Use of Clark and Luhs Indicators 247 



THE USE OF CLARK AND LUBS INDICATORS FOR THE 



DETECTION OF ACID PRODUCTION BY THE 



COLON-TYPHOID GROUP. 



P. A. Tetrault, Purdue University. 



The incorporation, into a medium, of heat resisting indicators is one 

 of the common methods practiced for detecting and determining acid 

 production by bacteria.^ Clark and Lubs indicators lend themselves 

 especially well to this kind of work because they are heat resisting; 

 they do not inhibit bacterial growth; they resist the action of bacteria; 

 and with a number of them, the virage point is at or near the acid con- 

 centration of ordinary stock media. The purpose of the following ex- 

 periments was to differentiate between the various members of the colon- 

 typhoid group by means of their action on various sugars. No attempt 

 was made to measure the amount of acid produced in each case. 



The medium used was a 3 per cent nutrient agar made with ..3 per 

 cent Liebig's beef extract, 2 per cent Difco peptone and .5 per cent 

 sodium chloride. The ingredients were mixed and autoclaved at ten 

 pounds for one hour, filtered and the reaction adjusted to slight pink to 

 phenol red (Ph^6.8 — 7.5). Then the bulk was divided into workable 

 pcrtions and .1 per cent of the desired sugar added to each. This was 

 further divided into three portions and .002 parts of indicator added 

 to each portion. The prepared media was then tubed, sterilized in the 

 autoclave at ten pounds for ten minutes and slanted. Care was taken 

 in slanting to allow for a butt in each tube of at least one inch below 

 the bottom of the slant." After solidification the tubes were ready for 

 inoculation. 



The indicators used were broni cresol purple, brom thymol blue, and 

 phenol red. The dyes were all made up in 1 per cent alcoholic solutions 

 which were used for stock solutions. In making dilutions .1 cc. of this 1 

 per cent stock solution was added to a graduate and media poured into 

 it up to the 50 cc. mark. This gave a dilution of 1 part of dye to 2,000 

 parts of media. By this method the dye readily mixed with the media 

 and did not require further mixing. 



The sugars included dextrose, lactose, sucrose, maltose, raffinose, 

 mannose, dulcitol, xylose, rhamnose, arabinose, levulose, and galactose. 

 All these sugars went into solution very readily if added directly to the 

 hot medium. Some of these are classified as rare sugars and would not 

 be practicable for class room work if used in 1 per cent quantities. 



The cultures used were the stock cultures used for laboratory work 

 including B. coli, B. typhosiim, B. dysenteriae, B. paratyphosum A, 



'■ H. R. Baker. Substitution of B.T.B. for Litmus in Routine Laboratory Work. 

 Jour. Bact. Vol. VIL 2. 



.J. Bronfenbrenner, M. .J. Schlesinger and D. Soletsky. On Methods of Isolation and 

 Identification of the Members of the Colon-Typhoid Group of Bacteria. The Study of 

 Bactericidal action of C. R. Indicator. Jour. Bact. Vol. V. 1. 



- H. J. Conn and G. J. Hucker. The Use of Aaar Slants in detcctin.u' Fermentation. 

 Jour. Bact. Vol. V. 4. 



"Proc. 38th Meeting, 1922 (1923)." 



