MEDICAL AND BIOLOGICAL STAINING TECHNIQUES 



6. Staining and counting of bacteria, and differentiating 



living from dead cells, in soil 



Solutions required: 



A. Buffer solution pH 7 



B. Solution A . . 

 Acridine Orange, FS 



C. Solution A 

 Solution B 



D. Solution A 

 Solution B 



E. Solution A 

 Solution B 



F. Solution A 

 Solution B 



G. Solution A 

 Solution B 



200 ml. 



100 ml. 

 o-i gm. 



5 ml. 

 5 ml. 



5 ml. 

 10 ml. 



3 ml. 

 9 ml. 



2 ml. 



8 ml. 



I ml. 



9 ml. 



Technique : 



1. Place I gm. of the sifted soil in each of six test tubes, and 

 label them consecutively B, C, D, E, F, and G. 



2. Add 10 ml. of the appropriate solution to the five tubes 

 labelled B, C, D, E, and F (i.e. 10 ml. solution B to tube B; 

 10 ml. solution C to tube C, etc.). 



Note: As soils differ in their ability to absorb the stain, the same 

 soil must be stained with different concentrations of the acridine 

 orange to ascertain the most suitable concentration. Solution G 

 will be used for differentiating between living and dead bacteria. 



3 . Shake each of the five tubes thoroughly ; then leave them to 

 stand for five or ten minutes. 



4. The most suitable concentration for the particular soil is that 

 contained in the tube, which shows the least amount, compared 

 with the others, of excess dye, and this specimen should be used 

 for examination as follows: 



(a) For qualitative analysis of autochthonic bacteria : 



The dyed soil suspension is strongly centrifuged ; the upper 

 (liquid) layer is poured off and discarded, leaving a small quantity 



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