Introduction:
It is often necessary to determine the number of bacteria in a sample or compare bacterial growth. Quantifying organisms has particular importance in the food and water industry. A variety of methods are available to determine approximately how many organisms are present in a suspension. The method used will depend on whether total counts or counts of only viable organisms needs to be measured.
The Plate Count (Viable Count):
Usually the number of bacteria in a given sample is too great to be counted directly. In these cases, the sample is serially diluted, and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies. The number of colonies can be used to measure the number of viable cells in that known dilution. Because some organisms form multiple cell arrangements, the colony may consist of groups of bacteria rather than a single organisms. We generally refer to these as colony forming units (CFUs) in that known dilution.
Samples are generally diluted by factors of 10 and plated on agar. The number of colonies on a dilution plate, after incubation, will be between 30 and 300. This range is chosen because it is statistically significant. Small dilution errors will have a drastic effect on a plate with less than 30 organisms, while with more than 300 organisms there would be poor isolation and colonies growing together. Usually, you will determine the number of CFUs per milliliter of sample. This will be done by finding the number of colonies on a suitable plate and multiplying it by the dilution factor.
# CFUs per ml sample = # colonies (30-300 plate) X dilution factor of plate counted
Direct Microscopic Method (Total Cell Count):
In the direct microscopic count, a special coverslip with a ruled slide is used the count the cells in a known volume. The number of bacteria is counted directly under the microscope, and then the number of the bacteria in a large sample is calculated by extrapolation. This is useful to count both living and non-living organisms.
Turbidity:
Since growth of microorganisms in a liquid medium causes it to become turbid, the amount of light absorbed by the bacterial suspension can be used to estimate the number of bacteria present in the sample. The instrument used is a spectrophotometer, which only allows a single wavelength of light to pass through the sample. A photocell compares the light coming through the sample with the total light entering the tube. The percent of light transmitted in inversely proportional to the bacterial concentration., while the absorbance is directly proportional to the cell concentration.
Direct Microscopic Count:
In this technique, 1.0ml of the sample is pipetted into a tube containing 1.0ml of the dye methylene blue to give a ½ dilution of the sample. A Pipette is used to fill the chamber of a Petroff-Hausser counting chamber (a special slide with a counting grid) with this ½ dilution. A cover slip is places over the chamber and the slide is placed under the 40X objective. The number of bacteria are counted in 5 of the large double lines squares. For more accuracy, organisms on the upper and left lines are counted, but not on the right and lower lines. This total number is divided by 5 to find the average number of bacteria per large square. The number of bacteria per cc is calculated as follows:
The number of bacteria per cc =
The average number of bacteria per large square X
The dilution factor of the large square (1,250,000) X
The dilution factor of any dilutions made prior to placing the sample
in the counting chamber, such as mixing it with dye (2 in this case). This is essentially the same as a random sampling technique that can be used to approximate the count of other organisms (including large mammals) when counting all of them is impractical.
Procedure: Serial Dilutions for Viable Plate Count
Procedure: - Viable plate count
You will need 6 dilution tubes containing 9.0 ml of sterile saline. Use aseptic technique to dilute 1.0 ml of a sample of bacteria. You will be using a new sterile pipette for each 1.0ml that you dilute.
Place 1.0 ml of sample aseptically into 9.0 ml of sterile saline. Mix the tube thoroughly by holding the tube in 1 hand and vigorously tapping the bottom with the other hand to assure even distribution of the bacteria throughout the liquid.
Use the same procedure to aseptically withdraw 1.0ml from the first dilution tube & dispense it into the second dilution tube. Continue to do this from tube to tube until the dilution is completed. Be sure to use a new pipette for each dilution.
Using a new pipette, aseptically transfer 0.1 ml from each of the last dilution tubes onto the surface of the corresponding labeled trypticase soy agar plate. Since only 0.1ml of the bacterial dilution rather than the desired 1.0ml is placed on the plate, the actual dilution of the plate is 1/10 the dilution of the tube from which is came.
Use a sterile cell spreader to immediately spread the solution over the entire surface of the agar plate.
Replace the lid and resterilize the glad rod with alcohol and flame.
Repeat this process for each of the plates.
Once the plate has dried, incubate the plates upside down at 37 degrees Celsius.
Counting the colonies:
Choose a plate that appears to have between 30 and 300 colonies.
Calculate the number of CFUs per ml of original sample as follows
CFUs / ml sample = # of colonies X dilution factor of the plate
___________ number of colonies
___________dilution factor of the plate counted
___________ number of CFUs per ml.
Practice Examples:
Using the following diagram, how many CFUs per ml are in the original sample?
http://www.sciencebuddies.org/mentoring/project_ideas/MicroBio_img019.gif
Examples of plates after going through series of dilutions:
Top row: plate on the far row offers most reasonable number of colonies to count
Bottom row: middle plate has enough to count to be valid, but not too few or too many.
Website with sample calculations:
