7. When the air is displaced, shut off the stop-cock of the entry tube, then that of the exit tube b. Screw down the clamp and remove the glass tube from the rubber connection and connect up the short tube a to the air pump by means of pressure tubing. 8. Open the stop-cock of tube a and with two or three strokes of the air pump, aspirate a small quantity of gas, so creating a slight vacuum. Then shut off the stop-cock and disconnect the air pump. 9. Fill the 10 c.c. bulb pipette with water; insert its point into the rubber tubing on the long tube b as far as the screw clamp. Open the screw clamp and run in water until stopped by the internal pressure. Shut off stop-cock. (The water dissolves the soda and pyrogallic acid converting the latter into alkaline pyro. and so bringing its latent capacity for oxygen into action). [Illustration: FIG. 137.--Bulloch's jar.] 10. Reverse the tubes from the tubulures so that they meet, out of harm's way, over the top of the bell glass; again see that all joints are tight and transfer the apparatus to the incubator. This last method is the most satisfactory for anaerobic cultivations, as by its means complete anaerobiosis can be obtained with the least expenditure of time and trouble. FOOTNOTES: [8] See also method of opening and closing culture tubes, pages 74-76. [9] If compressed tablets of pyrogallic acid cannot be obtained make up a stock "acid pyro" solution Pyrogallic acid, 10 grammes Hydrochloric acid, 1.5 c.c. Distilled water, 100 c.c. and at step 4, run in 10 c.c. of the solution. XV. METHODS OF ISOLATION. The work in the preceding sections, arranged to demonstrate the chief biological characters of bacteria in general, is intended to be carried out by means of cultivations of various organisms previously isolated and identified and supplied to the student in a state of purity. A cultivation which comprises the progeny of a single cell is termed a "pure culture"; one which contains representatives of two or more species of bacteria is spoken of as an "impure," or "mixed" "cultivation," and it now becomes necessary to indicate the chief methods by which one or more organisms may be isolated in a state of purity from a mixture; whether that mixture exists as an impure laboratory cultivation, or is contained in pus and other morbid exudations, infected tissues, or water or food-stuffs. [Illustration: FIG. 138.--Hæmatocytometer cell, showing, a, section through the centre of the cell, and b, a magnified image of the cell rulings.]