8 BACTERIOLOGY OF THE EYE 



There are a few details requiring attention if good Gram preparations are to be 

 obtained : 



(a) The discharge must be spread out on a perfectly clean slide in as thin and 

 uniform a layer as possible. When the thickness of the film varies, those parts 

 should be examined which are uniformly thin. At places where the material is 

 heaped up on the slide the reagents do not penetrate, and organisms which really 

 are Gram-positive (e.g., Xerosis) may appear Gram-negative, being stained by the 

 contrast dye. At such places positive and negative individuals of the same species 

 may lie one over the other. At other times in a clump of discharge a Gram- 

 negative organism may appear blue where the decolorizing alcohol has not been 

 able to penetrate. 



(b) The whole film should be rapidly covered with the stain. 



(c) Do not overdo the iodine. 



(d) Thoroughly wash away the alcohol with water. 



(e) Avoid overstaining with the contrast dye, excess of which must be thoroughly 

 washed away. Acid-safranin and fuchsin spoil the blue gentian colour; and we 

 often'no'tice after several weeks that the'blue colour has disappeared, and has been 

 replaced by red. 1 



(/) The anilin-gentian-violet must be often renewed best every three to four 

 weeks. Czaplewski's carbol gentian is more stable. 



To be absolutely certain when examining a new organism we should 

 place on the slide beside the film some well-known Gram-positive 

 bacterium, such as a fresh culture of Stapliylococcus or B. xerosis, 

 When this standard remains deep blue after the staining, then the 

 solutions are satisfactory, and the less well-marked or negative 

 staining of the other organism is demonstrated. 



When bacteria stain but slightly positive ('amphoter '), and the 

 colour is partly reddish or red, a film should be stained without any 

 contrast colour, for in the case of these microbes the violet is perhaps 

 only loosely fixed by the iodine, and is easily overcome by the 

 contract dye. 



There are undoubted variations in the action of the Gram stain. 

 Involution forms often do not take it up ; e.g., in exudations containing 

 Subtilis, individuals are seen which are Gram-negative. 



Amongst the negative bacteria the Gonococcus most rapidly loses 

 its colour ; it is completely decolorized (i.e., shows the contrast 

 stain) when the cell nuclei and the mucin in the film are still blue. 

 None of the other negative organisms give up the colour so rapidly. 

 Diplobacillus, Bact. coli, Bac. Friedldnder are usually not completely 

 decolorized so long as the blue remains in the tissues around. 

 Koch-Weeks bacillus, influenza bacillus, Meningococcus, and Micr. 

 catarrhalis decolorize rapidly, like the Gonococcus. Many authors con- 

 sider this rapid loss of colour as of value in differential diagnosis, 



1 This occurs, as a rule, in old films, which therefore are unsuitable for demonstration. 

 The balsam can be removed, and the film stained afresh. 



