252 THE DIPHTHERIA BACILLUS 



alkaline methylene blue [1 to 4 of water], Roux's blue or with dilute Ziehl's 

 fuchsin. [The first of these is the stain strongly recommended.] 



[The method recommended is that devised by Cobbett. Spread films, 

 dry and fix in the ordinary manner. Wash in 10 per cent, acetic acid. Wash 

 in water. Mount in a drop of Lceffier's blue diluted 5 times with water. Blot. 

 Examine.] 



When stained with methylene blue [and especially with a dilute blue] 

 the bacilli are found to be irregularly stained, granules being seen which stain 

 more deeply than the protoplasm : these granules or metachromatic bodies 

 of Babes are sometimes called polar bodies. German writers, in the deter- 

 mination of the diphtheria bacillus, attach great importance to their presence. 

 [But though the majority of diphtheria bacilli show these Babes' bodies, 

 some types stain uniformly ; moreover some bacilli other than diphtheria 

 bacilli also show deeply-staining granules.] The diphtheria bacillus, especi- 

 ally in old cultures, frequently shows irregular vacuolated spaces which do 

 not stain, whatever dye be used ; it is to be noted that these are not spores. 



(b) Gram's method. The diphtheria bacillus is gram-positive. Decolour- 

 ization must not be pushed too far because the organism will not resist a 

 prolonged action of alcohol. Gram's method gives beautiful preparations 

 with smears and sections of false membranes. 



(c) Special methods, (a) Neisser's stain. To bring out the polar bodies the 

 method of Ernst-Neisser may be applied. 



Two solutions are necessary 



A. Methylene blue (Griibler), - 1 gram. 

 96 per cent. Alcohol, - - 20 c.c. 

 Distilled water, - 950 

 Glacial acetic acid, - - - 50 ,, 



Dissolve the blue in the alcohol, then add the water and acid. 



B. Vesuvin, - 0'5 gram. 

 Boiling distilled water, - - 250 c.c. 



A cover-glass preparation is made with a drop of an emulsion of an eigh teen- 

 hour-old culture on serum. This is left in the acid solution of blue for 1-3 



hours, washed in water, stained for a few seconds 



':';":. r ' in the vesuvin solution, washed again and 



' . ' mounted. [The method may be modified by 



V^ staining for 1 minute in each of the blue and 



*^' $' ,-" *.'":.:'" brown solutions, washing in water between the 



.* two operations.] 



.*. : . v ." x .'. So stained the diphtheria bacillus is brown, 



\ v^ * '/-- with deep blue [or violet] granules situated as 



f/ . '"*'**{ ' r a rule at the poles or ends. (The pseudo-diph- 



, rt theria [Hofmann] bacillus, on the other hand, 



<T. according to Neisser, never shows polar bodies, 



but is stained uniformly brown or has some blue 



staged bv'L^r'fmSd. "Sf" gj a if ^regularly distributed through the body 

 Obj. Ath Zeiss. ot the organism. It is now admitted that this 



reaction is not characteristic.) 



[(/?) The following is a modification of Neisser's stain which apparently 

 gives better results than that just described. 

 Prepare two solutions : 



A. Methylene blue (Grubler), 1 gram. 



Absolute alcohol. - - - . . . . . 50 c c 



Glacial acetic acid, - - - . . . . . 50 



Distilled water, ----... 1000 





