MATERIALS. 315 
seen with a i /6 inch. Examine the bacteria and sketch. In this way 
make a microscopic examination of all of the cultures isolated and puri- 
fied, and compare with Figs. 7 and 9. If it is desired to preserve the speci- 
men place a drop of Canada balsam on the bacteria after drying, and 
then cover with a cover-glass. 
Motility. To determine the motility of bacteria transfer a small quan- 
tity from an agar slant to a bouillon tube, and allow to grow for 24 hours. 
Place a drop of the 24-hour-old bouillon culture on a slide and put upon it 
a cover-glass. Examine this with a i /6 inch objective and with the dia- 
phragm nearly closed. The best light for the purpose is artificial light (elec- 
tric) placed near the microscope and reflected through by the plain surface 
of the mirror. It will be very difficult at first to see the bacteria, but with 
careful focusing they will appear as transparent dots or rods. Examine 
carefully to determine whether they are stationary or motile, calling 
only those motile that move back and forth across the stage and not those 
that simply dance back and forth without locomotion (the Brownian 
motion). It is sometimes desirable to keep the specimen under observa- 
tion for some time in which case a hanging-drop method may be used. 
A concave slide is to be used and a ring of vaseline painted around the 
depression. The drop containing the living bacteria is placed in the 
middle of a large cover-glass and inverted over the concavity of the slide. 
By pressing it firmly into the vaseline ring it will be sealed so as to prevent 
evaporation and may be kept under observation for hours. 
No. 9. Bacteria in the Mouth. With a clean knife scrape a little of 
the material attached to the teeth and spread it in a very thin layer over a 
slide. Dry, fix and stain, and with a microscope note the large numbers 
of bacteria present. Sketch the varieties seen. 
No 10. Gram Stain. Prepare the following: 
Anilin Oil Gentian Violet. 
Saturated alcoholic solution of gentian violet, 6 c.c. 
Absolute alcohol, 5 c.c. 
Anilin water,* 50 c.c. 
Grams lodin Solution. 
lodin, . i gm. 
Potassium iodid,^ 2 gm. 
Distilled water, ~ 300 c.c. 
Spread and fix on a slide, a little of one of the cultures of bacteria, 
and stain for one and one-half minutes in the gentian violet solution. Pour 
off stain, without washing, and place in the iodine solution for one and one- 
half minutes. Apply 95 per cent, alcohol until the drippings do not stain 
white filter-paper. This will take about three minutes and the specimen 
will be largely decolorized. Wash in water and study with microscope to 
*Made by adding 2 to 3 c.c. anilin oil to 50 c.c. of water and shaking thoroughly, with subse- 
quent filtering. 
