broth cultures filtered free from bacteria.
12. Causation of disease as ascertained by the injection of animals with the bacteria themselves, and recovery of the organism from the animals.
13. Formation of specific antibodies as determined by the proper injection of animals with the organism or its products and the subsequent testing of the blood serum of the inoculated animals.
For special kinds of bacteria other activities must be determined (oxidation, nitrate and nitrite formation, action of sulphur and iron bacteria, etc.).
The first nine activities are determined by inoculating the different culture media already described and observing the phenomena indicated, making chemical tests where necessary.
In addition to those changes that are associated with the manifestation of different physiological activities, many bacteria, show characteristic appearances on the various culture media which are of value in their identification.
Too much stress should not be laid on these appearances alone, however, since slight variations, particularly in solid media due especially to the age of the medium, may change decidedly the appearance of a colony. This is true of variations in the amount of moisture on agar plates. Colonies which are ordinarily round and regular may assume very diverse shapes, if there chance to be an excess of moisture on the surface.
Also in slope and puncture cultures on the various solid media much variation results from the amount of material on the inoculation needle and just how the puncture is made, or the needle drawn over the slope. These variations are largely prevented by the use of standard media and by inoculating by standard methods. The Laboratory Committee of the American Public Health Association has proposed standard methods for all culture media and tests and for methods of inoculation, and these have been generally adopted in this country for comparative work.
Likewise the Society of American Bacteriologists has at different times (1904, 1914, 1917) adopted “descriptive charts” for detailing all the characteristics of a given organism. A committee is at present working on a revision of the 1917 chart to be presented at the 1920 meeting. One of the earlier charts which includes a glossary of descriptive terms is inserted in this chapter.
Among the cultural appearances the following are of most importance:
In broth cultures the presence or absence of growth on the surface and the amount of the same. Whether the broth is rendered cloudy or remains clear, and whether there is a deposit at the bottom or not (Fig. 144). An abundant surface growth with little or nothing below indicates a strict aërobe, while a growth or deposit at bottom and a clear or nearly clear medium above, an anaërobe. These appearances are for the first few days only of growth. If the broth is disturbed, or after the culture stands for several days many surface growths tend to sink to the bottom. So an actively motile organism causes in general a cloudiness, especially if the organism is a facultative anaërobe, which tends to clear up by precipitation after several days when the organisms lose their motility. Non-motile facultative anaërobes usually cloud the broth also, but settle out more rapidly than the motile ones.
In gelatin and agar punctures the oxygen relationship is shown by surface growth for aërobes, growth near the bottom of the puncture for anaërobes, and a fairly uniform growth all along the line of inoculation for facultative anaërobes. In the case of these last organisms, a preference for more or less oxygen is indicated by the approach to the aërobic or anaërobic type of growth.
Along the line of puncture the commonest types are filiform (Fig. 145), which indicates a uniform growth; beaded (Fig. 146), or small separate colonies; villous (Fig. 147), delicate lateral outgrowths which do not branch; arborescent, tree-like growths branching laterally from the line. In agar these branchings are usually short and stubby, or technically, papillate.
Further, in the gelatin puncture the liquefaction which occurs is frequently characteristic. It may be crateriform (Fig. 148), a shallow saucer at the surface; or funnel-shaped (Fig. 149); or it may be of uniform width all along the puncture, i.e., saccate (Fig. 150); or it may be stratiform, (Fig. 151), i.e., the liquefaction extends to the sides of the tube and proceeds uniformly downward.
On agar, potato and blood serum slope tubes the amount of growth, its form and elevation, the character of the surface, and the consistency should be carefully noted, and in some few cases the character of the edge. Figures 152 to 158 show some of the commoner types.
On agar and gelatin plates made so that the colonies are well isolated, the form of the latter, the rate of their growth, the character of the edge and of the surface, the elevation and the internal structure as determined by a low-power lens are often of almost diagnostic value. Also in the case of the gelatin plates, the character of the liquefaction is important. Figs. 159 to 167 show some of the commoner characteristics to be noted.
Colonies of mold frequently appear on plates. These are readily differentiated from bacterial colonies after a little experience. With the naked eye usually the fine radiations of the edge of the colony are apparent. The surface appears duller and by reflected light more or less “fuzzy.” With the low-power objective the relatively large, branching threads of the mold (mycelia) show distinctly. Also the large fruiting bodies (sporangia) are easily distinguished. Figs. 168 to 171 illustrate a common black mold (Rhizopus nigricans).