340 LABORATORY WORK 
the milk may be measured out with a capillary pipette calibrated to deliver o.o1 
c.c., and smeared over an area of exactly rsq.cm. Then it can be examined 
with a microscope that has a field of known size. With the size of the field, the 
amount of milk, and the size of the smear definitely known, it is possible to compute 
how many bacteria per c.c. each organism seen with the microscope represents. 
No. 11. Gram Stain.—Prepare the following: 
Anilin Oil Gentian Violet 
Saturated alcoholic solution of gentian violet, 6 c.c. 
Absolute alcohol, 5 Cc. 
Anilin water,* 50 C.C. 
Grams Iodin Solution 
Todin, t 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 see if the bacteria are still stained blue or have been 
decolorized. Different species differ in this respect. If they are still stained they 
are Gram-postitve; if decolorized, they are Gram-negative. 
No. 12. Microscopic Study of Yeast—Rub up a bit of an ordinary yeast 
cake in a little water. Place a dilute drop on a slide and proceed to stain exactly 
as above described for bacteria. Study with 1/2 immersion lens and compare with 
bacteria as to size and shape. Look over the specimen and find some cells that 
show buds. 
No. 13. Gelatin Culture Medium.—(a) Weigh out the same ingredients as 
directed in No. 2, omitting the agar. After the mixture has dissolved add 12 per 
cent. of first-grade gelatin. Allow to soak till soft and almost melted. Weigh the 
dish with its contents. Heat gently (preferably in flowing steam or in a double 
boiler over a saturated salt solution) for at least 15 minutes after the gelatin is com- 
pletely dissolved; then add water to restore original weight. 
(6) Determine the acidity and adjust the reaction as described in No. 2. 
(c) Heat 15 minutes. Cool and add the white of an egg dissolved in a little 
water. Heat slowly and allow to simmer till the egg is coagulated and the liquid 
clear; usually about 30 to 45 minutes. Replace the evaporated water. 
Filter through absorbent cotton. Place ro c.c. in each of about fifty tubes and 
the rest in a flask, plugging both with cotton. Sterilize in the steam sterilizer for 
*Made by adding 2 to3 c.c. anilin oil to so c.c. of water and shaking thorough! i 
subsequent filtering. 5 g ghly, with 
