140 SUMMARY OF CURRENT RESEARCHES RELATING TO 



colour is apt to go altogether. Some of these coloui-s are unsuited 

 to certain bacteria, e. g. Bismarck-brown does not stain the bacillus 

 of lepra at all, and that of sjilenic fever but badly. In order. to stain 

 the bacteria, the sections are placed in a 1 per cent, watery solution of 

 the dye ; in a few moments they are deeply coloured, and may then be 

 " differentiated " (the term ap^jlied by Professor Weigert to the process 

 of removal of the colour from the body of the cell), by means of 

 alcohol, in which they may be allowed to lie more than an hour ; if 

 oil of cloves is used, they maybe left in it half an hour and upwards ; 

 then if there is not time to examine them, they may be put into water 

 for as much as a day without losing their colour. The ixse of absolute 

 alcohol for the washing is specially recommended ; for gentian-violet, 

 which strongly resists the washing-out process, treatment with oil of 

 cloves, and then with alcohol, transfeniug back to the oil, is the 

 quickest way. Gentian-violet is particularly useful when it is uncer- 

 tain what fonn of bacterium is present, but the colour is removed 

 fi'om the nuclei when placed in glycerine. 



Douhle-staining is very useful for colom-ing the nuclei and the 

 bacteria differently ; of all combinations picrocarmine was found to 

 be the best ; it is used as made in the following way, in preference to 

 commercial specimens of this reagent, which Professor Weigert finds 

 are seldom entirely satisfactory : — 



Over 2 grammes of carmine are poured 4 grammes common 

 ammonia, and the whole left 24 hours in a place protected against 

 evaporation ; 200 grammes of a concentrated picric acid solution are 

 then poured in ; the mixture is left 24 hours until all soluble matters 

 are dissolved. Very small quantities of acetic acid are then added 

 until a slight precipitate comes dowTi even after stii-ring ; a rather 

 copious precipitate is usually thrown down in the course of the next 

 24 hours ; it should be removed by filtration. 



A picrocarmine which does not stain readily may be improved by 

 addition of acetic acid. After staining, the sections should be washed 

 in pure water or water containing only a trace of acid. 



In applying double-staining to bacteria contained in the tissues, 

 the sections should first be treated with gentian violet and alcohol, 

 placed for a moment in water to remove the alcohol and then placed 

 in the picrocarmine and kept there for half an hour or an hour ; the ■ 

 superfluous picrocarmine is washed away with water, and the specimen 

 well washed with alcohol and mounted in Canada balsam, after passing 

 through oil of cloves. This method may be applied to Micrococci, 

 care being taken not to stain them red by a too protracted sojourn in 

 the picrocai-mine. 



Actinomyces is not stained by the usual nucleus-staining prepara- 

 tions ; orseille is used as prej)ared by Wedl, and the sections left in 

 it for about an houi* ; they are then washed superficially with alcohol 

 and transferred to gentian-violet. The wall and contents of the cell 

 of Molluscum contagiosum are differentiated by this method. 



Sections of tissues containing bacteria may be made by Eoy's 

 microtome. The frozen sections are examined either fresh or in salt 

 solution ; if they are to be stained and mounted they are sjiread out 



