1. Define very briefly:
• serial dilution
• dilution factor
• optical density
2. A bacterial culture with 300 cells/ml is diluted so that it has 3 cells/ml. What is the
dilution factor?
3. What are the minimum and maximum number of colonies that can be counted on
a Petri dish (for both pour plate and spread plate methods) and be statistically
significant?
4. Draw a dilution scheme to describe how you would prepare a dilution of a culture
broth with 3000 bacteria/ml to obtain plates with approximately 200 colonies/plate.
5. You wish to determine viable counts on a culture of Bacillus subtilis. You begin by
pipetting 1 ml of culture into 99 ml of sterile water. After shaking the dilution well
you make a series of 2 further dilutions but of 10-1 each. From the most dilute you
make three pour plates using 1 ml in each. After incubation you find the plate counts
are 243, 256 and 222. What is the viable count (cells/ml) in the original culture?
6. Your TA gives you a bacterial culture and claims there are 6.3 x 107 bacteria/ml in
the culture. To check up on him, you plate (1ml) of two dilutions of the culture, one
plate at a dilution of 10-6 and one at 10-5. If your TA is right, how many colonies do
you expect to find on the 10-6 and 10-5 dilution plates?
7. Sam Smart found 324 bacterial colonies on his plate; dilution factor = 108. He
originally had 5 ml of a Streptococcus lactis culture suspension from which he made
appropriate dilutions so that 1ml produced the plate described above. How many
organisms were in one ml of the original culture? How many organisms were there in
the original 5ml of culture broth? Describe how he may have made a 10-8 dilution in
4 steps. Were his results valid?
8. Suppose you were given a tube containing 10 ml of frozen Staphylococcus aureus
culture, known to contain 4.5 x 105 bacteria/ml. You were told to thaw it by heating
at 70°C for 20 min, and then prepare a 10-4 dilution. How many colonies do you
expect to find on the plate?