Practice the following calculations for creating serial dilutions and calculating cell density. 1. You add 0.1 ml of undiluted cell culture to 9.9 ml of sterile broth. What is the dilution factor? 2. Transfer 1.0 ml and 0.1 ml of your dilution from step 1 onto separate plates (A and B). Report the final dilution factors of the plates on Table 1. 3. You take 0.1 ml of the cell suspension you made in question 1 and add it to 9.9 ml of sterile broth. By how much did you dilute it at this step? What is the total dilution of the original culture after this second step? 4. Transfer 1.0 ml and 0.1 ml of your dilution from question 3 onto separate plates (C and D). Report the final dilution factors of the plates on Table 1 5. You take 1 ml of the cell suspension you made in question 3 and add it to 9 ml of sterile broth. What is the total dilution of the original sample after this third step? 6. Transfer 1.0 ml and 0.1 ml of the suspension you made in question 5 onto separate plates (E and F). Report the final dilution factors of the plates on Table 1 After 24 hours of incubation you count the number of colonies on Plates A β F (see Table 1). Calculate the original density of the starting culture in colony forming units (CFU) Plate | Final Dilution Factor | Number of Colonies | CFU of original culture --------------------------------------------------------------------------- A | | TNTC | B | | TNTC | C | | 267 | D | | 58 | E | | 69 | F | | TFTC |
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You perform serial dilution. First, dilution pipette 1 ml of culture into 99 ml of water. Second, dilution pipette 1 ml of the first dilution into another 99 ml of water. Third, dilution pipette 1 ml of the second dilution into 9 ml of water. Diagram this procedure. What is the total dilution? You perform serial dilution starting with a culture containing 1,000,000 organisms/ml. First, dilution pipette 1 ml of culture into 99 ml of water. Second, dilution pipette 1 ml of the first dilution into another 99 ml of water. Third, dilution pipette 1 ml of the second dilution into 9 ml of water. What is the total dilution at each step? What is the number of organisms/ml at each step? In the previous question, you prepare pour plates from the second dilution by pipetting either 0.1 ml or 1 ml samples. How many organisms would you expect on these plates? You have a culture containing 10^9 organisms/ml and wish to dilute it to 10^2 (100) organisms/ml. Design a serial dilution. You have performed the following serial dilutions: 1:100, 1:100, 1:10. Diagram this dilution scheme and include the overall dilution in each tube. From each dilution, you then prepare pour plates using either 0.1 ml or 1 ml samples. After incubation, you count the plates and get the following results: What was the number of organisms/ml in the initial culture?
Jenny W.
Practice your understanding of dilutions with the following problems: 1. You perform a serial dilution. First dilution - pipette 1 ml of culture into 9 ml of water. Second dilution - pipette 1 ml of first dilution into another 9 ml of water. Third dilution - pipette 1ml of the second dilution into 99 ml of water. Diagram this procedure. What is the total dilution? 2. You perform a serial dilution starting with a culture containing 1,000,000 organisms/ml. First dilution - pipette 1 ml of culture into 99 ml of water. Second dilution - pipette 1 ml of first dilution into another 99 ml of water. Third dilution - pipette 1ml of the second dilution into 9 ml of water. What is the total dilution at each step? What is the number of organisms/ml at each step? 3. In the previous question, you prepare pour plates from the second dilution by pipetting either 0.1 ml or 1 ml samples. How many organisms would you expect on these plates? 4. You have a culture containing 109 organisms/ml and wish to dilute it to 10 2 (100) organisms/ml. Design a serial dilution. 5. You have performed the following serial dilutions: 1:100, 1:100, 1:10. Diagram this dilution scheme and include the overall dilution in each tube. From each dilution, you then prepare pour plates using either 0.1 ml or 1 ml samples. After incubation, you count the plates and get the following results: What was the number of organisms/ml in the initial culture? Final dilution | No of colonies 1 ml | No. of colonies 0.1 ml 1:100 | TNTC | TNTC 1:10,000 | TNTC | 63 1:100,000 | 59 | 7
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Serial Dilution Problems These problems present you with all the necessary information you need to solve and were designed in a way that you should not need a calculator to complete them. If you are stuck it may help to draw the culture tubes and dilutions out. 1. You dilute a bacterial culture by adding 10 Β΅l of culture to 990 Β΅l of sterile saline. From this dilution, you perform an additional dilution using the same method as in the first dilution. What is the Total Dilution of the resulting suspension? 2. You dilute a culture by adding 100 Β΅l of culture to 900 Β΅l of saline. From this dilution, you perform an additional dilution using the same method as in the first dilution. What is the Dilution Factor of the resulting suspension? 3. If the two solutions made in Q.1 and Q.2 are made from the same parent culture, which would have less cells in the final dilution? How many less cells would you expect in this culture than the other? 4. You perform serial dilutions and generate a culture with a dilution factor of 10^7. After plating 50 Β΅l of this dilution on an agar plate and counting 36 colonies following overnight incubation. Determine the concentration of bacteria in the original culture. 5. Using the culture in Q.4 you plate a 50 Β΅l aliquot of undiluted original culture on an agar plate containing rifampicin antibiotics. After incubation 18 colonies are observed to grow. What is the concentration of rif-resistant mutants present in the culture? 6. Using the results from Q.4 and Q.5 above, what is the frequency of antibiotic resistant mutants in the original culture? 7. You perform the following serial dilution of a culture: 1. 1st dilution - Transfer 10 ml of parent culture to sterile 90 ml and mix culture thoroughly. 2. 2nd dilution - Transfer 1 ml from 1st dilution to sterile 999ml and mix culture thoroughly. 3. Plate 100 Β΅l from 2nd dilution on an LB-agar plate. 4. The next day, you count 54 colonies on the agar plate. What was the concentration of bacteria in the original culture? 8. You were given a bacteria culture that has been estimated to have a concentration of 5 x 10^6 cells/ml. For an experiment you require an average of 50 cells on every plate, how many test tubes will you need in your serial dilutions? Assume you can only perform serial dilutions as 1:10 and you only have the means spread 1 ml of the inoculum into your plate at a time.
Dominador T.
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