108 LABORATORY MANUAL OF MICROBIOLOGY 



2. Eryihrosin 



(a) Erythrosin 5.0 gm. 



Alcohol (70 per cent) 100 . cc. 



(6) Erythrosin 1.0 gm. 



Carbolic acid (5 per cent) 100 . cc. 



This stain is especially recommended for root nodule bacteria. 



1. Place a drop of the fresh culture on a glass slide, tilt the 

 slide to allow drop to spread. Dry the film in an oven at 45°C. 

 and fix in absolute alcohol. 



2. After the alcohol evaporates, flood the mount with (a) and 

 allow to stain for 10 minutes. 



3. If the stain is not deep enough, wash off this alcohol Ery- 

 throsin and stain with (6) for 10 minutes. 



Exercise 19 

 Cultural Characteristics of Root Nodule Bacteria 



Select three cultures from the Brom thymol blue slope cultures 

 of Exercise 18, and make subcultures on the various kinds of 

 culture media given below. 



(a) Inoculate into litmus milk (Medium 90). 



(6) Inoculate into bean extract in shallow layers (Medium 82). 



(c) Inoculate on potato slopes (Medium 91). 



{d) Inoculate on slopes of xylose, glucose, and sucrose-nitrate- 

 mineral salts agar to which Brom thymol blue has been added 

 (Medium 78). 



After 1, 2, and 3 weeks, at 28°C., record growth and reaction 

 changes. In the milk tubes, note the effect on reaction and 

 serum zone formation. In the bean extract, note gum produc- 

 tion. After 4 to 5 weeks at 28°C., test for gum. Add 10 cubic 

 centimeters of alcohol (95 per cent) or 5 cubic centimeters of 

 acetone to 2.5-cubic centimeter portions of the bean extract 

 culture. Note the thickness of the layer of gum on top of the 

 liquid. 



On potato, note growth and pigment formation. In the case of 

 Brom thymol blue agar, note the changes in reaction. Alfalfa 

 bacteria produce a strong acid reaction with all of the carbo- 

 hydrates listed. Clover bacteria produce a strong acid reaction 

 from glucose, little or no acid from sucrose and xylose. Pea 



