Before the introduction of solid media the only method of obtaining pure cultivations was by "dilution"--by no means a reliable method. "Dilution" consisted in estimating approximately the number of bacteria present in a given volume of fluid (by means of a graduated-celled slide resembling a hæmatocytometer, 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° 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 tube No. 1a and agar tube No. 1. Flame the plugs and remove them from the tubes (retaining the plug of the agar tube in the hand); flame the mouths of the tubes, pour the serum into the tube of liquefied agar and replace the plug of the agar tube. 3. Mix thoroughly and pour plate No. 1 _secundum artem_. 4. Treat the remaining tube of agar and serum in a similar fashion, and pour plates Nos. 2 and 3.