III.—Brief Notes on General Protozoological Technique

The object of this book is to give accounts of the structure and life-histories of the numerous parasitic organisms that affect man more particularly. It is, therefore, inappropriate to devote much space to a consideration of technique, regarding which many volumes have already been written. Methods of procedure are largely matters of opinion, and the technique that gives brilliant results when used by one investigator may be a complete failure in the hands of another. In the present appendix, brief notes regarding certain relatively simple methods only can be given, because the number of fixatives in use is very great; there are also large numbers of stains as well as many modifications of them, while the methods of applying both fixatives and stains are, perhaps, still more numerous. There are so many, in fact, that confusion frequently arises from the multiplicity of choice presented to the worker. Those desiring more information on the subject of technique are advised to consult the treatises of Bolles Lee and of Langeron.

Fresh Material.

(a) Simple Examination.

Fluid Substances, such as Blood and Sputum.—A small quantity of the substance to be examined is taken on a sterile platinum loop and transferred to a perfectly clean glass slide. A clean cover-slip is gently lowered on to the drop, air bubbles being avoided. The preparation is luted with vaseline or paraffin and examined first with a low power and then with a high power objective. The light is cut down by partly closing the diaphragm of the substage of the microscope.

Skin Ulcers and Similar Sores.—Scrapings are made from the edge of the sore, mixed with sterile physiological salt solution, and prepared and examined as above.

Fæces.—A small portion of fæces, or flakes of mucus (which may be blood-stained) from the same, is removed on a sterile platinum loop, spread out thinly after dilution, if necessary, with physiological salt solution on a slide, covered and examined as before.

Alternatively, hanging drop preparations of blood, ulcerative tissue, or fæces, appropriately diluted if necessary with sodium citrate or physiological salt solution, may be made on a cover-slip, which is inverted over a slide with a well in it. The cover-slip is then luted and examined.

For the elucidation of the developmental processes of such organisms as trypanosomes, spirochætes and piroplasms, fresh preparations may be often kept under observation longer by the use of a thermostat, maintained at or near blood heat, in which the microscope is inserted.

(b) Intra vitam Staining of fresh Preparations.

Intra vitam staining is of service on some occasions, more particularly for the study of the nucleus and other chromatoid substances of the living organism. Two methods are in common use. In the first case, the stain, employed usually in very dilute solution, is mixed with the medium containing the organism. The latter takes up some of the stain, the amount of coloration depending on the organism concerned and on the stain employed.

The commoner intra vitam stains are pure, medicinal (zinc-free) methylene blue and neutral red, used in aqueous solutions. A solution of methylene blue of 1 per 1,000 of water may be tried, while neutral red in the proportion of 1 per 3,000 parts of water has proved of service.

The second method of vital colouring consists in placing a drop of 1 per cent. solution of methylene blue on a slide or cover-slip, slightly spreading it, and allowing it to dry. The living organism is then placed in a drop of saline on the prepared slide or cover-slip, which is then mounted and examined under the microscope. Progressive staining of the organism occurs and its internal structure can be seen. A similar procedure may be followed for neutral red. Intra vitam staining is useful for relatively large and easily deformed protozoa such as ciliates, as well as for amœbæ and flagellata of the gut.

When examining very actively motile organisms, it is sometimes useful to endeavour to restrict their movements by adding a little gum or gelatine to the medium.

(c) Examination by aid of the Paraboloid Condenser.

The use of one of the dark-ground illuminators (so-called ultra-microscopes) is of service for the detection of minute living organisms or of organisms present in small numbers only. The forms of paraboloid condenser manufactured by the firms of Zeiss and Leitz can be recommended. For details of their methods of employment, reference should be made to the leaflets of the firms supplying the said instruments. By the use of the paraboloid condenser, the finer details of certain stages of life-cycles, such as the formation of granules in spirochætes and treponemata, can be observed more readily than by using the ordinary substage of the microscope. The use of the paraboloid condenser for the detection of small numbers of living organisms renders it of value for rapid diagnostic purposes.

Stained Material.

Fuller accounts of the technique of fixed and stained material will be found in Bolles Lee and in Langeron, already mentioned.

Thin Films.—For the examination of blood-inhabiting Protozoa, it is necessary to make first thin films or smears of blood. There are many ways of doing this, and opinions differ as to their respective merits. A simple method is to take a straight surgical needle about 2 in. long, the eye of which has been removed, and a clean glass slide. The patient’s skin is pricked, and when the bead of blood reaches the size of a small pin’s head, the slide is applied to the surface of the blood, about 1/3 in. from the far (left-hand) end of the slide. The shaft of the needle is laid across the drop of blood, which spreads between the slide and the needle. The latter is drawn evenly along the slide towards the right. The film is dried by waving it in the air. The film should possess a straight edge parallel with that of the slide and should be as uniform and thin as possible. Another glass slide may be used as a spreader, or a cover-slip or thin glass rod may be employed.

Thick Films.—These are of service in detecting malarial parasites or trypanosomes, especially when the parasites are few. The method of Ross, or a modification thereof, has been much used. A small drop of fresh blood is spread evenly and quickly with a needle-point over a square area somewhat less than that of an ordinary square cover-glass. The blood is allowed to dry. The film is then carefully dehæmoglobinized in water in which there is a trace of acetic acid. The dehæmoglobinizing fluid is then carefully drained off and the film again dried. It is fixed in absolute alcohol and stained with Romanowsky’s solution. A cubic millimetre of blood divided into quarters may be thus dehæmoglobinized and stained. The parasites in such a cubic millimetre of blood may be counted. Such a procedure was followed by R. Ross and D. Thomson, in determining the periodic variation of the numbers of trypanosomes in the blood of a patient, as mentioned and figured on pp. and 79.

For cytological details of various Protozoa, thin film preparations on cover-slips or slides are often useful. Cover-slip preparations are preferable, unless the organisms under investigation are extremely scanty. The medium containing the organisms, such as blood, lymph, intestinal contents, sputum, scrapings of ulcers, and urine, is spread thinly, either alone or diluted with a little physiological salt solution, on the cover-slip. Fixation while still wet is necessary. Various methods are employed.

Fixatives.—A useful procedure is to fix the wet film by exposure to 4 per cent. osmic acid vapour for ten to thirty seconds, then place in absolute alcohol for five minutes to harden. Grade down from absolute alcohol through 90 per cent., 70 per cent., 50 per cent., and 30 per cent. alcohols to water. Stain wet with a suitable stain such as hæmatoxylin, and gradually dehydrate by grading through the necessary strengths of alcohol, clear in xylol or other oily clearing medium and mount in Canada balsam.

Other fixatives may be employed, such as are also useful for fixing pieces of tissue for sectioning. Films or smears on cover-slips while still wet are floated on the surface of the fixative in a watch glass. Some good fixatives of wide application are:—

Schaudinn’s Fluid.—This consists of a mixture of

Saturated aqueous solution of corrosive sublimate

2

volumes

Absolute alcohol

1

volume

Two modifications of Schaudinn’s formula may be found useful. A saturated solution of corrosive sublimate in physiological salt solution may be substituted for the aqueous one, and the addition of a few drops of glacial acetic acid to either of the preceding mixtures may be made.

Some workers prefer to use hot fixatives, raised to a temperature of about 50° C.

Fixation by corrosive sublimate solutions must be followed by thorough removal of the mercury salt by washing repeatedly in 30 per cent. alcohol or with iodine-alcohol.

Bouin’s Fluid, or modifications thereof, is also very useful for wet fixation. Bouin’s picro-formol solution consists of:—

Saturated aqueous solution of picric acid

30

volumes

Formalin, 40 per cent.

10

Acetic acid, glacial

2

The best-known modification is one due to Duboscq and Brasil, and often known as Bouin-Duboscq Fluid. Its formula is as follows:—

Alcohol, 80 per cent.

150

c.c.

Formalin, 40 per cent.

60

Acetic acid, glacial

15

Picric acid

1

grm.

Thorough washing of the smear or cover-slip preparation with 70 per cent. alcohol until the yellow colour disappears is necessary to remove excess of fixative.

Other fixatives, which may be of use, more especially for fixing small pieces of tissue for sectioning, are the solutions of Flemming (chromo-aceto-osmic acids) and of Zenker (sublimate-bichromate-acetic, with sodium sulphate).

Regarding the time of fixation, there is much difference of opinion. Usually, exposure to or contact with the fixative for five minutes is sufficient in the case of films or smears. Material for sections should be cut into small cubic pieces, of a thickness of about 5 mm. ( 1/5 in.). One or two hours should be sufficient time for the fixation of such pieces of tissue, though some, as Langeron, prefer a longer time of fixation. On the other hand, Gustav Mann recommends a short fixation period. The excess of fixative should be thoroughly washed out of the tissue in the manner appropriate to the particular fixative used. If it is desired to keep the tissue for some time before sectioning and staining, it should be transferred to 70 per cent. alcohol.

When fluid fixatives are employed, large quantities of the fixing media are necessary. The volume of the fixative should be at least ten to twenty times that of the object, and the latter should be suspended in the middle of the fixative. The tissue should be fixed as soon as possible after the death of the host.

For sectioning tissue parasitized by Protozoa, embedding in paraffin is generally recommended. Microtome sections should not, if possible, exceed 5 µ in thickness. Details of special procedures must be sought in larger works.

Staining.—Here, as with fixatives, much choice is presented. The various modifications of the Romanowsky stain have aided greatly in the detection of various Protozoa parasitic in the blood. Such stains, however, leave something to be desired in the revealing of finer cytological details. Other stains, more especially the hæmatoxylins, must be employed for cytological purposes.

Formulæ of some of the principal Romanowsky and hæmatoxylin stains may now be given.

The underlying principle of the Romanowsky Stain is the reaction between alkaline methylene blue and eosin, forming the so-called eosinate of methylene blue which stains chromatin purplish-red. A solution of medicinal methylene blue after having been subjected to the action of an alkali, such as sodium carbonate, becomes partly converted into certain derivatives, the chief of which are methylene azure and methylene violet. These substances are also present in matured polychrome methylene blue.

The formula of a slightly modified Romanowsky Stain which gives excellent results is given below:—

Two stock solutions are required—

Solution A.— Methylene blue, pure medicinal

1·0

grm.

Sodium carbonate

0·5

Water

100·0

c.c.

Keep in a warm incubator for two or three days, until the solution is distinctly purple in colour. It improves with age.

Solution B.— Eosin, water soluble, extra B.A.

1·0

grm.

Water

1,000·0

c.c.

This solution must be kept in the dark, in dark-tinted (amber-coloured) bottles, as unfortunately it is decolorized by light.

Before use each stock solution must be diluted. Thus, make up 5 c.c. of each stock solution to 100 c.c. by adding distilled water. For staining, 1 volume of solution A is added to 2 or 3 volumes of solution B. Mix thoroughly by shaking, pour the mixture over the film, previously fixed in absolute alcohol, and stain for ten to fifteen minutes. Wash carefully in running water, then dry. The cytoplasm of a protozoan parasite will be stained blue, the chromatin purplish-red and vacuoles or very tenuous protoplasm will remain colourless.

The exact proportions of solutions A and B, which must be mixed together, should be determined by experiment. Freshly mixed stain must be used on each occasion.

Leishman’s Stain is the precipitate resulting from the interaction of alkaline methylene blue and eosin. The washed and dried precipitate is collected and dissolved in pure methyl alcohol, which acts as a fixative; 0·015 grm. of Leishman powder may be dissolved in 10 c.c. of methyl alcohol for staining films. The film is covered with the solution for one minute, twice the volume of water is then added and mixed with the stain on the slide. The staining is then continued for five to ten minutes, and the film is finally washed with water.

Giemsa’s Stain.—This should be procured ready made. Azure II is a mixture of methylene azure and methylene blue. (Methylene azure is sometimes known as Giemsa’s Azure I.) The formula given by Giemsa himself in 1912 is:—

Azure II-eosin

3·0

grm.

Azure II

0·8

Glycerine, pure

125·0

Methyl alcohol, pure

375·0

The film is first fixed in absolute alcohol. The proportion of stain usually used is one drop of stain to 1 c.c. of water. Stain for about ten minutes and then wash in water.

The details of the application of the Giemsa stain to films fixed wet and to sections must be sought in larger works on technique. These works should also be consulted for information regarding the use of Pappenheim’s Panchrome mixture.

There are numerous formulae of stains containing ripened Hæmatoxylin or its essential principle, Hæmatein. A mordant is necessary, one of the alums being usually employed. The mordant may be included as an ingredient in the staining mixture, or it may be used separately as in the case of the so-called iron-hæmatoxylins, wherein ferric ammonium alum is used separately and is followed by staining with hæmatoxylin or hæmatein. A few of these stains of general application may now be mentioned.

Delafield’s (or Grenachier’s) Hæmatoxylin.

Hæmatoxylin crystals

4

grm.

Absolute alcohol

25

c.c.

Saturated aqueous solution of ammonia-alum

400

Mix these ingredients, and leave exposed to light and air for three to four days. Filter and add—

Glycerine

100

c.c.

Methyl alcohol

100

Allow the mixture to stand until the colour is sufficiently deep, then filter and place in a stoppered bottle. The solution should be allowed to ripen for at least two months before use. Dilute aqueous solutions of the stain are of service for films and for sections. A trace of acetic acid may be added at the moment of use, for sharp differentiation.

Ehrlich’s acid hæmatoxylin, Mayer’s hæmalum, and Mayer’s glychæmalum are also useful. Their formulæ will be found in larger works.

The chief Iron-Hæmatoxylin Stain is that devised by Heidenhain. Unfortunately the procedure involved is a long one, and various modifications have been made to obviate this disadvantage. Hæmatein may be used instead of ripened hæmatoxylin.

One efficacious modification of Heidenhain’s stain is that of Rosenbusch. The smear or tissue, after fixation, must be graded downwards through the alcohols to water. Mordant for one and a half hours in a 3 1/2 per cent. aqueous solution of ferric ammonium sulphate. Stain for about three minutes in 1 per cent. solution of ripe hæmatoxylin or hæmatein in absolute or 96 per cent. alcohol, to which a drop of saturated aqueous solution of lithium carbonate, sufficient to produce a wine-red colour, has been added. Differentiate under the microscope with a very dilute solution of the ferric ammonium sulphate. Wash, gradually dehydrate, clear and mount in balsam. It must be remarked that iron-hæmatoxylin is a regressive stain, hence great care must be exercised in differentiating with the iron alum.

Gentian Violet.—A 1 per cent. alcoholic solution of gentian violet, or of methyl violet, or of crystal violet, will be found useful for staining spirochætes.

Methyl Green.—This substance is considered to be a chromatin stain, for either fresh or perhaps recently fixed tissues. A concentrated aqueous solution contains about 1 per cent. of the stain. This should be added to a 1 per cent. solution of acetic acid. It may be used for demonstrating the nuclei of ciliates.

In conclusion it is essential to remember that the actual magnification of figures of Protozoa should be given, and not merely the combination of objective and ocular that has been used, for unless the tube-length and distance of the drawing board from the ocular be also given, it is not possible to compute the magnification from such information. Drawings should always be made with the aid of a camera lucida, drawing prism or other form of projection apparatus.