HANGING DROP PREPARATIONS 133 



having a circular cup-shaped hollow about 15 mm. in diameter ground in 

 its centre (fig. 121). Sterilize the slide as well as the cover-glass with which 



FIG. 121. Koch's hollow-ground slide. 



it is to be covered by rapidly passing them through the flame several times 

 just before they are about to be used. 



(a) In the case of cultures already incubated, take a drop of the culture 

 and place it in the centre of the previously heated and cooled cover-glass, 

 invert the cover-glass over the hollow in the slide and ring the edges with a 

 little vaseline to prevent evaporation. The drop of culture hangs from the 

 lower surface of the cover-glass into the cavity ground in the slide. 



The drop of culture placed on the cover-glass should be small enough to prevent 

 it touching the sides of the cavity otherwise the liquid will run by capillarity between 

 the slide and cover-glass and the hanging drop will disappear. 



When examining a hanging drop under the microscope great care must be exer- 

 cised in lowering the tube, because the cover-glass is only supported at its edges 

 and the least pressure on it will break it. It is best to use a No. 8 or No. 9 objec- 

 tive and No. I. or No. II. eyepiece (Reichert's lenses). 



The small quantity of air contained within the cell is quite sufficient to provide 

 all the oxygen necessary for several days. 



(b) Most frequently a hanging drop is used to study the development of 

 an organism. In this case the culture must be sown in the cell. It can be 

 done thus : Put a drop of sterile broth or sterile aqueous humour on the 

 cover-glass and sow it with the organism under investigation. 



It is absolutely essential in doing this that only a very few organisms be sown. 

 A trace of the culture may be picked up on the end of the straight wire and the 

 drop then very lightly touched with the latter, but it is better to adopt the dilution 

 method : thus, sow a broth tube (No. 1) with a loopful of the culture and shake; 

 sow a second broth tube (No. 2) with one or perhaps two drops from tube No. 1, 

 and then transfer a drop of the broth from tube No. 2 to the cover-glass to form 

 the hanging drop. If tube No. 2 still contain too many organisms, sow a third 

 tube (No. 3) with a few drops from No. 2. The hanging drop is then made with a 

 drop of broth from No. 3. 



The successive steps, then, are as follows : 



1. Flame the slide and cover-glass and allow them to cool. 



2. Place a drop of sterile broth in the centre of the cover-glass and sow it 

 with a trace of the culture (or, better, take a drop of broth from a tube sown 

 by the dilution method). 



3. Invert the cover-glass on the hollow-ground slide and lute the edges 

 with paraffin. 



4. Examine the hanging drop on a warm stage (vide post), or if a warm 

 stage be not available, incubate it in the ordinary incubator and examine 

 at frequent intervals on the ordinary stage, using a No. 8 or No. 9 objective 

 and a No. I. or No. II. eyepiece. Make certain that at the time when the 

 hanging drop is made there are not more than two or three organisms in each 

 field of the microscope. 



The culture can be kept for examination for 1 to 3 days. The air present 



