BESSON'S METHOD 405 



After filtration, test the reaction again thus : mix 10 c.c. of gelatin with 100 c.c. 

 of distilled water, add a few drops of phenol-phthalein solution, and from a 1 c.c. 

 pipette graduated in tenths of a cubic centimetre run in a half-normal solution of 

 soda ; the red colour should appear when 0*2 c.c. of the solution have been added. 



The desired degree of acidity being obtained, dissolve 2 '5 grams of magnesium 

 sulphate for every litre of gelatin. Tube in quantities of 10 c.c. and sterilize on 

 three successive occasions at 100 C. 



Immediately before use add to each tube 1 c.c. of a sterile 35 per cent, solution of 

 lactose and O'l c.c. of a 2'5 per cent, solution of carbolic acid. 



Method of sowing. Remy's gelatin is used in the same way as Eisner's. It is 

 advisable, first of all, to sow the suspected water in a broth containing 0*5 per cent, 

 of sulphuric and carbolic acids, and after incubating at 30 C. for 24 hours to use 

 this culture for sowing the gelatin plates by the dilution method. Colonies of the 

 colon and typhoid bacilli appear in the plates after incubating for 2 days. 



Cultural characteristics. The colon 'bacillus. Colonies in the depth of the medium 

 are rounded, ovoid or fusiform and of a yellowish- brown colour. Minute bubbles 

 of gas are occasionally formed. Colonies on the surface which are sometimes trans- 

 parent and bluish at first, rapidly become opaque : some of them are hemispherical 

 and of a yellowish- brown colour while others have irregular margins and tend to 

 spread. 



The typhoid, bacillus. In the depth of the medium the colonies are bluish- white, 

 smaller than the colonies of the colon bacillus and form no gas. Surface colonies 

 are not well seen until the third day : at first " they are rather like moulds in appear- 

 ance " but later spread out, become more bluish in colour and may attain the size 

 of a threepenny-piece. 



The differences between the colonies of the typhoid and colon bacilli are frequently 

 very slight and many sub-cultures may have to be made before the nature of the 

 organism can be definitely determined. 



D. Besson's method. 



Eisner's gelatin method has two great disadvantages. In the first place, 

 it is only available for the analysis of small quantities of water even though 

 the number of plates used be large which is in itself a disadvantage and, 

 secondly, the medium does not prevent the growth of saprophytic organisms, 

 which sometimes liquefy the plates as early as the second day and so put an 

 end to the experiment. With the object of simplifying and at the same time 

 rendering the method more efficient as a means of water analysis, Besson, 

 in 1896, introduced certain modifications which he claims improve it in that 

 they rapidly eliminate saprophytes and permit the use of large volumes 

 of water. 



1. Weigh out 30 grams of peptone (Chapoteaut) and 5 grams common salt, 

 add a litre of water and dissolve in the steamer ; then, without neutralizing, 

 heat in the autoclave to 115 C., filter, tube in quantities of 10 c.c. and 

 sterilize at 115 C. 



2. When an experiment is to be done, take ten tubes of the peptone water 

 and to each add 20-30 drops of freshly prepared Gram's iodine solution 

 (p. 143) and 10 c.c. of the water to be examined. 



The amount of iodine solution to be added varies a little with the composition of 

 the peptone. The first few drops will be rapidly decolourized but when about 

 20-25 drops have been added the medium assumes a pale brownish-pink colour 

 which disappears in 5-6 minutes. When this occurs sufficient iodine has been 

 added. 



3. Incubate the tubes at 37-38 C. Under these conditions the colon 

 bacillus produces a visible growth in 8-12 hours and the typhoid bacillus 

 in about 15-20 hours while other organisms do not appear until later. The 

 tubes should be examined at frequent intervals. 



4. After incubating for 18 hours pick out the tubes which are cloudy and 

 sow sub-cultures in iodine-peptone-water. 



