402 ISOLATION OF THE TYPHOID BACILLUS 



It is not a matter for surprise therefore to find that much experimental 

 work has been done with a view to perfecting a method or methods of detecting 

 with certainty the presence of the typhoid bacillus in material in which it 

 may be suspected to occur. 



SECTION I. THE ISOLATION OF THE TYPHOID AND COLON 



BACILLI. 

 1. Original methods. 



Under this heading will be briefly considered various methods which though 

 in use until recently do not give dependable results, being practically useless 

 for detecting the typhoid bacillus when the latter is mixed with the colon 

 bacillus. These methods are now almost entirely discarded. 



(a) Rodet's method. Rodet showed that the typhoid and colon bacilli would grow 

 at 45 C. while most other organisms failed to do so, and on this fact based the following 

 method of analysis. To a flask containing sterilized broth he added 20-100 c.c. of the 

 suspected water and incubated at 45 C. for 20-24 hours. If on taking the flask out of 

 the incubator the broth was cloudy a strong presumption was raised that the typhoid 

 or colon bacillus or both were present in the water. Microscopical examination of the 

 culture and, if need be, isolation on gelatin plates removed all doubt. 



(6) Method of Chantemesse and Widal. Chantemesse and Widal found that both 

 typhoid and colon bacilli would grow in artificial media containing 2'5 grams of carbolic 

 acid per litre, and utilized the fact in order to detect these organisms in water. 



To tubes containing 20 c.c. of liquefied gelatin add 1 c.c. of a 5 per cent, solution of 

 carbolic acid and a few drops of the water to be examined and pour plates. Unfortunately 

 a certain number of organisms develop in the plates which, as they grow, liquefy the 

 medium and consequently soon put an end to the experiment. A large number of plates 

 must be sown with each of the suspected samples because only a very small amount of 

 water can be used for each plate. 



(c) Vincent's method. Vincent devised a method, which for a long time was in general 

 use, based upon a combination of the two preceding observations. He used broth con- 

 taining O'l per cent, of carbolic acid as the culture medium and incubated the cultures 

 at41-5-42C. 



To each of half-a-dozen tubes containing 10 c.c. of broth add, immediately before use, 

 5 drops of a 5 per cent, solution of carbolic acid. Sow with O'5-l c.c. of the suspected 

 water, cover with india-rubber caps to prevent evaporation of the carbolic acid, and 

 incubate at 41 '5 or 42 C. If the medium in any of the tubes becomes cloudy after 

 incubating for 12 or 20 hours, transfer a little of the culture to a fresh tube of carbolic- 

 broth and incubate it similarly at 41 '5 C. As a rule, when the suspected water contains 

 the colon bacillus the first sub-culture yields a pure growth of the latter organism. It 

 must, however, be borne in mind that some saprophytes (Bacillus subtilis, Bacillus mesen- 

 tericus, B. luteus, the white streptococcus of water, Proteus vulgaris, etc.) will also grow 

 under these conditions. These latter organisms cannot be excluded by further sub- 

 cultivation in carbolic -broth because once they become accustomed to carbolic media 

 they grow in them just as well as the colon bacillus. A watered silk appearance in the 

 tubes is a fairly reliable indication of the presence of the colon or typhoid bacillus, but 

 the investigation must always be carried further by microscopical examination and 

 isolation on gelatin. It is well to remember that in carbolic-broth the colon and typhoid 

 bacilli often occur as very short rods (cocco-bacilli) arranged in pairs and devoid of 

 motility. 



(d) Method of P6re". This is merely Vincent's method modified in such a way as to 

 allow large quantities of the suspected water to be examined. 



Prepare a concentrated broth (meat, 1000 grams,, water 1000 grams, and peptone 

 50 grams), distribute in quantities of 50 c.c. in a series of flasks, and autoclave. 



To each flask add 3 c.c. of a 5 per cent, solution of carbolic acid and 100 c.c. of the 

 suspected water. Sow five or six flasks and incubate them at 41 C. As soon as the 

 medium becomes cloudy (15-20 hours) sow a series of broth tubes each containing O'l per 

 cent, carbolic acid with a trace of the growth from any of the flasks that may be cloudy. 

 Incubate at 41 C. and continue the experiment as in Vincent's method. 



(e) Method of Pouchet and Bonjean. This also is a modification of Vincent's method. 

 To each of a series of flasks containing 100 c.c. of sterile broth add 150 c.c. of the water 

 to be examined and 5 c.c. of a 5 per cent, solution of carbolic acid. Incubate at 42 C. 



