MICROORGANISMS AND FERMENTATIONS 25 



stains most used are alcoholic solutions of methylene blue or 

 fuchsine. The former, which must not be diluted too much with 

 water, is specially suited for preparations from milk, as it does 

 not stain the casein strongly ; if fuchsine is used, a red patch may 

 be formed in which it is impossible to distinguish the bacteria. As 

 fuchsine stains very^deeply, it is used in dilute solution ; its 

 staining capacity is increased by the addition of phenol (carbol 

 fuchsine). Tubercle bacteria, which are coated with wax, and 

 intracellular spores, which are very resistant to staining, may be 

 stained by warming with carbol fuchsine, and when thus stained 

 they retain the colour so persistently that mineral acids fail to 

 remove it. Methylene blue is also suited better than fuchsine for 

 staining broth cultures. Acid cultures should, however, first be 

 neutralised, unless the methylene blue solution has been treated 

 with a little alkali. 



Gram's method of staining is especially useful in the identification 

 of bacteria ; it depends on the fact that microorganisms after 

 treatment with gentian violet are stained a violet black by a 

 solution of iodine in potassium iodide, and retain this stain more 

 or less completely on treatment with absolute alcohol l . Bacteria 

 which retain the stain are known as Gram- positive, and those 

 which lose it as Gram-negative. Most yeasts and moulds and all 

 true lactic acid bacteria are Gram-positive, while most of the 

 harmful milk bacteria are Gram-negative. The Gram method 

 lends itself well to the examination of lactic acid cultures and 

 sour milk preparations in general, as the casein is completely 

 Gram- negative. 



Instead of staining, Bum's Indian ink method may be adopted ; 

 a drop of the liquid to be examined is placed on a cover slip, mixed 

 with a little sterile liquid Indian ink, allowed to dry, mounted in 

 water, and examined. If the liquid is acid it should be neutra- 

 lised, as the colloidal Indian ink is coagulated by acid. Treated 

 in this way, the organisms show up white on a black background. 

 Permanent preparations may be made by mounting in Canada 

 balsam instead of water. 



1 V. Jensen (" Hospitalstidende," 1912, No. 20) recommends a 0-5 per 

 cent, solution of methyl violet in water, and a solution of 1 gram of iodine 

 and 2 grams of potassium iodide in 100 c.c. of water. 



Notes to page 17. 



1 100 gm. sugar free casein (precipitated by acid) were digested for a 

 week at blood heat with a litre of water containing 4-6 per cent. HOI and 

 2 gm. pepsin. The resulting solution contained, after neutralisation, 

 sterilisation and filtration, about 1 per cent. N and 1-2 per cent. NaCl. 



2 At 50 C. the digestion is completed in 24 hours. The sugar dis- 

 appears at the same time. The highly acid solution contains about 2 per 

 cent. N. 



