322 MEDICAL BACTERIOLOGY 



a. Loeffler's methylen blue, or Koux stain.^ 



6. Gram's stain. 



c. Neisser's stain: a. 1 gram methylen blue dissolved in 20 ce. 

 of alcohol (96%), is added to 950 ce. of distilled water and 50 ee. 

 of glacial acetic acid; h. 2 grams of bismarck brown dissolved in a 

 liter of distilled water. Films are stained in a. 5 to 8 seconds, washed 

 in water, stained in &. 3 to 5 seconds, dried and mounted. The 

 Crouch^ stain may be similarly employed. 



Usually, however, mere microscopical examination is not sufS- 

 cient, and culture methods must be employed. In fact this method 

 ought always to be used. In this case make smears on Loeffler's 

 blood serum and incubate them at 36 to 38° C. for 12 to 24 hours and 

 then examine the growth in cover-glass preparations. The diph- 

 theria organism if present, should show : 



a. Characteristic appearance with Loeffler's methylen blue. 



i. Positive Neisser stain. 



c. Positive Gram stain. 



Occasionally micro-organisms (pseudo-diphtheria bacilli among 

 others) are met with that very closely resemble the Klebs-Loeffler 

 bacillus and render a positive diagnosis doubtful. In such cases 

 attention to following table will be helpful : 



^Eoux stain— Solution A: Dahlia 1, alcohol 10, and distilled water 90 

 parts; Solution B: Methyl green 1, alcohol 10, and distilled water 90 parts; 

 mix 2 parts of A with 1 of B. 



2 Crouch stain— Aqueous solution of dahlia (1%) 1 part, aqueous solution 

 lof methyl green (1%) 5 parts, and distilled water 4 parts. 



