8. Flame the plug of tube No. 1, remove it, then flame the lips of the tube; slightly raise the cover of Petri dish No. 1, introduce the mouth of the tube; then, elevating the bottom of the tube, pour the liquefied medium into the Petri dish, to form a thin layer. Remove the mouth of the tube and close the "plate." If the medium has failed to flow evenly over the bottom of the plate, raise the plate from the levelling platform and by tilting in different directions rectify the fault. 9. Pour plates No. 2 and No. 3, in a similar manner, from tubes Nos. 2 and 3. 10. Label the plates with the distinctive name or number of the inoculum, also the date; the number of the dilution having been previously indicated (step 3). 11. Place in the cool incubator for three or more days, as may be necessary. In this way colonies may be obtained quite pure and separate from each other. In plate No. 1, probably, the colonies will be so numerous and crowded, and therefore so small, as to render it useless. In plate No. 2 they will be more widely separated, but usually No. 3 is the plate reserved for careful examination, as in this the colonies are usually widely separated, few in number, and large in size. _Agar plates_ are poured in a similar manner, but the agar must be melted in boiling water and then allowed to cool to 45° C. or 42° C. in a carefully regulated water-bath before being inoculated, and the entire process must be carried out very rapidly, otherwise the agar will have solidified before the operation is completed. NOTE.--In pouring plates, since tube No. 1 (for the first dilution) rarely gives a plate that is of any practical value it is frequently replaced by a tube of bouillon or sterile salt solution, and in such case plate No. 1 is not poured. ~Surface Plates.~-- This method of pouring what may be termed "whole" plates (since colonies may appear both on the surface and in the depths of the medium) is essential to the accurate study of the formation of colonies under various conditions, but when the main object of the separation of the bacteria is to obtain subcultivations from a number of individual bacteria, "surface" plates must be prepared, since here colony formation is restricted to the surface of the medium. The method adopted varies slightly according to whether the medium employed is gelatine or agar, or one of the derivatives or variants of the latter. (a) ~Gelatine Surface Plates.~-- 1. Liquefy three tubes of nutrient gelatine. 2. Pour each tube into a separate Petri dish and allow it to solidify. Then turn each plate and its cover upside down. [Illustration: FIG. 126.--Surface plate spreader.] 3. When quite cold raise the bottom of plate 1, revert it and deposit a drop of the inoculum (whether a fluid culture or an emulsion from solid culture) upon the surface of the gelatine with a platinum loop--close to one side of the plate; replace the bottom half of the Petri dish in its cover.