-inch objective through the bottom of the plate and the layer of
medium.

b. If gross differences can be detected mark a small circle on the
bottom of the plate around the site of each of the selected colonies,
with the grease pencil.

c. If no obvious differences can be made out choose nine colonies
haphazard and indicate their positions by pencil marks on the bottom of
the plate.

(B) Fishing Colonies.--

a. Take a sterile Petri dish and invert it upon the laboratory bench.
Rule two parallel lines on the bottom of the dish with a grease pencil,
and two more parallel lines at right angles to the first pair--so
dividing the area of the dish into nine portions. Number the top
right-hand portion 1, and the central bottom portion 8 (Fig. 139).
Revert the dish. The numbers 1 and 8 can be readily recognised through
the glass and by their positions enable any of the other divisions to be
localised by number. This is the stock dish.

b. Slightly raise the cover of the dish, and with a sterile
teat-pipette deposit a small drop of sterile water in the centre of each
of the nine divisions.

c. With the sterilised platinum spatula raise one of the marked
colonies from the "plate 3" and transfer it to the first division in the
ruled plate and emulsify it in the drop of water awaiting it. Repeat
this process with the remaining colonies, emulsifying a separate colony
in each drop of water.

(C) Preliminary Differentiation of Bacteria.--

a. Prepare a cover-slip film preparation from each drop of emulsion in
the "stock dish" and number to correspond to the division from which it
was taken. Stain by Gram's method.

b. Examine microscopically, using the oil immersion lens and note the
numbers of those cover-slips which morphologically and by Gram results
appear to be composed of different species of bacteria.

[Illustration: FIG. 139.--Diagram for stock plate.]

(D) Preparing Isolation Subcultures.--

a. Inoculate an agar slope and a broth tube from the emulsion in the
stock dish corresponding to each of these specially selected numbers.

b. Ascertain whether the cover-slips from the nine emulsions in the
stock dish include all the varieties represented in the cover-slip film
preparation made from the original mixture before plating.

c. If some varieties are missing prepare a second stock dish from
other colonies on plate 3, and repeat the process until each
morphological form or tinctorial variety has been secu