APPENDIX 471 



water examination) nutrient gelatin gives a relatively larger number of colonies than 

 distilled-water gelatin, nutrient gelatin should be used when one gelatin only is 

 employed. At the same time, it is recognised that cultures in distilled-water 

 gelatin compared with cultures in nutrient gelatin often give useful indications. 

 Thus with an unpolluted water the number of colonies is usually relatively larger 

 in distilled-water gelatin than in nutrient gelatin ; with a polluted water the con- 

 verse is the case. Therefore the use of both gelatins (distilled-water and nutrient) 

 is desirable, sets of plates being made with each medium. 



Similarly, it was felt by many members of the committee that a comparison of 

 the ratio of the number of organisms developing at room temperature to those 

 developing at blood-heat gives useful indications. With a pure water this ratio is 

 generally considerably higher than 10 to 1, with a polluted water this ratio is 

 approached, and frequently becomes 10 to 2, 10 to 3, or even less. The actual 

 number of organisms growing at blood-heat is also of considerable value apart 

 from any question of ratio. Therefore it is suggested that plates of nutrient agar 

 should also be employed and incubated at blood-heat. 



In certain instances it is true that this ratio may be unreliable. Thus with surface 

 waters, especially in tropical countries (as pointed out by Major Horrocks), varieties 

 of the B. fluorescens liquefaciens and non liquefaciens and B. liquefaciens may be 

 abundant and grow well at blood-heat. 



Preparation and Reaction of Media for Enumeration 



(a) Distilled-Water Gelatin. Ten per cent, gelatin in distilled water, and brought 



to a reaction of + 10 (Eyre's scale). 



(6) Nutrient Gelatin. Ten per cent, nutrient gelatin, preferably made with meat 

 (beef) infusion and Witte's peptone, and brought to a reaction of + 10 

 (Eyre's scale). 



In hot weather it may be necessary to increase the percentage of gelatin. 

 Some members of the committee advocate the use of meat extracts in place of 

 meat infusion, on the score of convenience and uniformity of composition, Brand's 

 Essence being recommended as the best. It is the general opinion, however, that 

 Liebiy's Extract is less suitable for this purpose. 



(c) For enumeration at blood-heat it is recommended that nutrient agar should be 



employed, being prepared with the same constituents as nutrient gelatin, 

 but substituting 1J per cent, of powdered agar for the gelatin. Reaction 

 + 10. 



(d) Distilled-Water Agar. Powdered agar 1^ per cent., dissolved in distilled 



water, and brought to a reaction of + 10. 



Owing to the changes which occur in the reaction of the medium on keeping, the 

 media employed should preferably be not more than three weeks old. 



Amounts to be Plated, Size of Dishes, etc. Gelatin. For an ordinary water 

 amounts of 0'2, 0'3, and 0*5 c.c. may be plated in Petri dishes of not less than 10 

 centimetres diameter, preferably done in duplicate. 



Agar. Two plates may be made with O'l and I'O c.c., and are preferably 

 duplicated. 



In dealing with an unknown water, and in all cases of doubt, additional sets of 

 plates should be prepared with a dilution of the water (made with sterilised tap- 

 water) often or hundred fold, according to circumstances. 



The amount of the medium in a plate should be 10 c.c. 



The sample must be thoroughly shaken and mixed in all cases before plating. 



Temperature of Incubation. (a) Room temperature = 18-22 C. ; (6) blood-heat = 

 36-38 C. 



Counting. Counting to be done with the naked eye, preferably in daylight, any 

 doubtful colony being determined with the aid of a lens or low-power objective. 



Time of Counting. Gelatin plates should be counted at the end of seventy-two 

 hours ; but in all cases the plates should be inspected daily, in order that the count 

 may be made earlier should liquefaction render this necessary. 



The blood-heat agar plates should be counted at the end of forty to forty-eight 

 hours. 



