Exercise 16: Serial Dilution & Standard Plate Count

Introduction

The standard plate count is a quantitative technique used to estimate the population size of a sample. A portion of the sample is diluted several times prior to plating it on media. After incubation the number of colonies on each plate is counted. An accurate estimation requires that the exact dilution used to produce countable colonies be determined. This can be mathematically calculated because a series of standard dilutions is used – a serial dilution. Serial dilutions reduce the number of bacteria in regular repeated steps. Tenfold dilutions simplify the math used to estimate population size of the original sample.

A serial dilution is made by aseptically transferring a sample through a series of dilution blanks. Blanks are tubes containing a known volume of sterile buffer or water. Tenfold dilution series use blanks containing 990 or 900 µl of buffer. The volume of sample added to the blank will always add up to 1000 µl (or 1.0 ml) of total volume. Therefore, students will add 10 µl of sample to 990 µl blanks and 100 µl to 900 µl blanks. Adding 10 to 990 represents a 1:100 dilution (10^-2). Adding 100 to 900 represents a 1:10 dilution (10^-1). Once the sample has been added to the blank this tube becomes the sample for the next blank. This is repeated several times resulting in a very dilute mixture containing only a few cells (or sometimes none) in the last tube.

Each step in a serial dilution is assigned a dilution factor. This value is determined by dividing the sample volume by the total volume in the tube after adding the sample. A dilution containing 1 mL of sample and 99 mL of diluent (water) has a dilution factor of 10^-2 (or 1:100) because it is now 1/100 of its original concentration. The diluted sample now becomes the “sample” for the next step.

D = V1/V2 = 1/100 = 0.01 = 10^-2

V1 = the volume of sample being diluted (10 µl)

V2 = the total combined volume of sample and diluent (1000 µl)

A small portion (usually 100 µl) from the last several dilutions is then spread onto agar plates. The objective is to produce at least one plate with countable numbers of colonies. The countable range is between 30 and 300 colonies on the plate. Fewer than 30 colonies will result in inaccurate estimates. More than 300 and colonies will start to overlap causing counting errors. Once the number of colonies is determined the population size (or density) of the original sample can be calculated in CFU/ml. Colony Forming Units (CFU) is used as the unit measure instead of cells because it is impossible to know how many cells “started” the colony.

The cell density can be determined by the following formula:

OCD= CFU/FDF

Original cell density (OCD) = concentration of cells in the original sample/culture

Colony Forming Units (CFU) = colonies on plate

Final Dilution Factor (FDF) = sum of all dilution factors (don’t forget the 10^-1 onto the plate)

Course Intended Outcomes

Properly handle, prepare and maintain bacterial cultures, and isolate pure microbial species from a mixed culture.

Explain and apply the scientific method to answer scientific questions and solve problems in the laboratory.

Collect, organize and present data from an experiment, as well as draw appropriate conclusions and report finding in written form using the scientific method.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Bunsen burner
• Glass spreader
• Test tube rack
• Sterile test tubes, small
• Micropipettes and tips
• Additional materials and cultures will be assigned by your instructor

Procedure, Day 1

Pipetting Technique:

1. Practice using the micropipettes prior to beginning the serial dilution steps.
2. Hold the pipette so the hook is over your index finger and your thumb is on the plunger.
3. Open the box of tips and press down over a tip so that it firmly attaches to the end of the pipette. It helps to tap once or twice. Make sure you are using the right size tips for your pipette.
4. Press the plunger down slowly stopping at the first point of resistance. This is the stop for the specific volume shown on the side of the pipette. Do NOT press the plunger all the way down.
5. Immerse the tip in the bottle of sterile water and slowly release the plunger drawing up the water sample. Look at the water level in the tip.
6. Dispense the water back into the water bottle. This time press the plunger all the way down. This should evacuate the last drop of water remaining in the tip.
7. Repeat steps 4 – 6. Each time the water should stop at the exact same point on the pipette tip. Continue to practice until your results are consistent.
8. To dispose of the tip, press the button under your thumb (and next to the plunger). This will eject the tip so that you can pick up a new, sterile tip for the next inoculation.

Serial Dilution Technique:

1. Refer to Exercise 2 for proper use of instruments and aseptic technique.
2. Use dilution and inoculation schedule provided by your instructor or see figure 16.1 below.
3. To prepare your blanks use a preset micropipette to transfer 990 µl of buffer/water to the appropriate sterile tubes. Then use the preset micropipette to transfer 900 µl to the remaining sterile tubes.
4. Mix or vortex the E. coli culture provided before beginning dilutions.
5. Use the micropipette to transfer 10 µl of E. coli culture to dilution blank #1. Mix the sample using the vortex or by flicking the tube with your finger. Hold the middle and top of the tube firmly to prevent spills.
6. Use the 10 µl or 100 µl micropipette to transfer the appropriate amount of diluent to the next blank in the serial dilution series. Always use the last blank inoculated as the sample for the next blank. Always mix after each transfer.
7. When you have inoculated all 5 blanks the dilution series is complete.

Spread Plating Technique:

1. Use micropipette to remove 100 µl of sample from the appropriate dilution tube.
2. Dispense 100 µl aliquot onto the surface of the appropriate plate. (This represents a 1:10 dilution because 100 µl is 1/10^th of one milliliter)
3. Use dilution and inoculation schedule provided by your instructor (or see figure 16.1 below) to determine which dilution tube should be used for plates A – D.
4. Use a sterile glass spreader to distribute the 100 µl sample over the surface of the plate without touching the edges. Place the used glass spreader into the beaker provided.
5. Repeat steps 1 – 6 for the remaining plates making sure to use a new sterile glass spreader each time.
6. Allow plates to dry for 5 minutes.

Sampling/Inoculation:

1. Your instructor will assign the cultures to be used for inoculation by your group.
2. Collect the required materials for the lab.
   • Place all tubes in a tube rack.
   • Never hold a culture tube by its cap.
   • Bacterial culture tubes are always to be shared with your table group.
3. Clearly label plates (on the bottom side) with your group name or initials, organism name(s), and section day/time.
4. Clearly label tubes using label tape. Do not write directly on tubes with sharpie markers.
5. Place inoculated plates in the rack labeled “to be incubated” on the back bench. Always place the plates with lids down and bottoms up.

Fig 16.1 long description

Procedure, Day 2

1. Collect your group’s plates from the back bench area labeled “last week’s lab”.
2. Examine plates A through D and count the number of colonies on each plate.
3. If there are more than 300 colonies on the plate, stop counting and record the total as TNTC (too numerous to count).
4. One of the four plates should have counts in the ideal range of 30 – 300 colonies. If not, choose the plate closest to the ideal range to use for your calculation.

Lab Clean-up

Return bacteria cultures to the front bench after inoculations are complete.

Remove labels from tubes and place in the tube rack ABOVE the biohazard container.

Plates are disposable and should be thrown away in the biohazard container when everyone in the group has completed the exercise.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

1. Record your colony counts for plates A – D in your lab notebook along with the dilution series used.
2. Select the plate that is closest to the ideal range of 30 – 300 colonies.
3. Using the sum of the dilution factors from your dilution tubes + an additional 1:10 dilution for the 100 µl sample onto the plate estimate the density/population size of the original E.coli sample.

Critical Thinking Questions

When examining and counting your plates did you get 10-fold differences in the numbers of colonies on each plate? What does this indicate?

What are some common lab errors that would lead to a miscalculation of the original sample density?