752 LACTOBACILLUS 



and has little or no differentiating value amongst the members of the group. 

 Growth in gelatin is either poor or absent ; liquefaction never occurs. 



Resistance and Methods of Isolation from Natural Sources.— The organisms are 

 not particularly resistant to heat, and are generally destroyed by an exposure to 

 60° C. for half an hour. One of their most striking features, which gives to them 

 the names acidophilic, acid-resisting, or aciduric, is their ability to survive in 

 concentrations of acid that usually prove fatal to other non-sporing bacteria. It 

 is this characteristic that is generally made use of in their isolation. 



One of the most successful methods is to mcubate the material for 1 to 3 days in 

 0-5 per cent, acetic acid broth, and subsequently to plate on 2 per cent, glucose agar. 

 Mcintosh and his co-workers (1922) recommend incubating the material in broth of 

 pH 3-5 for 24 hours, then subculturmg mto a series of broth tubes varying in pH from 

 30 to 4-5, and plating after a further 24 hours. Kendall (1910) made three consecutive 

 subcultures in acid broth, and plated the last on dextrose agar containing 0-2 per cent, 

 sodium oleate, which is said to improve the growth. The acid may be added to the 

 tubes directly, or the organisms may be seeded into a medium containing a fermentable 

 carbohydi-ate ; the acid produced in this medium is usually sufficient to kill off most 

 other micro-organisms. Cruickshank (1925) recommends, for the isolation of L. bifidus, 

 inoculating the faeces into a deep tube containing 20 ml. of 1 per cent, glucose or lactose 

 broth together with a small piece of fresh sterile rabbit kidiaey ; the kidney is added to 

 promote anaerobiosis, but does not appear to be essential ; the medium is covered with 

 a vaseUne seal. The culture is incubated for about a week at 37° C. and then plated 

 on to 1 per cent, glucose agar or Loeffler's serum, which is incubated aerobicaUy and anaero- 

 bicaUy. After 48 hovu-s on the anaerobic plates, greyish pinhead colonies appear. The 

 only other organism that is Ukely to develop under these conditions is the enterococcus, 

 which forms larger whitish colonies. Another method that may be ebiployed for the 

 isolation of the acid-resistant bacteria is the use of VeiUon tubes (Veillon and Zuber 1898, 

 Rettger and Chephn 1921), containing 2 per cent, glucose agar or whey agar ; the different 

 organisms adapt themselves to the varying oxygen pressure in the medium, and form 

 characteristic colonies, which may be picked off with ease. The tomato broth medium 

 described by Kulp (1927) has given very favourable results ; if the primary cultures are 

 plated on tomato agar, single colony isolations are frequently successful. 



In our experience (Crowley et al. 1941) direct platmg of consecutive dilutions on the 

 glucose hver blood agar medium recommended by Eggerth and Gagnon (1933) has proved 

 satisfactory for the isolation of lactobaciUi from fseces. Lewis, Bedell and Rettger (1940) 

 have described a glucose cysteine agar medium which, Ukewise, seems well suited for 

 this purpose. The presence of added COg is beneficial for growth (Kulp 1926, Cruick- 

 shank 1934, Lewis et al. 1940). 



Metabolism. — Most of the members are microaerophilic or facultative anaerobes. 

 For their isolation fairly strict anaerobic conditions are often, though not always, 

 nec.essary. After a few subcultures many of them can be brought to grow aero- 

 bicaUy, but others remain persistently obligate anaerobes. Peroxide is produced 

 without, or with only very small quantities of, catalase. This probably accounts 

 for the poor viability of the organisms in media not maintaining a low oxidation- 

 reduction potential. Gillespie and Rettger (1938) noted a difference between 

 different species in the intensity of their reducing activity. Oral lactobaciUi, for 

 example, were found in a particular medium to reduce the Eh to about — 240 mv., 

 whereas L. acidophilus of intestinal origin, L. bifidus, Doderlein's bacillus and 

 L. bulgaricus reduced it to only about — 140 mv. According to Curran, Rogers, 

 and Whittier (1933), the optimum growth temperature is 37°-40° C. ; the range 



