Bacteria Staining .'^0 



mordantinpj with iodine to lonn with the dye a precipitate which is 

 insohible in water and is neither too sohiblc nor itrsohiblc in alcohol, 

 the differentiator. 



For the mechanism of gram reactions see Bartholomew and iMittwer 

 (1950, 1951); Bartholomew et al. (1959); Mittwer et al. (1950). 



Gram-Weigert Method (krajian, 1952) 

 fixation: 10% formalin or Zenker's formol. 



solutions: 

 Eosin: 



eosin Y, C.I. 45380 1.0 gm. 



distilled water 100.0 ml. 



Sterlino's ^entian violet: 



crystal violet, C.I. 42555 (gentian violet) 5.0 gm. 



9b% ethyl alcohol 10.0 ml. 



aniline oil 2.0 ml. 



distilled water 88.0 ml. 



Mix aniline oil with water and filter. Add the crystal violet dissolved 



in alcohol. Keeps for several weeks to months. 

 Gram's iodine solution, see page 410. 



procedure: 



1. Deparaflfinize and hydrate slides to water. Remove HgCL. 



2. Stain in eosin: 5 minutes. 



3. Rinse in water. 



4. Stain in Sterling's gentian violet solution: 3 minutes for frozen 

 sections; 10 minutes for paraffin sections. 



5. Wash off with Gram's iodine and then flood ^vith more of same 

 solution: 3 minutes. 



6. Blot with filter paper. 



7. Flood with equal parts of aniline oil and xylene; reflood luitil 

 color ceases to rinse out of sections. 



8. Clear in xylene and mount. 



results: 



gram-positive bacteria, and fimgi — violet 

 gram-negative organisms — not ustially stained 

 fibrin — blue-black 



Brown and Brenn Method (1931) 

 fixation: 10% formalin preferred. 



