A number of procedures may be used to greatly facilitate the above methods of isolation by taking advantage of the different physiological properties of different organisms in a mixture such as ability to form spores, different resistance to antiseptics, special food requirements, and pathogenic properties. (a) If material contains resistant spores, it may be heated to temperatures high enough to kill all of the organisms except the spores (80° for half an hour, for example) and then plated out. Or (b) an antiseptic which restrains the growth of some organisms and not others may be placed in the culture media (carbolic acid, various anilin dyes, (p. 162), excess acid, or alkali, ox bile, etc.), when the more resistant organisms grow on the final plates, the others not. (c) Special food substances (various carbohydrates) from which the organism desired forms special products (acids, aldehydes) that may be shown on the plates by various indicators, is one of the commonest means. Or media in which certain organisms thrive and others not, so that the former soon “crowd out” the latter (unsterilized milk for lactic acid bacteria, inorganic media in soil bacteriology) may be used. A combination of the general methods (b) and (c) is much used in the separation of the organisms of the “intestinal group” in human practice. (d) The inoculation of a susceptible animal with a mixture suspected to contain a given pathogenic bacterium frequently results in the development of the latter in pure culture in the body of an animal, from which it may be readily recovered. In all of the above methods (except Barber’s) the first “pure culture” obtained should be “purified” by replating in a series of dilution plates to make sure that it is pure.

CHAPTER XIX.
STUDY OF INDIVIDUAL BACTERIA—STAINING.

When an organism has been obtained in pure culture by any of the methods described in the preceding chapter the next step is the study of its morphology as discussed in [Chapters II–IV]. This involves the use of the microscope, and since bacteria are so small, objectives of higher power than the student has presumably used will be needed. Doubtless only the two-thirds inch or 16 mm. and the one-sixth inch or 4 mm. objectives are all that have been used in previous microscopic work, while for examining bacteria a one-twelfth inch or 2 mm. is necessary. It will have been observed that the higher the power of the objective the smaller is the front lens or object glass and consequently the less is the amount of light which enters. With the use of the one-twelfth inch or 2 mm. objective it is necessary to employ two devices for increasing the amount of light entering it, with which the student is probably not familiar. One of these is to place a drop of cedar oil between the front lens and the object and to immerse the lens in this oil—hence the term “oil-immersion objective;” the other is the substage or Abbé condenser. The latter is a system of lenses placed below the stage and so constructed as to bring parallel rays of light—daylight—from an area much larger than the face of the front lens of the objective to a focus on the object to be examined, thus adding very greatly to the amount of light entering the objective. Since the condenser brings parallel rays to a focus on the object, the flat-mirror is always used with the condenser when working with daylight. With artificial light close to the microscope, the concave mirror may be used to make the divergent rays more nearly parallel and thus give better illumination.

The function of immersion oil is to prevent the dispersion of considerable light that would otherwise occur owing to refraction as the light passes up through the slide and into the air. The accompanying diagram will help to make this clearer ([Fig. 137]). A ray of light (A B) coming through the slide will be refracted in the direction B C if the medium has a lower refractive index than the slide, as air has, and hence will not enter the objective O. If, however, there is interposed between the objective and the slide a medium which has the same refractive index as the slide, as immersion oil has, then the ray will continue in the same direction (B D) at the point B and hence enter the objective. Evidently the immersion oil causes much more light to enter the front lens and makes the field brighter and at the same time prevents considerable refraction and dispersion of light from the object seen and hence this appears more distinct and sharply defined. The Abbé condenser and the oil-immersion objective are practically always used in the microscopic study of bacteria ([Fig. 138]).

Fig. 137.—Diagram of use of immersion oil.

Fig. 138.—Diagram of paths of rays of microscope.

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