Probably the best stain for plague bacilli is that recommended by Archibald. There are two solutions, one made by dissolving 0.5 gram of thionin and 2.5 grams phenol crystals in a 1% aqueous solution of formalin; the other solution is made by dissolving 0.5 gram methylene blue and 2.5 grams phenol crystals in a 1% formalin solution. Let these stock solutions stand 24 hours before using and then mix equal parts of each solution; filter, and stain smear for 10 seconds. Wash and dry.
In a case of suspected pneumonic plague we stain the smear of watery or thin blood-tinged sputum as above.
The same procedure may be followed with a rather heavy blood smear of a drop of the 5 or 10 cc. taken from a vein for culturing in a case suspected of septicaemic plague.
Plague is practically the only bacterial disease where there is likelihood of finding the causative organism in smears. In septicaemic plague the blood culture is the proper procedure and one should take 5 to 15 cc. of blood in 15 to 25 cc. of normal saline containing 1% of sodium citrate. This prevents coagulation and at one’s leisure 1 or 2 cc. can be added to tubes of melted agar and plates poured or other portions added to bouillon or 3% salt agar. This same blood emulsion can be used to infect guinea pigs subcutaneously or to infect them cutaneously by rubbing on the shaven surface.
In smears from material from buboes, from sputum, or in blood smears, as well as from blood or spleen smears from experimental animals, we obtain the typical morphology of a cocco-bacillus (1.5 × 0.5 microns) with very characteristic bipolar staining, there being an intermediate, unstained area. Very characteristic also is the appearance in these smears of degenerate types which stain feebly and show coccoid and inflated oval types. The presence of these involution forms associated with typical bacilli is almost diagnostic for one with experience. Inoculating tubes of plain agar and 3% salt agar with this same material, we obtain in plain agar cultures organisms which are, typically, small, fairly slender rods, which do not stain characteristically at each end and are not oval. The smear obtained from the salt agar presents most remarkable involution forms—coccoid, root-shaped, sausage-shaped forms, ranging from three to twelve microns in length, more resembling cultures of moulds than bacteria. Another point is that on the inoculated plain agar we are in doubt at the end of twenty-four hours whether the dew-drop colonies are really bacterial colonies or only condensation particles. By the second day, however, these colonies have an opaque grayish appearance, so that now, instead of questioning the presence of a culture, we consider the possibility of contamination.
Blood cultures in septicaemic plague may show from 5 to 500,000 bacilli per cc. Smears from the blood in such cases are positive in only about 17%.
The plague bacillus grows well at room temperature—its optimum temperature being 30° instead of 37°C., as is usual with pathogens. Next to the salt agar culture, the most characteristic one is the stalactite growth in bouillon containing oil drops on its surface. The culture grows downward from the under surface of the oil drops as a powdery thread. These are very fragile, and as the slightest jar breaks them, it is difficult to obtain this cultural characteristic.
Albrecht and Ghon have shown that by smearing material upon the intact, shaven skin of a guinea pig, infection occurs. This is the crucial test. Smear the material on a shaven surface about 1 inch square.
A pocket made by cutting the skin of a guinea pig with scissors and extended subcutaneously with scissors or forceps, into which a piece of the suspected plague tissue is thrust with forceps, is more practical than injecting an emulsion with hypodermic syringe.
Mice inoculated at the root of the tail succumb quickly. Rats, this being primarily a disease of rats, are of course susceptible.