DIAGNOSIS OF DIPHTHERIA 349 



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 smears 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 torulse, 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 

 there is very little growth, a ring may be made round the material 

 on the under surface of the slide so that the position of the film 

 may be located. The preparations are examined with a T V m - 

 oil-immersion lens magnifying not less than 800-1,000 diameters, 

 and the Klebs-Loffler bacillus is identified from the description 

 given in the text. 



If negative results are obtained after eighteen to twenty-four 

 hours' incubation the tubes should be incubated for a further 

 twenty to twenty-four hours and re-examined, and occasionally 

 a positive result may be obtained by this longer incubation. 



Loffler's methylene blue gives much more characteristic pre- 

 parations than Gram's method. 



Although eighteen to twenty hours is recommended for incu- 

 bating the cultures, a microscopical examination will sometimes 

 reveal the bacilli at a much earlier period. The author has found 

 them in as short a time as six hours, but if bacilli are then not 

 found the tubes must be incubated for the longer period. 



Neisser's method of staining is as follows : 



(a) One gramme of methylene blue (Griibler's) is dissolved in 

 20 c.c. of 96 per cent, alcohol, which is then mixed with 950 c.c. 

 of distilled water and 50 c.c. of glacial acetic acid. 



(6) Two grammes of Bismarck brown are dissolved in one litre 

 of boiling distilled water and the solution is filtered. 



The preparations are stained in (a) for half a minute, rinsed, 

 and stained in (b) for one minute, washed in water, dried, and 

 mounted. The bacilli are stained brown, and contain two, rarely 



