Journal of Applied Microscopy. 



191 



Water, 



Strong alcohol, 

 Water and examine, 

 Contrast color, eosin (ten to twenty- 

 seconds), 

 Water and examine. 

 Dry in air, 

 Canada balsam. 



DOUBLE STAINING OF SPORES. 



Many bacilli when grown under favor- 

 able conditions form spores. These 

 bodies, which are the analogues of the 

 seeds, in the higher plants, are formed 

 within the bacterial cells. As a rule but 

 one spore is formed within one bacillus. 

 When tho spore-containing bacillus is 

 stained by dilute fuchsin or gentian vio- 

 let, the bacterial cell proper will take the 

 stain, whereas the spore will remain col- 

 orless. This resistance to coloration on 

 the part of the spore is due to the dense 

 impenetrable wall which envelopes the 

 contents of the spore. Under certain 

 conditions the staining reagent can be 

 forced into the spore. Once inside, it 

 becomes as difficult to remove the dye as 

 it was to introduce it. On careful treat- 

 ment with alcohol the stain can be 

 washed out of the bacterial cell proper so 

 that if the specimen is examined the 

 spore will appear deeply stained within a 

 colorless bacillus. On treatment with a 

 suitable contrast color, the latter becomes 

 colored and shows the spore in marked 

 relief. 



Spores containing bacilli may be found 

 on potato cultures, prepared as described 

 in the first paper. They may be present 

 after the growth has developed for sev- 

 eral days. If available, sporulating hay 

 bacilli may be used. 



Cover-glass preparations are prepared 

 and fixed in the usual manner. The pre- 

 paration is held in the forceps in the left 

 hand with the specimen side up, and cov- 

 ered with fresh carbolic fuchsin solution, 

 or anilin water fuchsin. It is then held 

 over a low Bunsen flame so that vapors 

 are slowly given off. From time to time 

 the liquid lost by evaporation is replaced 

 by the addition of a drop or two of the 

 dye. Under no condition should the dye 

 be allowed to dry down on the cover- 

 glass. 



After heating the specimen in this 

 manner for two or three minutes, it 

 should be thoroughly washed in water 

 and examined under the one-sixth or one- 

 eighth inch objective. Colorless spores 

 should no longer be visible, but every- 

 thing should be stained a deep red. If 

 the spores are not colored, the specimen 

 should again be covered with carbolic 

 fuchsin and heated till they take on the 

 stain. 



The cover-glass with the deeply stained 

 spores is then placed in dilute alcohol 

 and gently moved about. From time to 

 time, it should be washed in water and 

 examined under the microscope. As soon 

 as the bacilli are decolored, the washing 

 in alcohol is discontinued. The specimen 

 then shows bright red spores within cells 

 that are almost or wholly colorless. The 

 cover-glass is then stained for a short 

 time with methyline blue, washed with 

 water and examined. The spores should 

 be stained a deep red while the bacillus 

 itself should be light blue. 



The method of double staining spores, 

 it will be noticed, is essentially the same 

 as that employed for staining the tuber- 

 cle bacillus. The method may be sum- 

 marized as follows: 



Cover-glass preparation, 



Dry in air. 



Fix in flame. 



Carbolic fuchsin (hot, two to five 

 minutes). 



Water, and examine. 



Dilute alcohol. 



Water, and examine. 



Contrast color, methylene blue (one- 

 quarter to one-half minute). 



Water, and examine. 



Dry in air, 



Canada balsam. 



STAINING OF FLAGELLA. 



Many bacteria possess active motion. 

 The organs which cause this movement 

 cannot be seen when the germs are 

 examined in hanging-drop. Moreover, 

 they are not rendered visible by the ordi- 

 nary methods of staining. In order to 

 demonstrate their presence, it is neces- 

 sary to resort to a special procedure. 

 The organism will then be seen to be sur- 

 rounded by a fringe of very delicate wavy 

 lines known as whips or flagella. The 

 number of flagella .will vary with the 

 different species of bacteria, but usually 

 a bacillus will possess from three to ten 

 or more of these delicate appendages. 



The method of staining flagella is, with 

 slight modifications, that proposed by 

 Loffler. Special attention must be given 

 in the first place to the preparation of 

 the specimen. Only fresh, vigorous, 

 active cultures should be employed. The 

 growth, therefore, should not be more 

 than one or two days old. In old mate- 

 rial the whips are liable to be torn off 

 from the cell. 



An excess of material should not be 

 placed on a cover-glass. It is advisable 

 to first prepare a suitable dilution of the 

 germs. For this purpose two or three 

 drops of distilled water are placed on a 

 slide and a very small amount of ihe 



