ELSNER'S METHOD 403 



If the medium becomes cloudy in any of the flasks sow sub-cultures for three generations 

 in O'l per cent, carbolic acid broth and incubate at 42 C. Finally, sow a tube of ordinary 

 broth from the last carbolic broth culture, incubate at 36 C. for 8 days and then inoculate 

 a guinea-pig with 0*3 c.c. of culture per 100 grams of animal. If the animal die sow 

 cultures with fragments of the internal organs and heart blood. 



The five methods just described are available for the isolation of the typhoid bacillus 

 provided that the colon bacillus is not also present but if, as is most often the case, the 

 two organisms are present together the isolation of the former is impossible by these 

 means. 



2. Eisner's method and its modifications. 

 A. Eisner's method. 



The method is available according to Eisner for the isolation of the typhoid 

 bacillus from sources such as water or stools in which the colon bacillus is 

 also present. 



The technique is based upon the fact that the typhoid and the colon bacilli 

 grow, to the exclusion of most other organisms, on a potato-jelly containing 

 iodide of potassium. Disappointing results are however frequently obtained ; 

 sometimes the plates are rapidly liquefied and the experiment brought to an 

 end ; at other times the typhoid bacillus cannot be found even though it 

 has been purposely introduced into a sample of water as a control. Several 

 attempts have been made to improve the method, and these will be considered 

 subsequently. 



Technique. A. Isolation from water. 1. Prepare and sterilize : (i) a number 

 of tubes each containing 10 c.c. of potato gelatin (p. 41). 

 (ii) The following solution : 



Distilled water, 50 grams. 



Potassium iodide, - 10 



2. Immediately before use, melt the potato-gelatin tubes and add 1 c.c. (20 drops) 

 of the iodide solution. 



The gelatin will then contain 1 per cent, of iodide. 



3. Sow ten to fifteen tubes each with 0*5 or 1 c.c. of the suspected water and 

 plate. 



4. According to Eisner, the colon bacillus appears on these plates as early as the 

 second day (at 22 C.) as circular, opaque, slightly brown colonies while the typhoid 

 bacillus does not develop until the plates have been incubated for 4 days and then 

 as smaller, transparent, barely visible colonies. Other organisms fail to grow. 



As a matter of fact, various organisms other than the typhoid and colon bacilli, 

 and some of which liquefy the gelatin, do grow on the medium ; and then again the 

 colonies of the typhoid bacillus are not so easily differentiated as Eisner makes out. 

 It must be distinctly realized that Eisner's medium possesses no specific property 

 which ensures the development of the typhoid and colon bacilli to the exclusion of 

 other organisms. Its only advantage is that it allows the typhoid bacillus an equal 

 opportunity with the colon bacillus to grow. It is necessary, therefore, to examine 

 carefully every colony on the plates which does not liquefy the medium and which 

 does not form pigment. This is easily done by transferring them each to a separate 

 tube of broth and then incubating at 37 C. After incubating for 24 hours the 

 morphology of the organisms is determined by examining the cultures microscopically 

 and only those tubes which show short, gram-negative bacilli with rounded ends 

 need be reserved for the further tests to be described later. 



If any of the broth cultures prove to be impure they must be plated out again on 

 Eisner's jelly. Sow a loopful of the broth in a fresh tube of the jelly, a drop of this 

 on a second tube, and three drops of the second into a third tube (p. 77). 



B. Isolation from stools. The technique to be adopted in this case is similar to 

 that just described. Dilute a loopful of the stool in a tube of sterile water and use 

 a drop of the dilution to sow a tube of Eisner's gelatin : mix thoroughly and transfer 

 a drop to a second tube and from the second tube two or three drops to a third tube. 

 Pour plates and incubate. All the non-liquefying colonies which develop must be 

 picked off for further investigation in the manner described above. 



