GRAM'S STAINS 



115 



GROWTH 



in acetone until no more color is given 

 off, 10-15 sec. Wash in aq. dost. 

 Counterstain in 0.05% basic fuchsin 

 in N/500 hydrochloric acid (see Normal 

 Solutions). Drain, do not wash, apply 

 1% aq. trinitrophenol, 5-I min. Wash 

 in aq. dest. Dehydrate and differen- 

 tiate in acetone 10-15 sec, clear in xylol 

 and mount in balsam. Gram + bac- 

 teria, violet; Gram—, red; nuclei, light 

 red; cytoplasm, yellow. 



3. For organisms in frozen sections 

 by Krajian, A. A., Arch. Path., 1941, 

 32. 825-827. Stain 7-Wix frozen sec- 

 tions for 2 min. in Harris' alum hema- 

 toxylin. Wash in tap water till blue 

 and destain quickly by dipping 5 to 7 

 times in acid alcohol. Rinse in tap 

 water and apply following solution for 

 3 min. — copper sulfate, 7 gm.; zinc sul- 

 fate, 4 gm. dissolved in 100 cc. aq. with 

 aid of heat. Pour off and apply 0.3 gm. 

 brilliant green in 10 cc. above copper 

 zinc mixture for 5 min. Rinse in water 

 and fortify with 5% aq. ammonium ni- 

 trate for 1 min. Rinse in tap water and 

 stain with carbol fuchsin (Ziehl-Ncel- 

 sen) for 2 min. Rinse in tap water, blot 

 and apply dioxane for 2 min. Pour off 

 and add equal parts creosote and xylol, 

 changing this mixture and agitating to 

 promote even differentiation until back- 

 ground appears clear red. Clear in 

 pure xylol (2 min.) and mount in damar. 

 Gram positive organisms bluish green; 

 gram negative ones red. 



The mechanism of the Gram staining 

 technique and the interpretation of the 

 findings has been concisely presented 

 by Dubos, R. J., The Bacterial Cell. 

 Harvard Univ. Press, 1945, 460 pp. 

 The Gram + bacteria differ from the 

 Gram — ones in being more acidic and 

 perhaps in possession of lipids with 

 higher content of unsaturated acids. 

 Their Gram positiveness depends on 

 intactness of their cell walls, for ero- 

 sions of the walls make them Gram 

 negative. When the outer layer of the 

 cell walls is removed by extraction with 

 bile salts they become Gram negative. 

 The Gram positive property can be re- 

 stored by "replating" the bacteria with 

 the extract of the outer layer. The 

 . outer la5''er apparently contains a pro- 

 tein ribonucleate complex, for Gram 

 positive organisms can be made Gram 

 negative by action of the enzyme, 

 ribonuclease. The quality of the cell 

 membrane conditions not only the en- 

 try and retention of stains but the 

 whole manner of life of the cells. See 

 Cell Membrane, Acid Fast Bacilli, and 

 Dead Cells. 

 Craven's Gold Chloride method for nerve 

 endings in muscle (Garven, H. S. D., 



Brain, 1925, 48, 380-441). This is Fis- 

 cher's modification of l^nvier's tech- 

 nique as used in Golgi's Laboratory. 

 Immerse small pieces of tissue in 25% 

 aq. pure formic acid and tease a little 

 to assure penetration 10-15 min. Blot 

 with clean cloth. Place in 1% aq. gold 

 chloride just sufficient to completely 

 cover tissue and shake. Avoid all iron 

 instruments. Cover dish with blue or 

 yellow glass. Leave 20 min. Blot 

 with clean cloth and repeat above treat- 

 ment with formic acid and gold leaving 

 this time in latter 24 hrs. in absolute 

 darkness. Repeat still again. Pass to 

 glycerin and leave in closed vessel in 

 ordinary light. The sharpness of the 

 intensely purple black nerves in a 

 lightly colored background increases 

 with time. Small pieces can then be 

 transferred to aq. dest. and the indi- 

 vidual fibers separated. This is facili- 

 tated by dissociation in dilute nitric 

 acid. Wash and make final mounts in 

 glycerin. The author used panni cuius 

 carnosus of hedgehog, striated muscle 

 of frog and lizard, extrinsic eye muscle 

 of rabbit and human pectoral muscle. 



Gray, R, B, BB, see Nigrosin, water soluble. 



Green PL, see Naphthol Green B. 



Grenacher, see Alum Carmine, Borax 

 Carmine. 



Grieves' method for undecalcified dental 

 tissues and bone as outlined by Shipley 

 (McCIung, p. 345) is: Fix small pieces 

 in 10% formalin 24-36 hrs. or any other 

 desired fixative. Wash in running water 

 24 hrs. Then pass through 2 changes 

 of aq. dest. 1 hr. each. Dehydrate 

 through ascending alcohols beginning 

 with 50% ale. Equal parts abs. ale. 

 and chloroform, 2 hrs. Chloroform, 2 

 hrs. 5% sol. of rosin in chloroform, 2 

 hrs. 10% sol. rosin, 2 hrs. Sat. sol. 

 rosin until it becomes transparent. 

 Imbed in melted rosin using one after 

 another the rosins in 3 small glass dishes 

 on a heated copper bar, 1 min. each. 

 The chloroform carried over evaporates. 

 The rosin containing the tissue is al- 

 lowed to cool. The block is ground 

 very thin by hand on a carboriundum 

 stone and polished on a fine hone all 

 grinding being done under luke warm 

 water. The smooth surface is then 

 mounted on a slide with a little melted 

 rosiii after which the surface is ground 

 and polished in the same waj-- and the 

 section is ready for mounting or for 

 staining. 



Gross Specimens, see Color Preservation. 



Ground Substance (intercellular), see Tis- 

 sue Fluid. 



Growth. Many techniques are now avail- 

 able for the measurement of growth of 

 tissues. Increase in number of cells 



