ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 31 
SECTION 10: QUANTITATIVE BACTERIOLOGICAL 
ANALYSIS 
EXERCISE 49 
QUANTITATIVE PLATING METHODS 
Samples “X” and “Y” which are furnished represent two samples of water. 
Sample “X” is very pure water with a small number of bacteria. Sample “Y” 
is a sample of impure water which contains a large number of bacteria. 
Melt the tubes of agar in flowing steam or in boiling water. Place the 
melted agar into a water bath at about 45°C. This should be done in time so that 
the agar will be cooled to the proper temperature when it is ready to be poured 
into the plates. When poured, the agar should be between 42° and 45° C. 
With a sterile pipette place 1 c.c. of sample “X” into a sterile Petri dish. 
Mark this plate “X-1”. With the same pipette place 1/10 c.c. of the same sample 
into another sterile Petri dish. Mark this plate “X-1/10”. 
Materials which contain a large number of bacteria cannot be “plated out” 
directly as was done with sample “X”, but must be diluted with sterile water. 
With a sterile pipette remove 1 c.c. of sample “Y” and place it into a 99 c.c. 
water blank. This bottle now contains 1 c.c. of the original sample diluted 100 
times; thus 1 c.c. of this dilution is equivalent to 1/100 c.c. of the original sample. 
Shake this bottle vigorously 25 times to insure a uniform mixing of the sample, 
and to break apart bacteria which may be hanging together in clumps. 
With another sterile pipette remove 1 c.c. from this bottle (the 1/100 di- 
lution) and place it into a sterile Petri dish. Mark this dish “Y-1/100”. With 
the same pipette place 1/10 cc. of the 1/100 dilution into another Petri dish. 
Mark this plate “Y-1/1,000". With the same pipette take another 1 c.c. amount 
from this bottle and place it into another 99 c.c. water blank. 
The second 99 c.c. water blank represents a 1/10,000 dilution of the original 
sample. Shake the second 99 c.c. water blank 25 times, and with another sterile 
pipette transfer 1 c.c. of it to a Petri dish. Label this dish “Y-1/10,000”. 
Pour the melted and cooled agar into the Petri dishes and mix thoroughly. 
Note especially the details of the technique involved as demonstrated. 
In pouring the melted agar into the plates observe all the precautions out- 
lined in Exercise 18. Be sure the temperature of the agar at the time of pouring 
is between 42° and 45° C. Thoroughly mix the melted agar and the dilution 
water in the plate by tilting and rotating the dish. In mixing, take especial care 
not to splash the agar on the sides and cover of the Petri dish. The mixing of the 
agar and the dilution water should be done immediately after the agar is poured 
into the dish before it has time to cool and harden. 
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