1. If you inoculate fresh Nutrient Broth with 100 cells of Escherichia coli and keep the culture at the optimal temperature of 37°C, assuming the generation time is 30 minutes, how many generations will take place after 7 hours?
How many bacteria will be present?
Will the number of bacteria continue to double every 30 minutes indefinitely? Why?
2. Starting with four bacterial cells per cm3 in a rich nutrient medium, with a 20-minutes generation time, how many cells will there be in 1 dm3 of this culture after 1 hour?
After 2 hours?
After 2 hours if one of the initial four cells was non viable?
3. After washing your hands, you decide to do a plate count on the soap. You dilute 1 g of the soap to 10-6 and plate it on nutrient agar. After 24 hours of incubation at 37C, there are 168 colonies. How many bacteria were on the soap?
4. You were given a 24-hour liquid culture of T2 coliphage. After aseptically performing the usual 10-fold serial dilution procedure, you plated 0.1 cm3 of the 10-5, 10-6, 10-7 and 10-8 dilutions in three replicates. Plates were incubated at 37ºC for 24 hours, and these are the results you obtained:
|
Dilution
|
Replicate 1
|
Replicate 2
|
Replicate 3
|
|
10-5
|
TNTC
|
TNTC
|
TNTC
|
|
10-6
|
245
|
234
|
284
|
|
10-7
|
23
|
28
|
29
|
|
10-8
|
2
|
3
|
4
|
TNTC: Too numerous to count
Calculate the average number of plaque forming units per cm3 present in the original culture.
5. Most vaccines licensed for use in the UK are prepared from attenuated (live) viruses. Attenuated viruses are capable of limited replication in the target cell but cannot replicate sufficiently to cause disease. It is important that each dose of the vaccine contains the correct number of virus particles. Too few may not be sufficient to stimulate a protective immune response and too many may cause disease. It is therefore necessary to check all batches of vaccines made for infectivity using a suitable assay.
Three batches of the attenuated Sabin poliovirus vaccine were tested for infectivity using a plaque assay on Vero cells (a cell line derived from monkey kidney cells); 0.1 cm3 aliquots of each batch were tested three times and the following results were obtained.
Batch 1
Dilution
|
Replicate 1
|
Replicate 2
|
Replicate 3
|
Batch 2
|
Dilution
|
Replicate 1
|
Replicate 2
|
Replicate 3
|
Batch 3
|
Dilution
|
Replicate 1
|
Replicate 2
|
Replicate 3
|
TNTC = too numerous to count
What is a plaque?
Suggest why only dilutions above 10-5 were tested.
Calculate the average number of PFU (plaque forming units) per cm3 of the original vaccine suspension for each batch.
If each dose of 0.1 cm3 should contain 500 PFU of attenuated virus, by how much should each batch be diluted?