2. Discharge one-quarter minute in nitric-acid alcohol (nitric acid I, alcohol 10).

3. Wash in water.

4. Contrast-stain in a saturated aqueous solution of methylene-blue.

5. Alcohol.

6. Xylol.

7. Balsam.

While leprosy bacilli stain readily by this method, tubercle bacilli will not stain in so short a time.

Syphilis Bacillus.-Lustgarten's Method.-I. Stain twenty-four hours at room-temperature and two hours in the thermostat at 40° in aniline-gentian-violet.

2. Wash off in absolute alcohol three to five minutes.

3. Decolorize (a) in a per cent. aqueous solution of permanganate of potassium, and then (b) a few seconds in an aqueous solution of pure sulphurous acid (strength not given).

4. Wash in water.

5. Alcohol.

6. Oil of cloves.

7. Canada balsam.

If the section is not entirely decolorized when the section is put into water, then the third step must be repeated until decolorization is complete. If desired, the sections can be stained in safranin after the fourth step.

Giacomi's Method.-I. Stain several minutes in hot aniline-fuchsin.

2. Wash out in very dilute aqueous solution of chlorid of iron.

3. Decolorize in concentrated solution of chlorid of iron. 4. Wash out in absolute alcohol.

5. Xylol.

6. Xylol balsam.

The smegma bacillus is stained by Lustgarten's method, but not by the methods given for tubercle bacilli in tissues, because in all of them alcohol is used as well as an acid to effect the decolorization.

METHODS OF EXAMINATION OF ANIMAL PARASITES.

Protozoa. Of the rhizopoda, the malarial organisms and the ameba coli are of much pathological interest.

Malarial Organisms.-Three varieties of the plasmodium malariæ have been described-namely, the tertian, quartan, and estivo-autumnal parasites. They develop within the red corpuscles and cause the destruction of the corpuscles affected. The earliest forms of the parasite appear in the blood during the latter part of the malarial paroxysm or shortly after it. At this time they appear as small, colorless, disc-shaped hyaline bodies which occupy but a small portion of the blood-corpuscles. They possess a varying degree of ameboid movement, the amount depending upon the type of the organism. These ameboid movements are best observed on the warm stage. During the process of development the parasites increase in size and more or less completely fill the red corpuscles containing them. Small particles of reddish-brown pigment are produced, during their growth, from the hemoglobin of the corpuscles in which the organisms are developing. These granules show varying degrees of motion, probably imparted to them by the movements of the parasites. At first the pigment appears to be scattered about in the corpuscle, but it is in reality in the extremities of the pseudopodia. Later it appears more evenly spread about in the periphery. Toward the end of the cycle of development the pigment collects in the center of the parasite; at this time the ameboid movements have ceased, indications of segmentation occur, and the parasite nearly or completely fills the corpuscle. Oftentimes at this stage only a small portion of the corpuscle is visible at some point on the edge of the parasite.

The beginning of segmentation is indicated by a number of radial lines extending from the periphery of the parasite toward the central clump of pigment. Segmentation takes place, and the pigment is surrounded by a number of distinct segments which vary with the type of the organism. Each of these segments shows a central refractive spot

At this time one notices

which probably is the nucleus. small hyaline bodies, like those of the early stage in the development of the parasite, in some of the red blood-corpuscles. Oftentimes such a regular process of segmentation is not observed, but enough has been said to indicate the manner in which reproduction occurs. Segmentation is the indication of an approach of a paroxysm. Extra-cellular forms of the parasites are not infrequently seen. They may be fully-grown organisms which have destroyed the corpuscles that contained them, or they may be partly-grown organisms which have left the corpuscles. These free parasites are indistinct in outline and contain pigment. They possess ameboid movements, and may be considerably larger than a red blood-corpuscle. Various changes are observed in them:

1. They may increase in size until they become nearly as large as polymorphonuclear leucocytes. With the increase in size there is a gradual cessation in the movement of the pigment-granules until finally the organisms present the appearance of misshapen masses of protoplasm containing motionless pigment-granules.

2. They may undergo fragmentation and give off several small circular pigmented bodies.

3. Vacuolization may occur.

4. Flagellate forms may develop. One or more threadlike processes are thrust out from the organisms. These flagella may contain pigment, and may break away from the organism and move about among the corpuscles, looking not unlike the spirilla of relapsing fever.

The three varieties of parasites differ from each other in a number of ways. The chief differences are the length of the cycle of development; the size of the full-grown organisms; the difference in the refractibility of the organisms; the quantity, size, and color of the pigment-granules; the degree of ameboid movement; and the number and shape of the segments into which the full-grown organisms divide. In the earliest stage the varieties of organisms cannot be distinguished from each other.

The tertian parasite completes its cycle of development in about forty-eight hours. When it has attained its fullest growth it almost fills the corpuscle, which has become larger than normal. This organism is less refractive than either of the other two. The pigment-granules are more numerous, finer, and more reddish-brown in color; the ameboid movements are much more active; the segments are more irregular in shape and more numerous than those of the quartan parasite, varying from twelve to twenty in number.

The quartan appears to complete its cycle of development in from sixty-four to seventy-two hours. The full-grown organism does not fill completely the corpuscle, and the latter is not increased in size. The organism is more refractive than the tertian parasite. The pigment-granules are fewer in number, coarser, and have a darker-red color. The ameboid movements are slower; the segments are pear-shaped, more symmetrical, and less numerous than those of the tertian parasite, varying from six to twelve in number. Segmenting organisms are more numerous in the peripheral circulation than in the case of the tertian parasite.

The estivo-autumnal parasite cannot be studied so thoroughly in the peripheral circulation, because the later development and segmentation take place in the internal organs. The length of time required to complete its cycle of development is not so definitely settled. It appears to require from twelve to twenty-four hours, more or less. The full-grown organism is smaller than the tertian parasite, and the corpuscle which contains it is often smaller than normal and more or less distorted. The parasite is quite refractive. The pigment-granules are few in number and coarse. The ameboid movements are slow. After the duration of fever for from five days to a week or more, elongated, ovoid, or crescentshaped bodies make their appearance. They are sometimes as large or larger than a red corpuscle. These bodies are not a result of segmentation, but appear to be a further development of the round hyaline bodies. They are highly refractive and contain granules of coarse pigment in the center. They lie usually at one side of the red corpuscles,

the latter more or less completely filling the concavity between the two horns of the crescent. They may lie in the center of the corpuscles. Some of the apparently free ovoid bodies are turned in such a way as to present a convex surface toward the observer.

Double infections occur quite frequently in both tertian and quartan fever, and in the latter not infrequently triple infections occur. In the double infections two groups of parasites reach maturity on successive days and cause daily febrile paroxysms. In the triple infection of quartan fever three groups of organisms mature on successive days and cause corresponding paroxysms.

Methods of Examining the Blood for Malarial Organisms. -The organisms of malaria can be detected in fresh specimens of blood or in specimens of blood which have been fixed and stained. The examination of fresh specimens of blood is simpler and more reliable, because the development of the parasites can be observed.

The method employed in making cover-glass preparations of the blood has been thoroughly described (see preparation of cover-glass specimens in the Examination of the Blood, page 336).

In examining a fresh specimen of the blood for the malarial organisms a glass slide is substituted for one of the coverglasses, and the cover-glass which has the drop of blood on its surface is dropped lightly upon the glass slide and allowed to remain there. The first four or five drops of blood should be quickly wiped away from the ear until a very small drop is obtained. Great care must be exercised to touch only the tip of the drop with the cover-glass, so as to avoid smearing the blood. If the blood is smeared on the cover-glass, the edges of the blood-drop will dry before the cover-glass can be transferred to the slide, and the blood will not spread. It is necessary that the blood should spread in a thin layer in order to study satisfactorily the individual corpuscles. If one desires to study the preparation for several hours, the edges of the cover-glass can be surrounded by melted paraffin or vaselin to exclude the air.