Protocol: Using sample A, aseptically lift a single colony from the culture plat

ID: 195180 • Letter: P

Question

Protocol: Using sample A, aseptically lift a single colony from the culture plate and add it to 200 mL of tryptic soy broth (TSB) in a 500 mL Erlenmeyer flask. At each time point indicated, take 1 mL from the culture flask, then return it to the incubator. Add that 1 mL to 9 mL PBS (tube1), mix, then add 1 mL from tube 1 to 9 mL PBS in a second tube (tube 2). Mix and add 1 mL from tube 2 into a 3rd tube with 9 mL PBS. Mix and add 1 mL from tube 3 into a 4th tube (tube 4). Add 0.1 mL to a blood agar plate and spread it

4 40% 10:04 PM Microbiology Journal Home × Pearson " WileyPLUS × M Iowa State University Mail X G how to screenshot samsung https://canvas.iastate.edu/courses/40509/assignments/540559 Measuring by Plate Counts (determining CFU): IOWA STATE 9. Protocol: Using sample A, aseptically lift a single colony from the culture plate and add it to 200 mL of tryptic soy broth (TSB) in a 500 mL Erlenmeyer flask. At each time point indicated, take 1 mL from the culture flask, then return it to the incubator. Add that 1 mL to 9 mL PBS (tube1), mix then add 1 mL from tube 1 to 9 mL PBS in a second tube (tube 2). Mix and add 1 mL from tube 2 into a 3rd tube with 9 mL PBS. Mix and add 1 mL from tube 3 into a 4th tube (tube 4). Add 0.1 mL to a blood agar plate and spread it (spread plate method - Figure 5.14) (2 pts) Tube # counted # of bacteria in flask at that time expressed as CFU/mL (show your work) # of bacteria Time on plate 2 hours #3 212 4 hours #4 42 6 hours #4 87 8 hours #4 144

Explanation / Answer

Time

Tube # counted

# of bacteria on plate

# of bacteria in flask at that time as CFU/ml

2hours

212

2.12 X 106

4 hours

4.2 X 105

6 hours

8.7 X 105

8 hours

144

1.44 X 106

One colony is added to 200 ml. 1 ml from 200 ml is added to 9 ml of PBS. This is 1: 0 dilution in tube # 1. In tube # 2, 1 ml + 9ml = 1: 10 dilution. Tube 2 is a 1: 100 dilution of original. Tube 3 is a 1: 10 dilution of tube 2. Hence, tube 3 is a 1: 1000 dilution of original. Tube 4 is still 1: 1000 dilution. 0.1 ml of either tube 3 or 4 is added for spread plate.

1) At 2hours.

0.1 ml of tube 3 is spread plated.

# of bacteria= Number of bacteria * dilution factor/volume on plate= 212 * 103 * 10= 2.12 X 106 CFU/ml

As 1 ml of sample from 200 ml was taken, the final count need not be multiplied with 200 to obtain CFU/ml

2) At 4hrs

# of bacteria= Number of bacteria * dilution factor/ volume on plate= 42 * 103 X 1* 10= 0.42 X 106CFU/ml

3) At 6 hours

# of bacteria= Number of bacteria * dilution factor/ volume on plate= 87 * 103 X 1* 10= 0.87 X 106 CFU/ml

4) At 8 hours

# of bacteria= Number of bacteria * dilution factorvolume on plate= 144 * 103 X 1* 10= 1.44 X 106 CFU/ml