ng approximately the number of bacteria
present in a given volume of fluid (by means of a graduated-celled slide
resembling a haematocytometer, Fig. 138), and diluting the fluid by the
addition of sterile water or bouillon until a given volume (usually 1
c.c.) of the dilution contained but one organism. By planting this
volume of the fluid into several tubes or flasks of nutrient media, it
occasionally happened that the resulting growth was the product of one
individual microbe. A method so uncertain is now fortunately replaced by
many others, more reliable and convenient, and in those methods selected
for description here, the segregation and isolation of the required
bacteria may be effected--

A. ~By Mechanical Separation.~

1. By surface plate cultivation:

(a) Gelatine.
(b) Agar.
(c) Serum agar.
(d) Blood agar.
(e) Hanging-drop or block.

[2. By Esmarch's roll cultivation:

This archaic method (see page 226) is no longer employed for the
isolation of bacteria.]

3. By serial cultivation.

B. ~By Biological Differentiation.~

4. By differential media.

(a) Selective.
(b) Deterrent.

5. By differential incubation.

6. By differential sterilisation.

7. By differential atmosphere cultivation.

8. By animal inoculation.

The selection of the method to be employed in any specific instance will
depend upon a variety of circumstances, and often a combination of two
or more will ensure a quicker and more reliable result than a rigid
adherence to any one method. Experience is the only reliable guide, but
as a general rule the use of either the first or the third method will
be found most convenient, affording as each of them does an opportunity
for the simultaneous isolation of several or all of the varieties of
bacteria present in a mixture.

~1. Surface Plate Cultivations.~--

(a) _Gelatine_ (_vide_ page 164).

(b) _Agar_ (_vide_ page 167).

(c) _Alkaline serum agar_ (_vide_ page 211).

These plates are prepared in a manner precisely similar to that adopted
for nutrient gelatine and agar surface plates (_vide_ pages 231-233).

(d) _Serum Agar._--

1. Melt three tubes of nutrient agar, label them 1, 2, and 3, and place
them, with three tubes of sterile fluid serum, also labelled 1a, 2a,
and 3a, in a water-bath regulated at 45 deg. C.; allow sufficient time to
elapse for the temperature of the contents of each tube to reach that of
the water-bath.

2. Take serum