102 CULTIVATION OF ANAEROBIC MICRO-ORGANISMS 



between two constrictions. Wrap the plug with the tubes in position in paper, and 

 sterilize separately from but at the same time as the dish. 



3. Keep the gelatin or agar liquefied in a water bath at 

 30-40 C. as the case may be, while sowing the medium. Take 

 the india-rubber plug out of its wrapper and fit it into the neck 

 of the dish as quickly as possible. 



4. Lute the plug with Golaz's wax, attach A to the hydro- 

 gen-generating apparatus, keeping the medium liquefied in the 

 water bath, and pass a stream of hydrogen through the medium 

 for a few minutes. Lay the apparatus horizontally so that 

 the medium flows into the dish and continue the current of hy- 

 drogen for several minutes. Exhaustion may be combined with 

 washing with hydrogen if it be thought necessary. 



5. Seal the ends of A and B beyond the wool plugs. 



D. Zinsser's method. Zinsser uses an apparatus 

 similar to a Petri dish, but deeper, and having an an- 

 nular space of 5-6 mm. between the dish and the cover. 

 The agar or gelatin, as the case may be, is sown and 

 poured into the dish, and after it is set is inverted on 

 to the cover, into which a little alkaline pyrogallol is 

 poured (p. 89). A layer of oil is poured on the surface 

 FIG. 94.-Bombicci' s dish. o f tne pyrogallate in the annular space. 



E. Tarozzi s method. Tarozzi uses an alkaline glucose- agar which has 

 been heated under a pressure of two atmospheres (p. 98). The medium is 

 poured to a depth of 1 cm. into Petri dishes with ground-glass covers, which 

 are luted with paraffin. 



F. Marino's method. 1. Take a number of Petri dishes, remove the lids, 

 and place the dishes, cavity upwards, over (and therefore partly within) 

 them. Wrap in paper and sterilize in the hot air sterilizer. 



2. Take a number of large test-tubes, and into each pour 30 c.c. of 

 O5 per cent, glucose-agar. Sterilize in the autoclave. 



3. Cool the agar to 40-42 C. in a water bath. Add to the contents of 

 each tube 1 c.c. of rabbit- or horse-serum previously heated at 55 C. for 

 20 minutes. 



4. Sow the tubes by the dilution method. 



5. Pour the contents of each tube into the lid of one of the sterile Petri 

 dishes, and cover with the other part of the dish in such a way that the agar 

 is contained between and compressed by two sterile glass surfaces, the cavity 

 of the dish being obviously upwards. Cover with a sterile glass plate large 

 enough to project beyond the edges of the Petri dish to protect it from 

 contamination. Incubate. 



6. After the colonies have grown, gently separate the agar from one of the 

 glass surfaces, leaving it adhering to the other, and pick of! with a fine- 

 pointed pipette, any colonies it is thought desirable to examine. 



When separating the medium from one of the glass surfaces it often happens that 

 the agar is torn ; so it may be that the colony which was wanted cannot be found, 

 or else that it has become contaminated, by rubbing up against another colony or 

 by contact with the water of condensation. 



Lief man, Fehrs and Sachs-Miike's modification of Marino's method obviates this 

 defect. Instead of the lower part of the Petri dish a plate of sterile glass is used 

 as a cover, and sufficient medium is poured into the lid to slightly overflow the 

 edges. In covering with the sheet of glass care must be taken that no air bubbles 

 are included. 



[G. Bulloch's apparatus. The technique as now generally adopted has 

 been explained at p. 96. The only modification required in the present 



