This protocol is about how to properly do a serial dilution in the context of calculating the concentration of a bacterial culture (ex. C. difficile spore stock).
Serial dilution refers to the idea of diluting a solution in a stepwise manner. By plating a portion of each dilution on a plate and performing colony enumeration, we can calculate the concentration of the bacterial stock based on the number of colonies on the plate at each dilution.
- 1X Phosphate Buffer Saline (PBS)
- TCCFA plates (or appropriate media)
- Pipettes and sterile tips
- Microcentrifuge tubes or 96-well plates
- Bring supplies into the anaerobic chamber a day ahead to remove the oxygen in the supplies.
- Spray the bench surface and gloves with isopropanol to sanitize.
- Add 900 uL of 1X PBS in 7 microcentrifuge tubes and label in tubes or 90 uL in a 96-well plate with 1e3, 1e4. 1e5, 1e6, 1e7, 1e8, 1e9, 1e10
- Transfer 100 uL of the stock culture into the tube (or 10 uL into 96 well plate) labeled as 10-3 and mix by pipetting up and down. Discard the tip. A 10 fold dilution is performed here.
- Transfer 100 uL (or 10 uL) from 1e3 to 1e4 with a new tip and mix by pipetting. This is another 10 fold dilution.
- Repeat serial dilution across the entire range changing tips each time.
- After all the dilutions are done, transfer 10 uL three times to have three technical replicates from each dilution and plate on the corresponding section on the plate (Figure 2). Only dilutions in 10-4 to 10-9 tubes will be plated. For each dilution, there should be three “dots” on the section of that plate. Please space out the three “dots” carefully to prevent colonies from growing onto each other.
- Make sure to dry the plates entirely to avoid each dot running and growing into the other. Flip the plate before incubation.
- Incubate the plate according to the appropriate growth condition of the plates and observe and enumerate colonies on the plates after the colonies are formed.
Figure 1. The pictorial illustration of serial dilution in a 96-well plate.
Figure 2. The pictorial illustration of how each solution is plated. 10 uL from each dilution is plated. The plating pattern on the right is recommended for doing smaller scale drop plates with single pipettors and the pattern on the right is recommended for doing drop plates for larger scales, for example, a full plate of dilutions and this can be down by using a multi-channel pipettor.
- It is easier to count the colonies pre-maturely since big colonies might grow into another one and obscure the counting.
- Carefully count the number of colonies in each section of the plate. A valid count for each dot should be within 3-30 colonies.
- Average the colony count for three technical replicates of all dilutions:
An example is provided below:
| Dilution | Colony Count 1 | Colony Count 2 | Colony Count 3 |
|---|---|---|---|
| 10-6 | TMTC | TMTC | TMTC |
| 10-7 | TMTC | TMTC | TMTC |
| 10-8 | 26 | 20 | 28 |
| 10-9 | 5 | 4 | TFTC |
**Average CFU/mL = ((26 ×108)＋(20 ×108)＋(28 × 108)＋(5 × 109)＋(4 × 109)) ÷ 5 = 3 × 109 CFU/mL