482 APPENDIX 



charge of a tetanic wound is not always easy. Make pveparationSj and 

 stain with carbol-fuchsin. Drumstick-shaped, spore-bearing bacilli are 

 to be looked for. If a small piece of tissue is available, sections should 

 be prepared and double-stained. Cultivations should also be made from 

 the discharge in blood serum or glucose agar incubated at 37° C. for 

 forty-eight hours. Then keep the culture at 80° C. for twenty to thirty 

 minutes, to kill all non-sporing bacilli. Sub-culture in glucose gelatine 

 in hydrogen at 22° C, and examine in five days. Animal inoculation 

 (mice and guinea-pigs) is generally necessary. 



To isolate the tetanus bacillus from soil, proceed as follows : — Make 

 an emulsion of the soil in sterilised water. Expose it to 80° C. for 

 twenty minutes. Add 1 c.c. of the emulsion to each of three tubes of 

 glucose-formate broth, and incubate anaerobically in Buchner's tubes at 

 37° C. After twenty-four hours' incubation, inoculate guinea-pigs sub- 

 cutaneously, using 0"1 c.c, and observe results. Also make glucose-agar 

 plates from the same emulsion (after heating to 80° C), and incubate 

 anaerobically in Bulloch's apparatus. 



3. Tuberculosis. — The tubercle bacillus is an acid-fast organism, 

 stained by Ziehl-Neelsen method. But several allied organisms possess 

 the same tinctorial properties, and therefore inoculation into a guinea-pig 

 is frequently necessary for diagnosis. Sputum, however, is generally 

 accepted as proved to be tubercular if bacilli having the morphology and 

 staining properties of the tubercle bacillus are present. 



4. Typhoid. — Widal's Application of Griiber's Reaction. This diagnostic 

 test depends upon the effect which the blood serum of a person suffering 

 from typhoid fever has upon the B. typhosus. The effect is twofold. 

 In the first place, the actively motile B. typhosus becomes imraotile ; and 

 secondly, there is an agglutination, or grouping together in colonies, of 

 the B. typhosus. Neither of these features occur if healthy human blood 

 serum is brought into contact with a culture of the typhoid bacillus. 



The method of using the test is as follows : — 



(a) Collection of Serum. — Wash the lobe of the patient's ear with 

 antiseptic (2 per cent, lysol), and by rubbing render the ear hyperaemic. 

 Wash with methylated spirit and dry. Puncture the vein of the lobe 

 with a sterilised needle or lancet, and collect the issuing blood in a 

 pipette. Hold one end in contact with the bleeding point, and lower the 

 other end. By gravity the blood will enter the pipette ; if not, gentle 

 suction may be applied. When full to the shoulder, remove the pipette, 

 and placing the clean end to the lips, draw the blood gently but com- 

 pletely into the body of the pipette. Now seal the ends in a flame, and 

 let the pipette lie horizontally till the blood is coagulated. 



(6) Dilution of Serum. — Place the pipette in the vertical position, 

 preferably in an ice-chamber, and in a few hours the clear serum, free 

 from corpuscles, will collect at the lower end, ready for dilution. If 

 necessary, centrifugalise to obtain corpuscle-free serum. There are 

 several methods of dilution used in practice, but broadly they are 

 divisible into two, a rough-and-ready dilution and an exact measured 

 dilution. 



