272 APPENDIX 



15. Filter through absorbent cotton and cotton 

 flannel, passing the nitrate through the filter 

 until clear. 



16. Titrate and record the final reaction. 



17. Tube, using 10 c.c. of medium in each tube. 



1 8. Sterilize 15 minutes in the autoclave at 120 

 degrees, or for 30 minutes in streaming steam 

 on three successive days. Put the gelatin at 

 once into ice-water till solidified. 



19. Store in the ice-chest in a moist atmosphere, 

 to prevent evaporation. 



LACTOSE LITMUS AGAR 



Lactose litmus agar shall be prepared in the same manner 

 as nutrient agar, with the addition of i per cent of lactose 

 to the medium just before sterilization. The reaction shall 

 be made neutral to phenolphthalein (see p. 267). 



If the medium is to be used in tubes the sterilized azo- 

 litmin solution shall not be added until just before the 

 final sterilization. 



If the medium is to be used in Petri dishes the steril- 

 ized azolitmin solution shall not be added to the medium 

 until it is ready to be poured into the dishes. 



More colonies and better general results are obtained 

 on the lactose litmus agar plates, when the litmus and 

 lactose are each sterilized separately and added to the 

 plate with the neutral agar at the time of planting. Good 

 results can, however, be obtained, if the agar and lactose 

 are mixed and sterilized in an autoclave at 120 C. for 

 15 minutes only. 



The azolitmin on the market varies considerably, much 

 of that sold being entirely unreliable for the purpose. A i 

 per cent solution of Kahlbaum's azolitmin, if boiled for 

 5 minutes, readily dissolves and needs no correction for 

 acidity if added to standard agar. Many bacteriologists 

 prefer pure litmus to azolitmin, and it is therefore sug- 

 gested that its use be made optional. Both total and 

 red colonies may be counted after from 18 to 24 hours 



