CULTIVATION ON SOLID MEDIA 99 



2. Solid media. 



(i) Stab cultures. 



A. In test-tubes. Method recommended, (a) Gelatin. 1. Heat a tube of 

 sterile gelatin to boiling, taking care not to let the medium froth and boil 

 over. Boil for several minutes. [This is best done in a water bath.] 



A few drops of sodium sulphindigotate solution may be added to the gelatin 

 before boiling it and if this be done the medium will be decolourized by the growth 

 of the organism. 



2. Cool the gelatin rapidly, and when it is set sow a stab cul- 

 ture with a fine platinum wire. 



A little air would ordinarily be introduced with the needle, and the 

 following arrangement is devised to obviate this. Mount the wire on 

 the wall of a piece of glass tubing, and connect the other end of the 

 latter to a hydrogen-generating apparatus by means of a piece of india- 

 rubber tubing (fig. 89). To use the needle, after flaming it, take up 

 the material to be sown, then turn on the hydrogen and sow in a current 

 of the gas. In this way the oxygen of the atmosphere is prevented from 

 reaching the needle track. 



3. After sowing, dip the gelatin tube into very cold water and 

 pour a layer of agar over the surface with a Pasteur pipette. 

 Replace the plug. The object of this procedure is to form a plug 

 impervious to air on top of the gelatin. Sterilized oil or liquid 

 vaseline, etc., may be used instead of agar. 



Note. The agar plug may be omitted if some very oxidizable 

 substance capable of absorbing oxygen be added to the culture 

 medium (Liborius, Kitasato). The best substances for the pur- 

 pose are glucose (2 per cent.), sulphindigotate of sodium (Crl per 

 cent.), sodium formate (O5 per cent.). 



Liborius recommends the following medium : wf re 8 f b~r 



Ordinary agar, ....... 1000 grams. sowing an- 



S 



. 

 bodium sulphindigotate, - 1 gram. 



Nearly fill the tubes with the medium, and sow deep stab cultures as 

 described above. 



(b) Agar. The method is the same as in the case of gelatin. 



B. Absorption of oxygen by an aerobic organism (Roux). Proceed as 

 above, and when the agar plug has set sow the surface with B. subtilis. This 

 organism is strictly aerobic and absorbs the oxygen present in the tube, 

 while growth below takes place under anaerobic conditions in an atmosphere 

 free from oxygen. 



To reach the anaerobic organism without contaminating it with the B. subtilis, 

 wash the outside of the tube with perchloride of mercury, cut it across about the 

 level of the middle of the growth, break off the lower part of the tube, and the 

 anaerobic organism can then be removed without contaminating it. 



C. Roux's pipette. 1. Flame and break off the point of a Roux's pipette. 

 Dip the end into a tube of sterile gelatin which has just been boiled. Draw 

 the gelatin into the tube until it reaches the constriction a, fig. 67, p. 75. 

 Seal the narrow end of the pipette and the upper end at the constriction. 

 Dip the whole tube into cold water to cool it quickly. 



2. When the gelatin has set, pass the upper part rapidly through the 

 flame, and then break off the point a with a pair of forceps. Through the 

 opening sow a stab culture with a fine wire. Seal the opening in a flame. 



