HANGING DROP SLIDE.
It is sometimes necessary to examine living bacteria and for this purpose the device known as the “hanging drop slide” is used ([Fig. 139]). The slide has a slight concave depression ground in the middle of one face. A ring of vaseline is placed around this depression with the loop needle. On a clean cover-glass, large enough to fit over the ring of vaseline, several drops of a broth culture, or of material from a solid culture suspended in broth or physiological normal salt solution are placed. The slide is inverted on the cover-glass in such a way that the ring of vaseline seals the latter to the slide. When the whole preparation is quickly turned cover side up, the drops are seen “hanging” to the under side of the cover over the depression in the slide. In examining such a preparation with the microscope great care is necessary in order to focus on the bacteria, without breaking the cover. To see the organisms distinctly the lower iris diaphragm of the condenser must be nearly closed, so that the light coming through consists mainly of parallel vertical rays, otherwise the transparent bacteria themselves refract and diffract the light and appear blurred and indistinct. By studying living bacteria with this device it can be determined whether they are motile or not. The motility should not be confounded with the familiar “Brownian movement” of all minute insoluble inert particles which non-motile living bacteria and also dead bacteria show. The hanging drop slide is of value in the measurement of bacteria, since this is properly done on the living organism. Measurement is done with a calibrated ocular micrometer as in other kinds of measurement with the microscope with which the student is presumably familiar. The direct effect of various agents on living bacteria as light, electricity, heat, etc., in the study of “tropisms” and “taxes” has been investigated on various modifications of the above-described hanging drop slide.
Fig. 139.—Hanging drop slide.
Cell forms and cell groupings may be studied in the same way but these features are best determined on stained preparations in many instances.
“Dark field” illumination and the ultramicroscope are of great value in the study of living bacteria and other minute objects, but apparatus of this type would scarcely be used by the student in an introductory course, so that they will not be discussed in the present volume.
STAINING.
The main use of the microscope in bacteriology is in the study of stained preparations of the organisms. Staining makes bacteria opaque and hence more easily seen than the transparent unstained forms. Some methods of staining also show morphological structures which are either imperfectly recognized in the unstained cell, spores, or are not visible at all—capsules, metachromatic granules, flagella. Finally certain bacteria are colored by special methods of staining which do not affect others, so that under proper conditions these bacteria may be recognized by staining methods alone—tubercle bacilli in the organs of animals.
The phenomena of staining are essentially chemical, though sometimes the chemical union is a very weak one, even resembling an absorption of the dye rather than true chemical union—most watery stains. In other cases the chemical compounds formed are decidedly stable and are not decomposed even by strong mineral acids—staining of tubercle bacilli and other “acid-fast” organisms. In still other cases the principal action is a precipitation on the surface of the object stained—methods for staining flagella.
In many methods of staining in addition to the dyes used other substances are added to the solution which assist in fixing the dye in or on the organism stained. Such substances are called mordants. The principal mordants used are alkalies, anilin, carbolic acid, iodine, metallic salts, tannic acid.