52 ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 
Derivation of the Factor for the Microscopic Count 
Diameter of microscopic field=0.16 m.m. 
wR2=Area of microscopic field. 
R =0.08 m.m. 
R2=0.0064 
0.0064 3.1416=0.02 sq. m.m.—area of microscopic field 
0.02 0.01=0.0002 (or 1/5,000) sq. cm.—area of microscopic field. 
Since 1/100 c.c. of milk was spread over 1 sq. cm., each microscopic field 
covers 1/100)<1/5,000 or 1/500,000 c.c. of milk. Therefore, each bacterium in 
a field represents 500,000 per c.c. of milk. - 
Total No, in 1 field <500,000=No. per c.c. 
Total No. in 10 fieldsx 50,000=No. per c.c. 
Total No. in 50 fields 10,000=No. per c.c. 
Total No. in 100 fieldsy< 5,000=No. per cc. 
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EXERCISE 77 
METHYLENE BLUE REDUCTION TEST 
Using sterile pipettes, place 1 c.c. amounts of the methylene blue solution in 
sterile test tubes. Add 10 c.c. samples of the milks under test. See that they are 
thoroughly mixed. 
The solution of methylene blues furnished is of such strength that the sample 
under test contains one part of the dry dye in 200,000 parts of the mixture. 
Place the samples in a water bath at 37° C. and observe the time which 
elapses before the color disappears. 
After the milk samples and dye are thoroughly mixed, the sample should not 
be agitated, which would allow the admixture of air which would retard the re- 
duction of the methylene blue. 
By the use of this test milks may be roughly divided:into classes, as given 
below. 
Bact. per c.c. Time of Reduction 
< 50,000 >10 hrs. 
< 500,000 >5% hrs. 
500,000 to 4,000,000 >2 hrs. <5% hrs. 
4,000,000 to 20,000,000 >1/3 hr. <2 hrs 
> 20,000,000 <1/3 hr. 
The Methylene Blue Solution 
There are on the market special tablets of methylene blue of such strength 
that when one is dissolved in 200 c.c. of distilled water, and 1 c.c. of the solu- 
tion is added to 10 c.c. of milk it gives approximately the above mentioned con- 
centration (1 part of methylene blue to 200,000 parts of the mixture). 
If tablets are not available, a stock solution may be prepared by dissolving 
