Dilution is the process of making a solution weaker or less concentrated. In microbiology, serial dilutions (log dilutions) are used to decrease a bacterial concentration to a required concentration for a specific test method, or to a concentration which is easier to count when plated to an agar plate.
I have created this guide to provide a better understanding of dilutions and should be used as a guideline, not a replacement for laboratory procedures.
Types of Dilutions
Log Dilutions
A log dilution is a tenfold dilution, meaning the concentration is decreased by a multiple of ten. To complete a tenfold dilution, the ratio must be 1:10. The 1 represents the amount of sample added. The 10 represents the total size of the final sample.
For example, a sample size of 1 ml is added to 9 ml of diluent to equal a total of 10 ml. Example: 1:10 dilution – if the concentration is 1,000 CFU, a one log dilution will drop the concentration to 100 CFU.
Multiple Dilutions
Multiple dilutions are required to decrease the sample concentration by multiple logs. If the concentration is 35,000 CFU/ml (104), and 35 CFU/ml is the target concentration, the following serial dilutions can be performed.
Serial Dilution
A serial dilution is the stepwise dilution of a substance in a solution. Usually, the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion.
Purpose of Serial Dilution
Like I mention above A serial dilution is a series of sequential dilutions used to reduce a dense culture of cells to a more usable concentration. Each dilution will reduce the concentration of bacteria by a specific amount.
Requirements
- Sterile Normal Saline
- Desired Strain (Bacterial culture)
- Multiple tubes with a screw cap
- MicroPipette
- Agar (Tryptic Soy Agar, Selective media)
- Broth (Tryptic Soy Broth)
Precautions
- Clean and sterilize your work area.
- Use either disposable inoculation loop or a metal loop that can be heat sterilized to inoculate plates, slants and liquid tubes.
- If using a metal loop, be sure to cool the loop by touching the sterile cooled liquid media.
Procedure
- Make dilution in the 1st tube by taking 2ml normal saline in a tube and inoculate the desired culture in it.
- Label 10 tubes and plates as 1,2,3……..,10.
- Add 9 ml in each test tube.
- After this, transfer 1 ml (known volume) of the culture from the previously made dilution into the 1st tube having 9ml normal saline.
- From 1st tube transfer 1ml (known volume) in 2nd test tube and repeat steps till 10th test tube.
- Discard 1ml from the 10th test tube.
- After making dilutions, pour 100ul with a pipette from 1st test tube into the respective plate.
- Repeat this procedure until the 10th plate.
- After following these steps, pour media TSA or desired media into the plates and let them solidify.
- Incubate at 35° ±2 in case of bacterial culture and for fungus incubate at 23°±2.
Results
Observe after 24 hours.
Calculations
- Dilutions are useful in science when making solutions or growing an acceptable number of bacterial colonies to count. There are three formulas used to work microbiology dilution problems: finding individual dilutions, finding serial dilutions, and finding the number of organisms in the original sample.
- To find a dilution of a single tube, use the formula: sample/(diluent + sample). The sample is the amount you are transferring into the tube, and the diluent is the liquid already in the tube. When you transfer 1 ml into 9 ml, the formula would be 1/(1+9) = 1/10. This could also be written as 1:10.
After you have calculated the individual dilutions for each tube, multiply the dilutions when using serial dilutions. Serial dilutions are the culmination of a number of diluted tubes used in order to get smaller dilutions. When a sample diluted 1/100 is added to a sample diluted 1/10, the final dilution would be: (1/100) x (1/10) = 1/1000.
Example of Calculation
Let’s think through a practice dilution: You will make several dilutions of a bacterial stock culture. For some dilutions, you will add 10µl of the more concentrated solution to 990µl of sterile diluent in a microfuge tube. For others, you will add 100µl of the more concentrated solution to 900µl of sterile diluent. Following is a graphic representation of these dilutions:
How did we get to those dilution values?
Here is an example:
10µl of sample put into 990µl of diluent gives:
10µl divided by (990 + 10) µl total volume = 10/1000 = 1/100 = 10-2
You plate (put subsamples onto nutrient agar) the following dilutions:
(A) 10µl of the 10-3 dilution
(B) 100µl of the 10-5 dilution
(C) 100µl of the 10-6 dilution
(D) 100µl of the 10-7 dilution
You incubate the plates for 24 hours, after which you obtain the following results:
Plate Colonies on
Plate A too many to count
B) 685
C) 52
D) 4