FIG. 70.-Providence Health Department outfit for diphtheria diagnosis. A pasteboard box containing a swabtube and a serum-tube, both with etched surface on which to write the name and address of patients, etc.

FIG. 71.-Bacterium diphtheriæ; agar culture (photograph by Dr. Henry Koplik).

swab made of non-absorbent cotton over the back of the throat, tonsils, diphtheritic membrane, etc.

2. Rub the swab over the surface of a Löffler blood-serum tube.

3. Place the tube in an incubator at 37.5° C. for sixteen hours or longer.

4. Examine the growth for the small grayish, slightly elevated diphtheria colonies.

5. Prepare films from a suspected colony.

6. Pass three times through the flame and stain in Löffler's methylene-blue for one minute.

7. Wash, dry, mount, and examine with the 12 inch oil-immersion lens. The characteristic diphtheria bacteria can easily be detected.

8. Isolate the diphtheria organisms in pure culture by inoculating tubes from a single colony which on examination proves to be diphtheria. If a single colony cannot be found, touch the needle

once to the growth on the serum-tube and make a series of strokes on 3 or 4 tubes. When the colonies develop, those in the last tube will be sufficiently distinct so that pure cultures may be made from them. Another method is to inoculate a bouillontube or a tube of sterile water or normal salt solution, and immediately from this make stroke cult

FIG. 73.-Bacterium diphtheria, colony twenty-four hours old upon agar; x 100 (Fränkel and Pfeiffer).

ures on serum-tubes.

The growth on these will probably be in individual colonies. Either of these methods obviates the necessity of making plates.

9. Study the morphology and biology of the diphtheria bacterium obtained above.

10. The following stains are diagnostic for the diphtheria bacterium:

Hunt's Stain.

(a) Prepare films as usual.

(b) Stain in aqueous methylene-blue for one minute.

(c) Wash in water and dry.

(d) Treat with a 10 per cent. solution of tannic acid for one minute.

(e) Wash in water and dry.

() Stain in aqueous methyl-orange for one minute.

(g) Wash, dry, and mount.

Neisser's Stain.

(c) Treat films with solution A for one to three seconds.

(d) Wash in water.

(e) Treat with solution B for three to five seconds. (f) Wash, dry, and mount.

II. Test the virulence of the diphtheria organism isolated as follows:

(a) Prepare a twenty-four to forty-eight hour bouillon culture.

(b) Sterilize a hypodermic syringe and needle by soaking in 2 per cent. carbolic acid and washing out in sterile water or bouillon, or by boiling for five minutes.

(c) Select and weigh a full-grown guinea-pig. (d) While the pig is held on its back on the table

FIG. 74.—Slide, 7 x 24 inches, for the routine examination of diphtheria cultures. Each square can be placed under the lens of the microscope without disturbing the equilibrium of the slide.

by an assistant, the hair is removed from a small area on the ventral abdominal wall.

(e) With the thumb and forefinger of the left hand pinch up a fold of the skin, and with the right hand insert the hypodermic needle between the skin and the muscular body-wall.

(f) Inoculate an amount of the culture equal to I per cent. of the weight of the pig.

(g) Record the daily weight of the pig until death.