272 THE DIPHTHERIA BACILLUS 



[The medium is adjusted so that on coagulation the reaction is but faintly alkaline. 

 When throat swabs are sown on surface slopes and incubated at 37 C. for 18 hours, 

 colonies of the diphtheria bacillus appear almost invariably to yield a bluish-pink 

 tint, with diffusion of like tint through the medium ; with Hofmann's bacillus the 

 growth is yellowish and alkaline with diffusion of a yellowish tint. Staphylococci 

 usually yield a straw-coloured raised growth with discrete colonies, but certain 

 varieties produce either discrete pink colonies with strictly local diffusion of tint 

 into the medium ; and again, other varieties, more especially such as are derived 

 from the throats of adults, yield acid with pink colouration of the medium in varying 

 intensity. The colonies of torulse are usually raised and straw-coloured but they 

 may be brownish or red.] 



Sow a tube of blood serum with a small piece of membrane held in a platinum 

 loop and after rubbing it all over the surface of the serum and without recharg- 

 ing the loop sow two other serum tubes (p. 82 B 1). In the absence of membrane 

 the spatula or cotton-wool swab, as the case may be, which has previously 

 been applied to the tonsils or pharynx is rubbed over the surface of the 

 serum. Should the cotton-wool swab be dry on arrival at the laboratory 

 wash it in a little sterile water and then use the latter for sowing cultures. 

 Dried membranes should similarly be softened in sterile water before being 

 sown. 



Incubate the cultures at 37 C. and examine about 20 hours later : colonies 

 of the diphtheria bacillus are easily recognized at this stage ; some cocci 

 indeed produce a very similar growth but it is moister and more homogeneous 

 than that of the diphtheria bacillus. A mere naked eye examination of the 

 growth is however insufficient, and must always be supplemented by micro- 

 scopical examination. If examination of the cultures be delayed beyond 

 24 hours difficulty may arise from the development of micro-organisms which 

 are either associated with the diphtheria bacillus or which are present as an 

 impurity. Select the culture which shows the greatest number of discrete 

 colonies. 



[In examining cultures sown with swabs from infected throats Cobbett picks off 

 single colonies one by one with a straight platinum wire, sows a separate tube of broth 

 with each colony, and then smears the wire in a straight line across a cover-glass. 

 The first colony is smeared along one edge of the cover-glass, the others at right 

 angles to it. In this way not only is it possible to make 4 to 9 separate preparations 

 on one cover-glass from the different colonies of a single culture-tube, but pure cul- 

 tures of each colony also are available for further examination. ] 



A diagnosis of diphtheria [infection] must be given in all cases in which 

 colonies consisting of organisms having the morphological appearance and 

 giving the staining reactions of the diphtheria bacillus are found on the 

 serum sown with the suspected material. [In cases of faucial diphtheria 

 such colonies will generally be present in large numbers ; in laryngeal 

 diphtheria they are sometimes few in number this is often the case also with 

 convalescents and " contacts."] 



C. Inoculations. When absolute confirmation of the positive microscopical 

 and cultural results is desired resort must be had to animal inoculation. 

 Several of the suspected diphtheria colonies must be inoculated, because 

 bacilli of different degrees of virulence may be present in the same membrane. 



Each colony is dealt with as follows : Sow a portion of a colony in broth 

 (taking every care that the needle touches no other colony), incubate for 24 

 hours, and after verifying the purity of the culture inoculate 1 c.c. sub- 

 cutaneously into an adult guinea-pig : the animal dies more or less rapidly 

 according to the virulence of the organism (p. 247). Should the organism 

 prove non- virulent for the guinea-pig test it on a small bird in a similar 

 manner. 



