212 



Journal of Applied Microscopy. 



familiarize him with the method of sim- 

 ple staining and with Gram's double 

 staining of bacteria. The concentration 

 of the dye, the time of exposure, and the 

 temperature of the liquid are important 

 factors which must not be lost sight of 

 by the operator. The concentration of 

 the acid or alcohol employed in the decol- 

 oration and the length of time that these 

 are allowed to act on the stained section 

 likewise affect the result. In the event 

 of failure, the beginner should ascertain 

 by systematic trial which of the factors 

 is the one at fault. Success in staining 

 sections requires an intelligent persever- 

 ence in and a study of the method 

 employed. 



SIMPLE STAINING. 



The section, after treatment with xylol 

 and alcohol, is transferred to water and 

 then to dilute fuchsin or gentian violet 

 stain. Carbolic fuchsin may be use 1 to 

 advantage. The section is allowed to 

 remain in the dye from five to fifteen 

 minutes. It is then washed for two or 

 three minutes in water in order to remove 

 the excess of dye; after which it is placed 

 in very dilute acetic acid (1 cc. of glacial 

 acetic to 1,000 cc. of water) for one-half 

 to one minute. The treatment with acetic 

 acid is not always necessary and should 

 be avoided if possible. The section is 

 now placed in strong alcohol for one-half 

 to one minute and is then transferred to 

 clean water. 



It is then transferred to a slide and 

 examined with a one-sixth or one-eighth 

 inch objective. This examination is made 

 in order to acquaint oneself with the con- 

 dition of the specimen. The bacteria 

 should be deeply stained and should be 

 differentiated as much as possible from 

 the surrounding tissue. If they are feeb- 

 ly stained, it is unnecessary to proceed 

 with the method. If the tissue is still 

 deeply stained, thus masking the bac- 

 teria, it should be again subjected to 

 decoloration with acetic water and 

 alcohol and reexamined. 



When the section shows the proper 

 degree of differentiation it should be 

 placed in absolute alcohol for a few sec- 

 onds in order to thoroughly dehydrate it. 

 Inasmuch as this treatment with alcohol 

 removes additional dye, it is well to stop 

 the decoloring process, as given above, 

 when the specimen is still slightly over- 

 stained. The treatment with alcohol, 

 when dehydrating, will remove the slight 

 excess of stain, and thus complete the 

 differentiation. 



The section is then placed in oil of 

 cloves or origanum for some minutes, 

 after which it is transferred to xylol and 

 then placed on a clean slide. The excess 

 of xylol is removed by the application of 

 a piece of filter paper. A drop of Canada 



balsam is now applied and a clean cover- 

 glass is placed in position. Gentle pres- 

 sure or slight warming will cause the bal- 

 sam to spread out evenly. 



Oil of cloves can be used to advantage 

 in the clearing up of sections. It dis- 

 solves some of the stain, and thus assists 

 in the differentiation. This is especially 

 true when it is used in Gram's method. 

 All trace of the oil must be removed from 

 the section by washing in xylol. Some 

 stains, like methylene blue, are readily 

 dissolved by the essential oils and in such 

 instances the oil can be omitted. The 

 dehydrated section in that case is placed 

 direct in xylol. The process of simple 

 staining as given is applicable to nearly 

 all bacteria. It must, therefore, be 

 resorted to whenever the organism does 

 not take the Gram's stain. 



The several steps in the method can be 

 summarized thus: 



Simple stain: 



Dilute anilin dye, 5 to 15 minutes. 



Water, 2 to 3 minutes. 



Acetic water, 1-2 to 1 minute. 



Strong alcohol, 1-2 to 1 minute. 



Water (and examine). 



Absolute alcohol, few seconds. 



Oil of cloves or cedar. 



Xylol. 



Canada Balsam. 



GRAM'S METHOD. 



This method of double staining bacteria 

 is easy of execution, and, when properly 

 carried out, it will give clean, beautifully 

 stained preparations. Unfortunately all 

 pathogenic bacteria cannot be stained by 

 this process. 



Fresh solutions of anilin water gentian 

 violet and of iodine are prepared accord- 

 ing to the directions given in the third 

 paper of this series. The stain may be 

 slightly warmed on the iron plate, but 

 this is not necessary. Care must be 

 taken not to stain too long, since in that 



