108 STAINING OF BACTERIA 



arrested by the paper filter, and a clear watery solution with the smell 

 of anilin oil obtained. This watery fluid is known as anilin water. 

 Add to the latter enough of a saturated alcoholic solution of gentian 

 violet until a metallic luster is produced on the surface. This indi- 

 cates that the gentian-violet stain has been added to the point of 

 saturation. The strong stain which has now been prepared is known 

 as anilin-water gentian violet. This stain does not keep well and must 

 be made fresh every few days. 

 B. Grain's Decolorizing Fluid. 



lodin 1 gram 



Iodide of potassium 2 grams 



Distilled water 300 c.c. 



The steps in staining by Gram's method are as follows: 



1. Obtain the cover-glass preparation held in forceps, from pus, 

 a culture, or any other secretion or excretion, as already described. 

 Allow it to become air dry and fix in the flame as usual. 



2. Cover the preparation with recently prepared anilin water 

 gentian violet and allow the stain to act for several minutes in the 

 cold, or, better, heat slightly over a flame. 



3. Pour off the stain and cover with Gram's iodin decolorizing fluid; 

 change this fluid once, and allow it in all to act one minute. 



4. Pour off the iodin solution and wash freely in 95 per cent, 

 alcohol until no more violet color is given off. 



5. Allow the alcohol to evaporate or dry between filter paper, and 

 now counter-stain the cover-glass preparation with a weak watery 

 solution of eosin (J- to ^ watery solution of eosin). 



6. Dry between filter paper, mount on a slide in Canada balsam, and 

 examine with oil-immersion lens. Certain bacteria, if treated by this 

 method, appear in a deep violet color, while others appear in a faint 

 eosin (yellowish pink) stain. The bacteria which appear in violet 

 are said to be stained by Gram's method, to hold the Gram stain, 

 or to be Gram positive. Those which are stained faintly pink do 

 not stain by Gram's method, lose Gram's stain, or are Gram negative. 



Gram Positive Bacteria. The following are some of the important 

 pathogenic bacteria which are Gram positive (appear deep violet if 

 stained by Gram's method) : 



Staphylococcus pyogenes aureus, albus, and citreus. 

 Streptococcus pyogenes. Pneumococcus. 



Micrococcus tetragenus. Bacillus diphtheria). 



Bacillus tuberculosis. Bacillus anthracis. 



Bacillus tetani. Actinomyces. 



Bacillus aerogenes capsulatus. 



Gram Negative Bacteria. The following are some of the impor- 

 tant pathogenic bacteria which are Gram negative (appear light 

 pink if stained by Gram's method) : 



