174 DIFGO MANUAL 



centration to determine motility, and stated that indole determination could be 

 made using the Ehrlich reagent in this medium. 



In the development of Bacto-S I M Medium it was determined that 0.0025 per 

 cent sodium thiosulfate added to the semisolid 3 per cent Bacto-Peptone medium 

 produced results comparable to those secured when hydrogen sulfide was deter- 

 mined in a 1 per cent Bacto-Peptone solution, using lead acetate paper strips as 

 an indicator of hydrogen sulfide. Smaller quantities of sodium thiosulfate did 

 not give a satisfactory response in the semisolid medium, while larger quantities 

 obscured motility and also permitted some false reactions. Any blackening along 

 the line of inoculation is considered as a positive hydrogen sulfide reaction. Hy- 

 drogen sulfide producing organisms generally give a positive reaction in 18-24 

 hours. 



Motile cultures in Bacto-S I M Medium show diffuse growth or turbidity away 

 from the line of inoculation. Hydrogen sulfide reactions are intensified by motile 

 cultures. Cultures vary in the amount of motility as pointed out by Friewer and 

 Shaughnessy.® Strains of Proteus and Salmonella often show diffuse growth 

 throughout the entire medium, while S. typhosa is not as actively motile. In 

 Bacto-S I M Medium motility of typical members of the enteric group is demon- 

 strated in 18-24 hours, or less, incubation at 37 °C. Green and co-workers^*^ used 

 Bacto-S I M Medium in 1-2 ml. amounts in 75 x 10 mm. tubes and reported 

 the detection of motility by a large series of cultures following incubation at 

 37°C. for 90 to 120 minutes. 



Bacto-Tryptone has been used universally in the test for indole production. It 

 has been demonstrated that Bacto-S I M Medium gives parallel indole production 

 in comparison with a 1 per cent Bacto-Tryptone solution after 18-24 hours in- 

 cubation at 37 °C. The oxalic acid paper test for indole production may be used 

 with this medium. This test has the advantage of making it possible to observe 

 motility, hydrogen sulfide production and indole formation after any desired 

 incubation period. Oxalic acid paper for testing is prepared by soaking filter paper 

 in a saturated oxalic acid solution, drying and then cutting the paper into strips. 

 For the performance of the test, after the medium has been inoculated, suspend 

 a strip of the paper in the mouth of the tube so that the strip projects one-half 

 inch below the cotton plug holding it in place. The paper must not become wet 

 as this may contaminate the medium. Indole production is shown by the formation 

 of a pink color on the paper during incubation. 



If desired, other tests for indole may be employed; the Kovacs or Ehrlich test 

 gives good results. When using these tests hydrogen sulfide and motility readings 

 should be made before testing for indole. The recommended procedure for the 

 Kovacs' test is to overlay the medium with 2 ml. of chloroform without agitation, 

 followed by the direct addition of 2 ml. of Kovacs' reagent. A pink to deep red 

 color is formed in the chloroform layer if indole is present. In a negative test no 

 color is formed in the chloroform layer. Kovacs' reagent consists of 75 ml. amyl 

 alcohol, 25 ml. concentrated hydrochloric acid and 5 grams /?-dimethylamino- 

 benzaldehyde. The indole test may also be performed in accordance with the 

 "Manual of Methods of Pure Culture Study of Bacteria," ^^ of the Society of 

 American Bacteriologists. To perform this test, add about 2 ml. of ethyl ether to 

 each tube. Shake gently, but do not break the agar. Let stand several minutes and 

 then add about 1 ml. Ehrlich reagent by dropping it down the side of the tube so 

 that it spreads out as a layer between the ether and the medium. The formation 

 of a purplish red color at the interface of the two liquids within 5 minutes indi- 

 cates indole production. In negative reactions, after the Ehrlich reagent has been 

 in contact with the medium for a period longer than 5 minutes, a slight pink 

 color may be observed on the surface of the medium. This should not be mistaken 

 for a positive reaction, since in a positive test the color must appear within 5 



