# GENERATION TIME CALCULATION AND GROWTH MEDIA: 1. You wish to chara...

## Question

GENERATION TIME CALCULATION AND GROWTH MEDIA:
1. You wish to characterize the growth rate of a new species of the cyanobacterium Oscillatoria that you isolated from an Antarctic freshwater pond. Because it is filamentous, you cannot use viable count, turbidity, or electronic counters to measure its growth; instead, you will track its dry weight over time. The protocol is to pipette 5 mL of a well-mixed liquid culture onto a preweighed filter paper disk, filter the cells onto the disk, desiccate the disk, and measure its weight.
The initial weight of the filter is then subtracted to get the dry weight of the cells. Given that on January 25, 2017 the cell weight was 0.0255 g, and on March 10, 2017 the cell weight was 2.0934 g, what is the growth rate of this species? SHOW YOUR WORK

COLONY FORMING UNITS (CFU) CALCULATIONS: (See Appendix F for help setting these up.)
2. In tube 1, 100.0 µL of apple juice was mixed with 9.9 mL of water. Next, 200.0 µL from tube 1 was added to 800.0 µL of water in tube 2. Finally, 150.0 µL from tube 2 was plated. If there were 83 colonies on the plate, what was the original CFU/mL in the apple juice? SHOW YOUR WORK

3. Calculate the CFU that are present per gram of soil in the experiment below.
Tube 1: 0.37 grams of soil was suspended in 5 mL of sterile saline solution. (Since soil doesn’t dissolve, consider the total volume to be 5 mL.)
Tube 2: 1 mL of the suspension (Tube 1) was pipetted into 9 mL saline.
Tube 3: 300 uL from Tube 2 was pipetted into 2.7 mL saline.
Tube 4: 0.1 mL from Tube 3 was pipetted into 99.9 mL saline.
Plating: 100 uL from each of Tubes 1-4 was spread onto a Sabouraud Dextrose Agar (SDA) plate (labeled “Plate 2” – “Plate 5”, respectively).
After the plates were incubated for 5 days at 25◦C, the following colony counts were observed:
Plate 1: TNTC Plate 2: 1231 Plate 3: 255 Plate 4: 21
a. What is the CFU/g in the soil? SHOW YOUR WORK
b. If you had plated from onto Nutrient agar (instead of SDA), how would this have changed your final CFU/g results, and why?
c. Notice that the number of colonies on Plate 3 is ~1/5 (255/1231 = 21%) of the number of colonies on Plate 2, even though there is a 10-fold dilution between Tube 2 and 3.
List 1 reason why CFU data are often biased for smaller colony counts:

4. A food inspector wants to check for bacterial contamination at a local restaurant. As part of her check, she moistened a sterile cotton swab and then swabbed a 10.0 cm2 surface of the meat preparation counter. The swab was suspended in 2.0 mL of sterile saline and vortexed to suspend the bacteria collected on the swab. 500.0 µL of this suspension was added to 9.50 mL of saline and 200.0 µL of this dilution was spread on a nutrient agar plate.
a. After overnight incubation, 289 colonies were visible on the plate. How many CFU/cm2 were present on the surface of the prep counter? SHOW YOUR WORK
b. At what temperature should the plates be incubated in order to culture potential human pathogens? Briefly explain your answer.

## Solution Preview

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If the organism is in its logarithmic phase of growth, then the number of cells (and approximately total biomass) is changing exponentially. As the cell mass is changing during generation time, the mass of all the cells is not the same, but there will be the cells in each phase of growth in any moment, so we can assume that the number of cells is directly proportional to the dry weight of cyanobacterium. The generation time –period between each successive binary fission, can be calculated by dividing some amount of time in the log phase of bacterial growth (t) with the number of generations during that period (n):...

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