V49-20 MANUAL OF METHODS FOR PURE CULTURE STUDY 



plate add a drop of dilute iodine solution and read reaction as follows: if blue, no 

 hydrolysis; if reddish brown, partial hydrolysis with production of erythrodextrin; if 

 clear, hydrolysis complete, with production of dextrin or perhaps glucose. The tubes 

 showing complete hydrolysis may be tested for reducing sugar with Fehling's solution. 



For bacteria that do not grow well in liquid media, no better 

 method has yet been proposed than the plate technic given in all 

 previous editions of the Manual with little modification. This 

 method has its disadv^antages, but is often useful; it is as follows: 



Use beef -extract agar containing 0.2% of soluble starch. Pour it into a Petri dish, 

 and after hardening make a streak inoculation on its surface. Incubate at optimum 

 temperature for the organism under investigation. Observations are to be made on 

 the second day for rapidly growing organisms but not until the 7th day for the more 

 slowly growing ones. To make the test, flood the surface of the Petri dishes with 

 Liigol's iodine or with a saturated solution of iodine in 50% alcohol. The breadth 

 of the clear zone outside of the area of growth indicates the extent of starch 

 destruction. By means of a simultaneous inoculation on another plate containing 

 the same medium with brom cresol purple as an indicator one may at the same time 

 learn whether or not acid is produced as an end-product. 



THE METHYL RED AND VOGES-PROSKAUER TESTS 



Special tests as to cleavage of glucose are commonlj^ made in the 

 differentiation of the organisms of the colon-aerogenes group. The 

 medium ordinarily employed is as follows: 5 g. proteose peptone 

 (Difco, Witte's, or some brand recognized as equivalent), 5 g. 

 C. P. glucose, 5 g. K2HPO4 in 1000 ml. distilled water. The dry 

 potassium phosphate slioidd be tested before using in dilute solution 

 to see that it gives a distinct pink color with phenolphthalein. Accord- 

 ing to Smith (1940), the K2HPO4 in this medium should be replaced 

 with the same amount of NaCl, if the tests are to be carried out on 

 aerobic spore-formers. Tubes should be filled with 5 ml. each and 

 each culture should be inoculated into duplicate (or triplicate) tubes 

 for each of the two tests. Incubation should be at optimum tempera- 

 ture of the organism under investigation, and tubes shoidd be in- 

 cubated 2-7 days, according to the rate of growth of the organism in 

 question. Although the same medium is used for both the methyl 

 red and the Voges-Proskauer tests, they must l^e performed in 

 separate tubes. The latter test depends upon the production of 

 acetyl-methyl-carbinol from the glucose; see Leaflet \T. 



A positive methyl red reaction is regarded as being present when 

 the culture is sufficiently acid to turn the methyl red (0.1 g. dissolved 

 in 300 ml. 95% ethyl alcohol and diluted to 500 ml. with distilled 

 water) a distinct red; a yellow color with the methyl red indicator is 

 regarded as a negative reaction, while intermediate shades should be 

 considered doubtful. 



