proportion of pararosaniline hydrochloride in the dye. When the 

 alcohol has evaporated to concentration of 20-25% a colloidal 

 precipitate forms which settles on the flagella making them thicker 

 and colored red. Freshly prepared stain usually has a variable 

 amount of coarse precipitate. On storage in the refrigerator this 

 precipitate settles and the supernatant used for staining is clear. 

 Do not disturb this precipitate when removing stain from the bot- 

 tle. By careful observation of the stain on the slide the formation 

 of the colloidal precipitate may be observed by the change from 

 a clear solution to an opaque and rust colored solution. With the 

 slide on a black background a strong beam of light readily shows 

 the formation of this precipitate. As soon as the precipitate has 

 formed the staining is completed and the slides are washed imme- 

 diately. 



Another method is to prepare one or two extra slides. When 

 the staining time appears to be about up the trial slide is washed. 

 If the smear is not macroscopically visible the time is too short. 

 After about 2 more minutes wash off the second slide and observe 

 the smear. With a little experience one trial slide is usually suffi- 

 cient and will serve as a guide to the staining time of a dozen or 

 more slides stained at the same time. When many slides are 

 stained at one time they should be placed on the board about 1 

 inch apart to allow the alcohol to evaporate at somewhat the 

 same rate from the middle and end slides. When the staining time 

 is up the slide is placed directly under the faucet or a stream of 

 water. Do not allow any of the stain to run off the slide before it 

 is placed under the faucet. After washing, the slide is allowed to 

 drain dry or carefully blotted. 



Cotinterstaining 



The soma of some species of bacteria characteristically stain 

 very faintly or not at all. With such organisms a counterstain may 

 be used for better visualization of the soma. A satisfactory coun- 

 terstain is the usual methylene blue stain diluted 5-10 times with 

 water and slightly alkalinized with sodium hydroxide, sodium bi- 

 carbonate, or sodium borate. Application of this stain for a minute 

 or so usually stains the soma blue while the flagella remain red. 



