Special Methods for Staining Micro-organisms and Blood.

It is impossible, within the limits of this work, to attempt any adequate description of the modern methods of bacteriological investigation. Some of these are very lengthy and complicated, and require much skill and practice before good results can be relied on. But those who do not desire to make a special study of bacteriology may often require to examine for the presence of organisms in sections, or in various excretions, and it is hoped that they may find the following short description of special methods sufficient for their purpose. For more elaborate work they must consult one of the many excellent textbooks on the subject.

The student should provide himself with the following dyes in powder:—

Methylene blue.

Gentian violet.

Methyl violet.

Fuchsine.

Bismarck brown.

The following solutions of these dyes are used:—

1. Saturated alcoholic solutions which may be kept in stoppered bottles.

2. One per cent. aqueous solutions. These must be freshly made each time of using.

In filtering either alcoholic or aqueous solutions it is well to moisten the filter paper beforehand with alcohol or water as the case may be.

The following special solutions will also be wanted:—

Löffler’s methylene blue.—In this solution a weak solution of caustic potash is employed as a mordant:—

Saturated alcoholic solution of methylene blue	3	volumes.
Caustic potash, aqueous solution 1 : 10,000	10	volumes.

Filter.

This solution is perhaps the most generally useful stain. It colours most bacilli and micrococci, and while rapid in its action rarely overstains. It must be made up fresh on each occasion. It is the best counterstain after staining tubercle bacilli, &c., with fuchsine.

Ziehl’s carbol-fuchsine.

Carbolic acid (5 per cent. aqueous solution)	100	volumes.
Fuchsine (saturated alcoholic solution)	11	volumes.

The solution must be filtered immediately before being used.

Gram’s iodine solution.—Sections are placed in this solution after being stained with aniline dyes. The iodine in some way fixes the dye in the organisms, so that they are not decolourised along with the rest of the tissues.

It is made thus:—

Iodine	1	grm.	1 1/2	grains.
Iodide of potassium	2	grms.	3	grains.
Distilled water	300	c.c.	1	ounce.

Ten per cent. aqueous solutions of nitric and sulphuric acids should be prepared and may be kept indefinitely.

The following are the general methods of employing these reagents for the purpose of staining organisms in sections. Special methods are required for special organisms, but one or two only can be given.

Weigert’s method.—The sections must be placed in a freshly made one per cent. aqueous solution of methyl violet, gentian violet, fuchsine, &c. The solution may be kept at the temperature of the body in an incubator. The organisms will often stain more readily if the section be passed through a 1 in 2000 solution of corrosive sublimate before putting it into the staining fluid. After staining the section is washed in distilled water and then in methylated spirit until it appears almost decolourised. Some prefer to decolourise the tissues by washing in a half per cent. solution of acetic acid instead of methylated spirit. Practice is required before the correct time for decolourising is accurately estimated. The beginner should float a section rapidly on the slide now and then, put on a cover-glass and examine it under a low power to see if the decoloration has been carried far enough. A contrast stain may then be used, such as picrocarmine, after which the section may be mounted in Farrant’s medium: or a weak solution of another aniline colour may be used as a counter stain, after which the section is clarified in xylol, and mounted in balsam dissolved in xylol.

Gram’s method.—Place some aniline oil in a test tube and add ten times its volume of distilled water. Close the end with the thumb and shake very thoroughly. Filter ninety drops into another clean test tube, and add ten drops of a saturated solution of gentian violet or some similar dye. Filter the mixture into a watch glass. Stain sections in it for from three minutes to half-an-hour according to the temperature,—the shorter time for the incubator at 100°, the longer when the sections are stained at the ordinary temperature of the room. Wash in distilled water, and transfer to Gram’s iodine solution until they become black, usually in a few minutes. They are then decolourised in absolute alcohol. This often takes some time. It may be hastened, as Crookshank suggests, by placing the section in clove oil, returning to alcohol, and so on.

Ehrlich’s modification of Gram’s method. The contrast stain is here used first.

Stain the section (e.g., that of a mitral valve in a case of ulcerative endocarditis), in an alcoholic solution of eosine (1 in 1500). Transfer to a solution of some aniline dye, such as gentian violet, dissolved in aniline oil water, exactly as in Gram’s method. The section floats on the surface and spreads out, owing to the alcohol diffusing out. Stain for about twenty minutes. Wash the section in water, and float out (p. [55]) on a glass slide. Allow the water to drain off and add Gram’s iodine slowly from a pipette so as not to disarrange the section. When the section has become quite black pour off the Gram’s solution. Remove all superfluous fluid from the slide with blotting paper, and dry the section by carefully and firmly pressing on it a folded piece of blotting paper. If this is done with care the section need not be injured in the least. Decoloration is effected on the slide with aniline oil, instead of alcohol as in the preceding method. The slide is rocked about so that the colour may be evenly discharged by the aniline. When no more colour comes away, the aniline oil is poured off, the section clarified in xylol, and mounted in Canada balsam.

As soon as the section is decolourised it may be treated with a contrast stain, the most suitable being alcoholic solutions of eosine or Bismarck brown if a blue stain has been employed, or methylene blue if fuchsine has been the first stain used.

The following will be found the most useful stains and contrast stains:—

Stains.	Contrast Stains.
Gentian violet. Methyl violet. Methylene blue.		Picrocarmine. Eosine. Bismarck brown. Safranine.
Magenta. Fuchsine.		Methylene blue.
And vice versâ.

Much practice is required in using either of the methods before one can judge accurately how long to leave sections in the staining reagents or decolourising agents, and the beginner must not be discouraged if at first he is unable to obtain good results although he follows the book directions most minutely.

Ehrlich method for tubercle bacilli.—Sections are stained for six to twenty-four hours in a one per cent. solution of gentian violet, methyl violet, methyl blue or fuchsine. They will stain more rapidly if the staining fluid be kept in an incubator at the body temperature. They should be removed from the staining fluid, and washed in distilled water, and then transferred (preferably on a glass section lifter) to a ten per cent. solution of nitric acid in distilled water until they are nearly decolourised. They should then be very thoroughly washed in distilled water. They may then be treated with some suitable contrast stain and mounted in Canada balsam.

Neelsen’s stain for tubercle bacilli.—Sections are placed in Ziehl’s carbol-fuchsine solution (p. [103]) which should be warmed for ten minutes to half-an-hour. They are then decolourised in a solution of sulphuric acid. Twenty-five per cent. is the strength originally recommended, but a ten per cent. solution does equally well and injures the section less. They are then very thoroughly washed in a large quantity of water, and afterwards may be treated with a contrast stain.

Gibbes’ double stain for tubercle bacilli.—

(1)	Rosaniline hydrochlorate	2	grms.	25	grs.
	Methyl blue	1	grm.	12·5	grs.

Triturate in a glass mortar,

(2)	Aniline oil	3	c.c.	37·5	grs.
	Rectified spirit	15	c.c.	3 1/2	drms.

Dissolve and add slowly to (1).

(3)	Lastly add slowly to the mixture
	Distilled water	15	c.c.	3 1/2	drms.

Some of the solution is filtered into a watch glass and warmed. The sections are placed in it and left for some hours. They are then washed in methylated spirit till they are sufficiently decolourised, and then rapidly passed through absolute alcohol and oil of cloves and mounted in balsam and xylol. It is a very useful stain for examining the sputum for tubercle bacilli.

In order to stain fluids, such as blood, pus, or sputum, for organisms, a very thin layer should be obtained by placing a little of the fluid between two clean cover-glasses and pressing them together. They are then separated and allowed to dry. The film is fixed by holding the cover-glass in a pair of forceps, and passing it slowly through the flame of a spirit lamp two or three times. Films of pus should be ‘cleared’ after fixing by placing them in a twenty per cent. solution of acetic acid for three minutes.

For clinical purposes it is often necessary to examine urine, fæces or vomited matter for bacilli. Films are prepared in the usual way and allowed to evaporate slowly, and then fixed by passing through the flame, and then washed in distilled water before staining. In the case of vomited matter and fæces this is usually done without difficulty. In the case of urine however it is often difficult to get the urine to evaporate completely. A syrupy layer remains, and if more heat be applied it decomposes and chars, and the products cause precipitation of aniline during subsequent staining processes. This may be partly avoided by gently washing the film in distilled water before staining.

Another plan is to mix the urinary deposit with a little gelatine free from organisms, such as that in unused culture tubes. The gelatin is liquefied by heat, and mixed with the deposit. Films are made from this mixture, and allowed to set, and then thoroughly washed in distilled water. The film is then dried thoroughly, and the cover-glass laid flat with the film uppermost, and a few drops of the staining fluid filtered on to it. After it has been stained sufficiently the stain is drained off, and the slip gently washed. The film may then be stained with some contrast stain in exactly the same way as sections, again washed, dried between folds of blotting paper, and mounted in balsam.

It is sometimes difficult to tell which is the side of the cover-glass which bears the film. This is readily done by holding the glass obliquely so that light from a window is reflected from its surface. The side which is coated appears dull; while the other is smooth and bright.