144 STAINED PREPARATIONS 



Notes. (a) Stage 5, decolourization, is a delicate manipulation. The length of 

 time during which decolourization must be continued varies with the intensity of 

 the stain used, the length of time during which it is allowed to act, the number 

 of organisms, etc. ; practice and a certain amount of skill are more than any rules 

 the secrets of success. It is obvious that insufficient decolourization of a gram- 

 negative organism may lead to mistakes ; on the other hand the most resistant 

 bacteria can be decolourized by prolonging unduly the action of the alcohol, and 

 such treatment might result in a gram-positive organism being classed with the 

 gram-negative group. 



[(6) In view of the difficulty as to the time of decolourization we have found 

 it useful, especially for beginners and in dealing with organisms such as the 

 meningococcus, to prepare on the same slide three separate films. At one end there 

 will be a film of a gram-positive organism e.g. Staphylococcus, at the other end a 

 gram-negative organism e.g. Bacillus coli communis, and in the centre the organism 

 whose reaction is to be tested e.g. Meningococcus. The films are stained and 

 decolourized as described above, and then examined in water. If decolourization is 

 sufficient, the staphylococci are all violet and the colon bacilli all pink (or brown). 

 If the films have been under- decolourized some of the bacilli will be stained violet, 

 and if over- decolourized some of the cocci will be pink (or brown). When it is 

 evident that the decolourization has been correctly done the organism whose 

 reaction is being tested is examined.] 



(c) Films prepared by Gram's method do not keep so well as when stained with 

 ordinary stains and ultimately become decolourized. 



4. Claudius' method. 



Claudius suggested a method of staining which, while having all the 

 advantages, has both a simpler technique and gives more constant results 

 than Gram's method. Thus the bacillus of malignant oedema and the 

 bacillus of quarter ill are somewhat readily decolourized by Gram's method 

 but retain the stain well by Claudius' method. 



The author repeated Claudius' experiments and obtained results which fully 

 confirm that observer's. Claudius' method has many advantages for the student ; 

 beginners using Gram's method never know when to stop decolourizing, sometimes 

 they leave the alcohol on too long and sometimes they do not let it act for long 

 enough, and in either case the results are unsatisfactory. These difficulties do not 

 arise in Claudius' method. 



The following solutions are required : 



(a) A 1 per cent, aqueous, solution of methyl violet 6B (or a solution of 

 carbol-gentian violet). 



(b) A solution of picric acid. 



Saturated solution of picric acid, - 1 volume. 



Distilled water, - 1 



Technique. 1. Prepare and fix a film in the ordinary way. 



2. Stain with violet for 1 minute. 



3. Wash in water, and blot up the excess. 



4. Treat with the picric acid solution for 1 minute and blot. 



5. Decolourize with chloroform or clove oil until the decolourizing agent is 

 no longer tinted blue. 



6. Examine in clove oil or mount in balsam. 



