Exercise 10. The Microscopic Examination of Bacteria 
in Pure Culture 
MATERIALS: 
3 plain agar slants 
Pure cultures made in Exercise 9 
9 plain slides 
1. Carefully label three plain agar slants and make new transfers of the pure 
cultures isolated in Exercise 8. Place them in the incubator for 48 hours. 
Save them for use in Ex. 11. 
2. Carefully clean 8 slides. Arrange three of them in a row and label each 
of the three the same as one of the pure cultures that have been isolated. 
Place a small drop of water, about 1 /8 inch across, in the center of each slide. 
Remove some of the growth from the agar culture with the sterile wire needle 
and mix it with the water on each slide, spreading the mixture as thin as 
possible. (See Fig. 8.) Permit each to air dry. Cause the bacteria to adhere 
to the slides by passing them three times through the top of the gas flame 
(fixing). (See Fig. 9.) Lay the slide upon the wire cover of the sink for stain¬ 
ing. (See Fig. 10.) Repeat with each of the other cultures. 
3. Stain one slide from each culture with methylene blue for 5 minutes. 
Stain another slide from each culture with carbol fuchsin for one minute. 
Stain the remaining slide from each culture by means of the Gram method as 
follows: 
(a) Stain with ammonium oxalate crystal violet for 2 or 3 minutes. 
( b ) Pour off the stain. 
(c) Do not wash. 
( d ) Treat with Gram’s iodin (Lugol’s) solution for one minute. 
Pour off and treat again for one minute. 
(e) Pour off iodine. Wash in water quickly. Blot off excess 
water quickly but do not permit the film to air dry. 
(0 Decolorize in equal parts of acetone and 95 per cent ethyl 
alcohol, until the stain no longer washes from the slide. 
(g) Dry between blotters. 
(/?) Counter stain one minute with aqueous one percent safranin. 
(i) Wash and dry between blotters or air dry. 
This method of staining is an aid in the classification of bacteria. Some 
bacteria do not lose the purple color imparted to them by the crystal violet 
when treated by this method; others do lose the color. The former are termed 
Gram-positive, the latter Gram-negative organisms. 
4. Examine the slides. Draw and describe one slide from each culture. 
(a) What is the form of the organisms from each culture? (Fig. 5.) 
(b) Are any of them yeasts? 
(c) Do all of the organisms stain by the Gram method? 
(</) Are there spores in any of the cultures? 
(e) What stain shows each organism best? 
(f) Under the microscope, how can bacteria be distinguished from 
yeast? 
(g) In a Petri dish culture there is a mixture of bacteria, yeast, 
and mold colonies. What should be done and what would be 
found in distinguishing them? 
