ROUTINE TESTS FOR THE IDENTIFICATION OF BACTERIA 163 



A satisfactory method has been proposed by Eckford (1927) for learn- 

 ing the type of action on starch brought about by organisms capable of 

 making good growth in broth. The same method may be adapted to 

 organisms which prefer some other liquid medium by substituting it for 

 broth in Eckford's method. The procedure, however, is not well adapted 

 to those bacteria that fail to grow well in liquid medium. The technic is 

 as follows: 



Add 0.2 per cent soluble starch to broth and incubate cultures a week to 10 days. 

 Examine on second, fourth, seventh and tenth days for hydrolysis of starch, produc- 

 tion of acid, and reduction of Fehling's solution. For this test a drop is placed in a 

 depression on a porcelain plate and a larger quantity in a serological test tube. The 

 latter is tested for acid production with an indicator of the proper pH range. To the 

 drop on the 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 erythro- 

 dextrin; 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 disadvan- 

 tages but is often useful; it is as follows: 



Use beef-extract agar containing 0.2 per cent 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 seventh day 

 for the more slowly growing ones. To make the test, flood the surface of the petri 

 dishes with Lugol's iodine or with a saturated solution of iodine in 50 per cent alcohol. 

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

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

 the same medium with bromcresol 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 commonly made in the 

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

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

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

 glucose, 5 g of K2HPO4 in 1,000 ml of distilled water. The dry potassium 

 phosphate should be tested before using in dilute solution to see that it 

 gives a distinct pink color with phenolphthalein. According 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 sporeformers. 



