SECTION SEVEN 



Stain, by means of the wire loop, over the smear. Allow the stain 

 to act for about one half to one minute. 



8. Wash off excess stain with distilled water. 



9. Shake off excess distilled water; then allow the smear to dry 

 thoroughly in the air before examining under the oil immersion 

 objective. 



Results: 



Cell walls stained red. 



Note : Walls in the early stages of formation are not stained. Cross 

 walls are stained in the older portion of a filament of B. cereus but not 

 in the younger portions. The end cell of a filament has relatively heavy 

 side walls but the wall becomes much thinner around the free end of 

 the cell. The stain does not show cross walls in chains of E. Coli. 



A further differentiation of the stained material may be made by 

 exposing the stained specimen to the fumes of hydrochloric acid which 

 will change the colour of the free congo red to blue. 



Reference: Chance, H. L. (1953), Stain Tech., 28, 205-7. 



NEWMAN'S STAIN 



A single solution for defatting and staining milk smears 

 for the enumeration of milk bacteria 



Solution required: 



Newman! s Stain 



Methylene blue . . . . . . i gm. 



Absolute alcohol . . . . • • 54 ml. 



Tetrachlorethane . . . . . . 40 ml. 



Acetic acid, glacial . . . . . . 6 ml. 



Place the methylene blue with the alcohol in a flask; then plug 

 the neck of the flask lightly with cotton- wool. Heat water till it 

 boils in a water bath ; then turn off the flame and place the flask 

 in the hot water until the alcohol just begins to boil. Allow the 

 solution to cool after swirling the liquid round inside the flask until 

 all the methylene blue has gone into solution. Filter into the stock 

 bottle; add the trichlorethane ; replace the stopper or cork and 

 shake well. 



387 



