30 General Bacteriology. 
EXERCISE XXIV. STUDY OF CELL GROUPING. 
Hanoine Drop PREPARATIONS. 
a. Make hanging-drop preparations from bouillon cultures prepared above and also 
from those supplied. 
b. Examine with oil-immersion objective and assign organisms to their proper place, 
as determined by cell grouping, in the following scheme: 
Name. Sketch. 
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Filaments 
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Masses | Irregular ow... cee cece eee ees wivicaeeeeti. chad a Seka eaar. « Suances 
| Zoogloea cnile wan static Ganiead epam ede women nes See Y AA ATEERNET ERA ete Oe 
IMPRESSION PREPARATIONS. The exact relation of cell to cell as they develop in the 
colony can frequently be determined with greater accuracy by studying a ‘‘contact prep- 
aration’’ which is prepared as follows: 
a. Melt a gelatin tube and slopeit, when solid make a streak culture of B. mycoides 
FLUEGGE and when growth has taken place dip the tube in hot water to loosen gelatin 
which is then slipped out of the tube. 
b. Lower gently a clean cover-glass over the surface. Apply a slight pressure by 
tapping glass. Raise cover-glass by one edge taking care that natural arrangement of 
adherent bacteria is not disturbed. 
c. Thoroughly air dry the same, then fix and stain in the ordinary manner. 
d. Examine the thinner layers noticing the arrangement of cells with reference to 
each other and draw a sufficient number to illustrate this relationship. 
AGcar Hanaine-Drop CULTURES. 
a. Melt a tube of agar and cool to 48° C. 
b. Sterilize a cover-glass by passing it two or three times through the flame quickly. 
c. With the needle make a streak on the cover-glass about 3mm. long of B. subtilis. 
d. With the loop place a drop of liquid agar so as to cover up streak. 
e. Seal cover-glass to hollow ground slide. Incubate and later examine and sketch. 
EXERCISE XXV. STUDY OF INVOLUTION FORMS. 
a. Grow Bacillus subtilis (EHRENB.) Mic. in bouillon and also in a solution con- 
taining 0.1% asparagin, 10% sugar, and by means of stained cover-glass preparations 
compare the individual organisms in each case in regard to theirform and size. The de- 
generated or involution forms are more apparent by staining. Draw several cells illus- 
trating a variety of involution forms. 
b, Examine a culture of Bacterium diphtheriae (LOEFFLER) Mia. on Loeffler’s blood 
serum. Read M. & R. 5. 
