ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 29 
SECTION 9: ISOLATION AND PURIFICATION 
OF BACTERIAL CULTURES 
EXERCISE 46 
LOOP DILUTION METHOD 
To isolate an organism so as to get it in a pure culture, free of all other 
types of bacteria, a transfer may be made from an isolated colony on an agar 
plate to an agar slope (or other suitable culture medium). Since each colony 
develops, as a rule, from one single cell, or a group of cells of the same species 
of organism hanging together, a subculture made from a colony growing well 
removed from the other colonies on the plate will usually contain only one kind 
of bacterium. In order to obtain the colonies of the various bacteria from a 
mixed culture it must be “plated out” on agar in the proper dilution. For this 
purpose what is known as the “loop dilution method” is commonly used. 
Procedure 
Label sterile Petri dishes number 1, 2, and 3. Melt three tubes of nutrient 
agar and cool them to 42° to 45° C. Label one tube number 1; one, number 2; 
and the remaining one, number 3. Transfer one loopful of the mixed culture to 
the agar tube number 1. Mix the organisms and agar thoroughly by rolling the 
tube between the palms of the hands. Transfer 1 loopful of the agar and organ- 
isms from tube number 1 to tube number 2. Mix thoroughly. Transfer 2 loopfuls 
from tube number 2 to tube number 3. Pour the agar in tube number 1 into Petri 
dish number 1, and the agar in tube number 2 into Petri dish number 2, and 
that of tube number 3 into Petri dish number 3. This gives a dilution of the 
organisms and enough scattered colonies on one of the plates so that the colonies 
may be studied in detail. 
When the agar has thoroughly hardened, invert plates and incubate at 
37° C. After two days make agar slope cultures from well-isolated colonies of 
different types. 
EXERCISE 47 
STREAK PLATE METHOD 
While not such a satisfactory method, bacteria may be isolated by simply 
streaking the surface of an agar plate with a little of the material on a loop. 
Make streak plates from the mixed culture furnished, as demonstrated. Note 
especially the precautions and details essential to this technique. 
