QUANTITATIVE DILUTION METHOD 55 



2. Shake the flask to secure an even suspension of the 

 microorganisms. 



Do not allow the liquid to touch the cotton plug. 



3. With a sterile pipette, transfer 1 c.c. of the first 

 dilution into a flask containing 99 c.c. of sterile water 

 and shake. The second flask now contains 100 c.c. of a 

 liquid containing 1/100 of the original sample, a dilution 

 of 1/100 in 100, or 1 in 10,000. 



4. If a higher dilution is required, 1 c.c. from the flask 

 containing the 1/10,000 dilution placed in a flask con- 

 taining 99 c.c. sterile water gives 100 c.c. of a liquid con- 

 taining 1/10,000 of the original sample, or a dilution of 

 1 in 1,000,000. 



If a lower dilution of the original sample than 1/100 

 is desired, make use of the 90 c.c. dilution flasks as follows: 



With a sterile 10 c.c. pipette place 10 c.c. of the original 

 sample into 90 c.c. of sterile water and shake. This flask 

 now contains 100 c.c. of liquid containing 10 c.c. of the 

 original sample, giving a dilution of 1 in 10. A dilution 

 of 1 in 1000 may be made either by placing 1 c.c. of the 1/10 

 dilution in 99 c.c. of sterile water, or by placing 10 c.c. of 

 the 1/100 dilution in 90 c.c. of sterile water. 



Note. Almost any desired dilution can be made by the use of these 

 flasks. 



5. For plating, transfer 1 c.c. with a sterile 1 c.c. pipette 

 from the flask containing the desired dilution to a sterile 

 Petri dish. 



Note. Never use less than 1 c.c. Run duplicates when absolute 

 accuracy is necessary. 



6. Liquefy the desired number of agar or gelatin tubes 

 in the water-bath or steam at 100 C. 



7. Cool to a temperature of 40 to 45 C. 



8. Pour the plates, tilting each carefully so that the 

 1 c.c. of the diluted sample may be mixed well throughout 

 Jhe medium. 



