DIAGNOSIS OF DIPHTHERIA 293 



stained with Loffler's methylene blue, another by Gram's method. 

 The bacilli will be found lying parallel to one another in larger or 

 smaller groups, together with involution forms. Films stained 

 with Neisser's or Pugh's stain (see below) may also be of assist- 

 ance. Another method is to stain the films for five seconds in 

 dilute carbol-methylene blue (seven drops to 10 c.c. water), 

 rinsing and drying, and counter-staining in dilute carbon-fuschin 

 (ten drops to 10 c.c. water) for one minute, rinsing and drying 

 (Higley). 



* II. Frequently the membrane is so crowded with different forms 

 of organisms that it is extremely difficult to recognise the diphtheria 

 bacilli with any degree of certainty. Recourse must then be had to 

 cultivation. 



For this purpose sloping blood-serum tubes, or tubes of serum- 

 agar, must be employed ; simple agar is unsuitable. * 



A piece of membrane or a swabbing from the throat is rubbed 

 over the surface of one or two serum tubes, care being taken not 

 to break up the medium. The tubes are incubated at 37 C. for 

 eighteen to twenty hours, and are then examined microscopically 

 whether there is any visible growth or not. If there be no visible 

 growth a scraping is taken by means of a sterilised platinum needle 

 from the whole surface and a film is prepared. If there is a visible 

 growth the film should be prepared from the most likely colonies, 

 or, if the growth be confluent, from the upper half inch or so. 

 A microscopical examination must always be made, for some 

 colonies certain staphylococci and torulae, for example simulate 

 those of the diphtheria bacillus very closely. The films may be 

 stained with Loffler's methylene blue for five to ten minutes, or 

 by Pugh's method, then washed and dried. If the films are made 

 on a slide, after staining, washing, and drying, a drop of cedar oil 

 may be put on the stained patch, which is then examined directly 

 without a cover-glass. If, however, there is very little growth, it 

 is better to make a cover-glass specimen, as the position of the 

 material is so much more easily located. The preparations are 

 examined with a iV m - oil-immersion lens magnifying not less than 

 800-1000 diameters, and the Klebs-Loffler bacillus is identified 

 from the description given in the text. 



Prausnitz considers that if negative results are obtained after 

 eighteen to twenty -four hours' incubation the tubes should be incu- 

 bated for a further twenty to twenty -four hours and re-examined, 



1 Various selective media have been devised, e.g. potassium-sulpho- 

 cyanide, neutral-red, glucose- blood-serum (Rankin, Journ. of Hyg. 

 xii, 1912, p. 60). 



