5.3: Lab Procedures- Viable Plate count
- Page ID
- Learn microbiological skills such as pipetting, performing serial dilutions, prepare spread plates for a viable plate count, and performing a colony count.
- Estimate the number of microbes in a sample using serial dilution techniques, including: a. correctly choosing and using pipettes and pipetting devices b. correctly spreading diluted samples for counting c. estimating appropriate dilutions d. extrapolating plate counts to obtain the correct CFU in the starting sample.
Cultures: Stationary phase broth culture of Serratia marcescens
Media: Dilution tubes of sterile water
Supplies: Nutrient agar plates, P-1000 Pipetman, sterile tips, Sterile L-shaped blue cell spreaders ("hockey stick” or “spreader”)
A. Sample Dilution and Spread Plating
1. Label one plate on the bottom for each of the following dilutions: 10-5,10-6 , 10-7, 10-8,and 10-9. See Image 1.
2. Label the dilution tubes as follows: label the two tubes (Navy blue caps) containing 9.9 mL sterile water as 10-2 and 10-4; label the four tubes (Green caps) containing 9.0 mL sterile water as 10-5, 10-6, 10-7, and 10-8.
3. Carefully and aseptically remove 0.1 mL (100µL) of the Serratia marcescens culture and pipette it into the tube marked 10-2. Mix the tube completely, being careful not to spill any of the contents. The vortex mixer will help you mix the contents of the tube.
• You have now prepared a 10-2 (1/100) dilution. Why is it 10-2? Because 0.1 mL of undiluted culture was diluted into 9.9 mL of water, giving a total volume of 10.0 mL in the dilution tube. That makes this a 10-2 dilution (0.1/0.1 + 9.9 = 0.1/10.0= 1/100= 10-2).
4. Change the pipette tip, and take 100µL (0.1 mL) from the 10-2 dilution and pipette it into the next 9.9 mL dilution tube marked 10-4. Mix well. You have now prepared a successive 1/100 dilution, resulting in 10-4 dilution of the original culture (1/100 multiplied by 1/100 = 1/10,000 = 10-4, which is the same as 10-2 x 10-2=10-4).
5. Change your pipette tip again.
• Why keep changing pipettes? Because any fluid left in the pipette tip from the previous dilution will contain many more cells per mL than any successive dilution and, if used, will grossly confuse the final results by indicating a higher number of cells than were actually present in the original sample.
Now we will start doing a series of 1/10 dilutions. Set your Pipetman to 1,000µL and remove a 1,000µL (1.0 mL) aliquot from the 10-4 dilution tube. Transfer it to the 9.0 mL dilution tube marked 10-5 and mix well. The original culture has now been diluted 1/100,000, or 10-5 (10-4 x 10-1 = 10-5),
• You have just prepared a 10-1 (1/10) dilution of the previously diluted culture. Why is it 10-1? Because 1.0 mL of diluted culture was further diluted into 9.0 mL of water, giving a total volume of 10.0 mL in the dilution tube. (1.0/1.0 + 9.0 = 1.0/10.0 = 1/10 = 10-1). Now you know why these series of dilutions are referred to as serial dilutions.
- Continue your dilution series, as indicated in Image 1, through to the 10-8 dilution tube.
Image 1: Serial dilution and plating
6. Using a new pipette tip, transfer 100µL (0.1 mL) of the 10-8 dilution onto the center of the agar surface of the plate marked 10-9.
Consider why this is a 10-9 plate after you put 100uL (0.1 mL) of inoculum from the 10-8 dilution tube on it. Remember that you are trying to determine the number of viable cells in each 1.0 mL aliquot of the original Serratia marcescens sample, and you only put 10% of 1.0 mL (0.1 mL) on the nutrient agar plate.
Repeat with the rest of the dilutions, transferring 100µL (0.1 mL) of each dilution tube onto the appropriate agar surface, using a new pipette tip between each dilution tube.
7. Spread plates: Using good aseptic technique, take one sterile blue L-shaped cell spreader out of the bag and reseal the bag. Starting with the plate marked 10-9 , using the spreader, gently push the liquid inoculum applied to the center of the plate, two or three times clockwise around the dish, then several times counterclockwise, turning the plate on the turn table as needed to obtain complete coverage.
Continue with the rest of the spread plates using the same cell spreader. Make sure you are continuing to work backwards (from 10- 9 , then 10-8 , then 10-7, then 10-6 etc.) from the most dilute suspension to the most concentrated suspension to minimize the amount of carryover from plate to plate. Again, use good aseptic technique, work quickly, do not touch the cell spreader on surfaces other than the agar plate you are using. If you accidentally “contaminate” the spreader by touching a surface (table or other), then dispose of it and get a new spreader. We are trying to minimize the use of the spreaders to reduce waste, but if it gets compromised, then replace it with a new sterile spreader.
Dispose of the cell spreader in the small orange biohazard bin on your bench when you are done with your spread plates.
Remember that the plates should be labeled as a ten-fold higher dilution than the dilution tube of the 0.1 mL sample being plated. For example, 0.1 mL of the 10-6 dilution tube should be plated on the agar plate marked 10-7. Again, if you need help visualizing this, see Figure 6-1.
It is generally desirable to make duplicate or triplicate plantings of each dilution and to average the resulting counts. However, since your lab sample comes from the same stock culture, the class average should give an accurate enumeration of the original stock culture.
8. After the spread plating, leave plates agar side down for at least 30mins. in order for the inoculum to absorb onto the agar, then invert the plates and incubate at 30°C.
B. Counting colonies on plates
1. Looking at your dilution plates prepared last period, choose the plates that have from 30-300 colonies on them. As this might take some practice in plate counting, you might need to choose all plates with what looks like a reasonable number of colonies to count.
a. Those plates that have no microbial growth can be recorded as 0 or NG, No Growth.
b. Those plates on which colonies are not individually distinct (their edges run together) can be recorded as TNTC, Too Numerous To Count.
c. Those plates on which you cannot distinguish any individual colonies, the entire surface is covered with microbial growth, can be recorded as confluent.
2. Count each colony to give a total colony count for each plate chosen. You will avoid counting a colony twice by marking off the colonies on the bottom of the plate as you count them. This requires, of course, that the plate be upside down. Be sure to count any small colonies. Record your results on the report sheet.
C. Calculation of number viable cells/mL in the original sample
• Choose the plate containing between 30 and 300 colonies.
• Multiply the number of colonies on the plate by the final dilution factor. This gives the total viable cells/mL in the original sample.
• Calculate the colony-forming units, CFU, per mL for the Serratia marcescens culture.
D. Watch this video on how to perform a serial dilution and make spread plates.
Watch Video 1: Dilutions and Plating at NC State Microbiology labs. URL: https://youtu.be/IJcw4fRsYnU