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).

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.

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.

While it is true that some bacteria may be stained by that standard histological nuclear dye, hematoxylin, it is of little value for this purpose. Practically all bacteriological stains are solutions of the anilin dyes. These dyes, as is well known, are of nearly every conceivable color and shade but relatively very few are used in bacteriological work. The beginning student will rarely use solutions of other than the three dyes fuchsin (red), methylene blue and gentian violet for staining bacteria, with occasionally Bismarck brown, or eosin, or safranin as tissue contrast stains.

The bacteriological dyes are kept “in stock” as saturated solutions in 95 per cent. alcohol which are never used as stains, but merely for convenience in making the various staining solutions.

The approximate percentages of the three common dyes in such solutions are indicated in the following table adapted from Woods Chemical and Microscopical Diagnosis, Third Edition, 1917, Appendix:

The stains made from these dyes which are in most common use are the following:

These stains rarely keep longer than ten days in the laboratory (unless kept in the ice-box) and must be made fresh on the first sign of a deposit on the glass of the container.

Anilin Water.—Anilin water is made by putting 3 or 4 cc of anilin “oil” in a 120 cc. flask, adding 100 cc of distilled water, shaking vigorously for a minute or so and filtering through a wet filter, in other words, a saturated solution of anilin in water.

Staining solutions are conveniently kept in square dropping bottles inserted in a block as shown in Fig. 140. This form of holder necessitates the use of one hand only in securing the stain and dropping it on the preparation.

The actual staining of bacteriological preparations can be learned only by repeated laboratory practice, yet the following methods have given such uniform results in class work that it is felt they are not out of place in a text-book.

Preparation of the “Film.”—The author learned to stain bacteria, on the “cover-glass” but does not recall having used this method in fifteen years and does not teach it to his students. All staining is done on the slide. To prepare a film from a solid culture medium the procedure is as follows:

First, be sure the slide is clean and free from grease. This is accomplished most readily by scouring a few minutes with finely ground pumice stone and a little water, then washing and drying with a grease-free cloth, handkerchief, or piece of cheese-cloth. With the “loop” needle place in the middle of the slide a small loop of water. This is best done by filling the loop by dipping in water, then tapping it gently so that all that remains is the water that just fills the loop level full, and this amount is placed on the slide by touching the flat side of the loop to the glass. Then the straight needle is sterilized, dipped into the culture and just touched once into the small drop of water on the slide. The remainder of the culture on the straight needle is then burned off and the needle is used to spread the drop of water containing the bacteria into a thin even film, which will result, provided the slide is free from grease. This is dried and then “fixed” by passing three times through the Bunsen flame at intervals of about one second, passing through slowly for thick slides and a little more rapidly for thin ones. If the culture is in a liquid medium, the use of the loop of water is unnecessary; a loop of the fluid from the surface, middle or bottom as the culture indicates is spread out to a thin film, dried and fixed.

After the film is fixed the stain desired is dropped on, allowed to act for the proper time, which will depend on the stain and the preparation, washed in water, dried thoroughly and examined with the oil-immersion lens, without a cover. If it is desired to preserve the preparation it may then be mounted in balsam. This is not necessary, as they keep just as well, provided the immersion oil is removed. To do this, fold a piece of filter paper so that at least three thicknesses result. Lay this on the slide and press firmly several times, when the surplus oil will be taken up by the paper. Slides not mounted in balsam are more apt to become dusty than those that are. This is the only disadvantage.

Gram’s Method of Staining.—It has been ascertained that some bacteria contain a substance, possibly a protein, which forms a compound with gentian violet and iodine, which compound is insoluble in alcohol, and other bacteria do not contain this substance. Consequently when bacteria are stained by Gram’s method (given below), those that contain this chemical remain colored, while if it is not present the dye is washed out by the alcohol and the bacteria are colorless and may be stained by a contrast stain. The bacteria which stain by this method are said to “take Gram’s” or to be “Gram-positive,” while those that decolorize are called “Gram-negative.” The method is:

1. 1. Prepare the film as above given.
2. 2. Stain with fresh anilin gentian violet 1 minute.
3. 3. Wash in tap water.
4. 4. Cover with Gram’s solution 1 minute.
5. 5. Wash in tap water.
6. 6. Wash with 95 per cent. alcohol three times or until no more color comes out.
7. 7. Dry and examine.

Gram’s solution is:

This method is excellent for differentiating Gram-positive and Gram-negative organisms on the same slide. First stain by this method and after washing with alcohol stain with a counter-stain, carbol-fuchsin diluted ten to fifteen times with water is excellent. The Gram-positive bacteria are violet and the Gram-negative are red.

It is also of great value in staining Gram-positive bacteria in tissues, but the sections should be stained about five minutes in the anilin gentian violet and be left about two minutes in the Gram’s solution. Sections are to be counter-stained in Bismarck brown, dilute eosin or safranin solutions and cleared in oil of bergamot, lavender or origanum and not in clove oil or carbol-xylol, as these latter dissolve out the dye from the bacteria.

Staining of Spores in the Rod.—Prepare the films as usual. Cover with carbol-fuchsin, using plenty of stain so that it will not dry on the slide; heat until vapor arises, not to boiling; cool until the stain becomes cloudy and heat again until the stain clears, and repeat once more; wash in tap water and then wash in 1 per cent. H₂SO₄ three times, dropping on plenty of acid, tilting and running this over the slide three times and then pour off and use fresh acid and repeat this once. Wash thoroughly in distilled water, then stain with Löffler’s blue one to three minutes. Wash, dry and examine. The spores should be bright red in a blue rod.

This method will give good results if care is taken to secure cultures of the right age. If the culture is too old the spores will all be free outside the rods, while if too young they will decolorize with the acid. For Bacillus subtilis and Bacillus anthracis, cultures on agar slants forty-eight hours in the 37° incubator are just right. For the spores of Clostridium tetani, the culture should be three days old, but may be as old as a week.

Staining of “Acid-fast” Bacilli.—Mycobacterium tuberculosis, Mycobacterium of Johne’s disease, “grass” and “butter bacilli,” Mycobacterium lepræ, Mycobacterium smegmatis.

1. 1. Prepare the film as usual.
2. 2. Stain with carbol-fuchsin as given above for spores.
3. 3. Wash with tap water.
4. 4. Decolorize and stain at the same time with Gabbet’s blue, two or three minutes.
5. 5. Wash, dry and examine.

The sulphuric acid in Gabbet’s blue removes the carbol-fuchsin from everything except the “acid-fast” bacteria, which remain red, and the blue stains the decolorized bacteria and nuclei of any tissue cells present.

1. 1, 2, 3, as in Gabbet’s method.
2. 4. Decolorize with 10 per cent. HCl until washing with water shows only a faint pink color left on slide.
3. 5. Wash thoroughly.
4. 6. Stain with Löffler’s blue one or two minutes.
5. 7. Wash, dry and examine.

The results are the same as with Gabbet’s method.

Staining of Capsules.—Räbiger’s Method.—Films of the organism to show capsules should be freshly prepared, dried but not fixed. Material is usually obtained from milk or blood. A drop of the fluid is placed on the middle of a slide about one-fourth of the distance from one end. The narrow edge of another clean slide is placed in this drop and then drawn lengthwise across the slide with firm pressure. This gives a thin layer which is necessary if good results are to be expected. The preparation is covered with a freshly prepared saturated solution of gentian violet in formalin and this allowed to stain for 30 seconds. Then wash lightly, dry and examine. The organisms appear deeply violet and much larger than with ordinary stains and capsules are well stained and show well.

Welch’s Method.—Prepare films as in the above method. Cover with glacial acetic acid for 10 to 20 seconds. Wash off the acid with carbol-fuchsin. Wash the stain off with physiological normal salt solution (0.85 per cent.) until all surplus stain is removed. Dry and examine. Capsules and bacteria are red.

Staining of Flagella.—The rendering of flagella visible is considered one of the most difficult processes in staining. Experience of a number of years during which whole classes numbering from one hundred to three hundred students accomplish this result shows that it is no more difficult than many other staining processes. The essentials are: (1) clean slides, (2) young cultures on agar slopes, (3) freshly prepared mordant and stain which are kept free from precipitate, (4) gentle heating. The author’s students are furnished only stock materials and make their own cultures, mordants and stains.

The slides are cleaned with pumice in the usual way. An agar slope culture of the organism to be stained from six to twenty-four hours old is selected. A bit of the culture is removed and placed in a watch-glass of water. The bacteria are allowed to diffuse of themselves without stirring. After several minutes a loop of this water is removed and three streaks are made across the slide, one in the middle and one on each side of this about one-quarter of an inch from it. This gives well scattered bacteria in one of the three streaks at least and very little other material on the slide to cause precipitates. The slide is carefully dried and fixed and then covered with an abundance of the mordant by filtering through a small filter onto the slide so that the mordant shows transparent on the slide. The preparation is then gently warmed and cooled three times, adding mordant if necessary. Do not heat to steaming. After mordanting for about five minutes the excess is washed off under the tap. It is a good plan to hold the slide level and allow the water to run into the center of the mordant and flow it off. Inclining the slide is apt to cause the film on the surface of the mordant to settle down on the slide and spoil the preparation. After the mordant is washed off and all traces of it removed with a clean cloth if necessary the stain is applied and gently heated and cooled the same way for from three to five minutes. The preparation is then washed, dried and examined.

The mordant used is a modification of Löffler’s which is somewhat simpler in preparation since the stock solution of FeCl₃ is more permanent than FeSO₄ solution.

Mordant sufficient for one student:

The solution of FeCl₃ is made up in the cold and must be perfectly clear. The tannic acid solution must be thoroughly boiled and filtered until clear. The iron and the acid are carefully mixed, boiled and filtered clear. The anilin fuchsin must be added slowly with constant stirring and the mixture boiled and filtered. The NaOH is added in the same way and this mixture boiled and filtered. The final mordant should not leave a film on a clean slide when poured on and allowed to run off. Unless the mordant is in this condition and perfectly clear, it should not be used, but a new one must be made up. Time and care in the preparation of the mordant are essential.

The stain to follow this mordant is anilin fuchsin.

Staining of Metachromatic Granules.—Neisser’s Method. Prepare the film in the usual way. Stain with Neisser’s stain a few seconds only. Wash and stain with Bismarck brown a few seconds only.

By the use of the hanging drop slide and the methods of staining just described all the various morphological features of the bacterial cell may be ascertained.

It is necessary when cell groupings as characteristic of definite modes of division are to be determined to make slides from a liquid culture, as broth. Place a drop of the material, preferably from the bottom of the tube in most instances, from the top in case a pellicle or scum is formed on the surface, on the slide and allow this to dry without spreading it out, fix, wash gently with water, then stain lightly with Löffler’s blue. Such slides also show characteristic cell forms as well. Slides should be made from solid media to show variations in form and size and involution forms. These latter are especially apt to occur on potato media.