## Part C: UV Experiments Serial Dilutions and Viable Cell Counts

The experiment Observing the Effects of Solar Ultraviolet Radiation on Cells shows that when cells are exposed to sunlight all, some, or none of them may be killed. Many experimental questions can be answered with qualitative answers like "all, some, or none." Other questions may require quantitative answers. For example, in the next experiment you will use the sensitive yeast strain to measure the intensity of solar UV radiation by measuring the fraction of cells exposed that survive. To get quantitative answers about yeast survival you must put a known numbers of viable (living) cells onto the agar plates and then count the number that remain after being exposed. You can determine the number of viable cells by counting the colonies that grow up on the agar growth medium in a Petri plate by assuming that each colony grows from a single viable cell. This is usually a reasonable assumption.

Experiment:
In the experiment that follows you will learn how to measure the number of viable cells on a Petri plate. You will be able to use this procedure whenever you need to measure the number of cells that survive an exposure to radiation or some other treatment. First you will estimate the number of cells in a liquid suspension in order to plate a reasonable number of cells. For this you will use one of the most sophisticated and sensitive optical instruments in existence, the human eye. With surprisingly little practice you can learn to estimate the number of cells in a suspension by just looking at it.

You can estimate cell density because of your eyes' fairly sharp threshold for observing turbidity (cloudiness). When viewed in a standard 13 100 mm glass tube, yeast suspensions of less than about 1 million cells per mL are not visibly turbid. Above this threshold density, the suspension is cloudy. When you adjust the number of cells in a suspension until just barely visible, you obtain a suspension of known density (approximately 1 106 cells/ml).

When you have a suspension that contains approximately 1 106 cells/ml, you will dilute it to get the right concentration for plating. You will make the dilutions in known steps so you can calculate the number of cells in each dilution tube. This procedure helps you plate a countable number of colonies.

Time Line:
Day 1: 50 min Serial Dilution and Plating Cells
Day 3: 30 min Counting Plates

Materials:

For each student or team:

12 to 15 sterile culture tubes, 13 100 mm with caps 5 to 10 polystyrene Petri dishes with YED agar 1 Alcohol wipe 1-11 sterile pipets, either 1-mL calibrated bulbed transfer pipets or (1mL disposable serological pipets calibrated in 0.1 mL steps and pipet pump)

Common Materials:

• Freshly grown subculture of yeast cells (See specific experiments for appropriate strain.)
• Sterile water
• Sterile toothpicks
• Marking pens
• Tape

Optional Materials:

• Glass spreader, alcohol in 100 ml beaker, and source of flame
• Micropipettor and sterile tips

Time Line:
Day before: 10 min To have a freshly-grown culture of yeast cells grow the yeast on YED growth medium overnight at 30o C or 1 to 2 days at room temperature.
1. Make a clean sterile work space by wiping the table or bench with an alcohol wipe. Because most contamination is airborne select a place free from drafts.
2. Open the yeast storage vial.
3. Using the broad end of a sterile toothpick, pick up a small amount of yeast from the slant.
4. Replace the lid. Tighten. ( Store in a refrigerator to keep viable for up to nine months.)
5. Open the YED Petri dish just enough so that you can reach into it with the toothpick full of cells.
6. Gently make several streaks of the culture on the surface of the agar. (Remember that you need not be able to see the streaks to have enough to grow into a visible culture overnight.)
7. Close the lid and incubate the culture overnight at 30oC, or 2 days at room temperature. (Most microbial cultures should be incubated with the agar side up to prevent condensation from dropping on the colonies.)
Teacher Tip

Technical Tip:
By starting with an obviously turbid cell suspension and diluting it in two-fold steps, you eventually reach a dilution that is not visibly turbid. We have found that for most people, the last, or "just turbid" tube will contain approximately 1 106 cells/mL.

Time Line:
Day 1: 50 min

### Making a "just turbid" suspension:

1. Use a sterile pipet to place 2 mL of sterile water into one tube and 1 ml into each of six more tubes.
2. Make a turbid suspension in the first tube. Take a small amount of yeast--less than the size of a pin head--on a toothpick and select the tube that contains 2 mL of water. Without touching the mouth of the tube, place the yeast as far down the inside wall of the tube as you can.
3. Mix the suspension thoroughly by thumping it. Holding the tube loosely near its top between thumb and forefinger, and thump it near the bottom with the palm side of one or more fingers of the other hand, imparting a swirling motion. The thumping motion approximates the gesture one would make to beckon someone to come hither. 4. Add yeast until the suspension is noticeably turbid or cloudy. 5. Make a series of two-fold dilutions until you have a tube that is clear. Transfer 1 mL of the suspension from each tube to the next, beginning with the one containing the cells. Mix each tube between steps by thumping it.
6. Compare each tube with a tube that contains 1mL of clear water. The last turbid tube will contain between 1 and 2 million cells/mL. Use this tube for the rest of the experiment.
Teacher Tip

Technical Tip:
This procedure is called a serial dilution. For making the "just turbid" suspension the dilution steps are 1 mL into 1 mL so that each tube is twice as dilute as the previous one. Each dilution step is two-fold. To dilute the cell suspension further, for plating, dilute 0.1 mL into 0.9 mL to obtain more convenient ten-fold dilution steps
Technical Tip: Three drops from the bulbed transfer pipet is equal to 0.1 mL.
Technical Tip: Label the plates using a marking pen (a Pilot SC-UF or a Sharpie). Use the same kind of pen for marking the colonies when you count them. Identify each plate with the dilution plated and your initials. Write in small letters on the edge of the bottom leaving the middle clear for marking the colonies when you count them.
Prepare and Plate a series of ten-fold dilutions: 1. Start with the most dilute turbid tube (just barely turbid tube from step 6) Use the following procedure to make six serial ten-fold dilutions from this tube into sterile water. (see Figure 2)
2. Set up a series of dilution tubes. Pipet 0.9 ml of sterile water into each of six tubes and label them. Make a diagram in your notebook showing the procedure and explaining your labels.

3. Dilute the just turbid suspension into the first tube. Thump the tube containing the cells. Remove 0.1 ml, and transfer the 0.1 mL to one of the tubes of water. Then thump that tube to suspend the cells.

4. Repeat the procedure serially, using a fresh pipet for each of the remaining tubes. The sixth dilution (which will be the seventh tube) should contain approximately 1 100 cells/ml (1 cell!).
5. Spread samples of the cells from each tube in your ten-fold dilution series on agar plates by the following procedure:
Resuspend the cells in each tube by thumping the tubes.
Pour the entire contents of each tube
onto an agar plate. Replace the lid and distribute the suspension over the surface by tilting and rotating the plate.
6. Incubate the plates for two days at 30 C or for three or four days at room temperature until the colonies are large enough to count.(Teacher Tips)

### Counting Colonies:

Time Line:
Day 3: 30 min 1. Count the colonies on each plate Mark the position of each colony on the bottom of the plate with a marking pen as you count it. Plates containing more than a few hundred colonies are considered "too numerous to count" (recorded as TNTC in data records).
2. Calculate ratio for each dilution step by dividing the number of colonies on each plate by the number on the next more dilute plate.

### Questions:

1. How well do your colony counts agree with the expected dilution steps of ten?
2. If the agreement isn't perfect (and we don't expect it to be, even with a "perfect" technician) do you think the errors could be explained by "random chance"? Explain.
3. Is there any pattern to the errors? Do the ratios systematically get larger or smaller as the number of colonies increases?
4. Write down any reasons you can think of for such systematic errors to occur in this procedure.
5. If you have a culture with 1x106 cells/mL and you wanted to dilute it to 1x102 cells/mL, would it be better to just make a single 10,000-fold dilution or four ten-fold dilutions? Explain the advantages and disadvantages of each method.(answers)