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How to quantify bacterial cultures - From CFU and OD to counting chamber

Lab Academy


Many standard procedures and assays rely on scientists first accurately counting the number or determine the density of bacterial cells. Cell counting is crucial for keeping track of cell growth, evaluating transformation and selection resistance, seeding cells for further studies, and preparing for cell-based assays. It is important that bacterial cell counts are accurate and consistent, particularly for downstream quantitative measurements.
In this article, we will outline each method for manually counting bacterial cells and go over some of their benefits and drawbacks. Whether you’re producing batch cultures for protein expression, preparing for a transformation experiment, or measuring quantitative cellular responses to different stimuli, you will almost certainly come across the need for at least one of these cell counting techniques. Through this article, you can find the method most suitable for your work.

Colony Forming Units (CFU)

CFU provides a direct measurement of viable bacterial cells, referring to the number of individual colonies of any microorganism that can grow on a plate of media. The standard unit of measure for CFU is the number of culturable microorganisms present per 1 mL of culture (CFU / mL), determined by serial dilution and spread plating techniques as depicted in Figure 1.
Quantification of bacterial cultures – colony forming units (CFU)
General method:
  • Dilute the batch culture through serial dilutions of 1:10 by transferring 1 mL of the previous dilution into the subsequent tube containing 9 mL of sterile, distilled water
  • Pipette and spread 100 µL onto agar plates of the correct growth medium using a cell spreader
  • Label plates with the Dilution Factor (DF) and incubate overnight (or as necessary). (DF = total volume of dilution/sample volume)
  • Choose a plate with a reasonable number of colonies (between 30 and 300 colonies)
  • Time to do some cell counting! Count how many colonies you can see on each plate.
  • Once you have the CFU for each plate, perform the following calculations:
Volume plated (mL) x DF of the plate = Total Dilution FactorNumber of colonies counted (CFU) / Total Dilution Factor = Total CFU / mL

For example, if you counted 150 colonies on the plate with the dilution factor of 1:100. You plated 100 µL onto that plate:

Total DF = 0.1 mL x 1/100 (or 0.01) = 0.001
Total CFU = 150 / 0.001 = 150,000 CFU / mL

Advantage: Simple method that doesn’t require specialist equipment.

Disadvantage: It is very slow due to the time required for colony formation.
Optical Density (OD) measurement
Optical density is determined by a spectrophotometer and provides a quick estimate of the number of cells present. This technique is widely used to estimate measurements of microbial growth, and is a valuable method for monitoring cell growth during fermentation. Cell growth isn’t linear and instead has different phases of growth that reflect the cells condition and environment. During batch culture, a typical bacterial growth curve shows four distinct phases of growth:

  • lag phase, the delay before the start of exponential growth;
  • exponential or log phase, where cells divide at a constant rate;
  • stationary phase, when conditions become unfavourable for growth and bacteria stop replicating;
  • death phase, when cells lose viability.

Measuring the OD of your sample is the easiest way to determine which phase of growth your culture is in.
The sample is placed in a transparent cuvette or microtiter plate and light scattering, or turbidity, is measured in relation to a media blank. The amount of light scattered by a culture sample will depend on the concentration of cells, the size of cell, and the configuration of the spectrophotometer. Therefore, to accurately estimate cell concentration from observed OD values, a spectrophotometer must be calibrated separately for each strain. This can be done by measuring the OD of several dilutions of a cell suspension and then measuring the CFU of those serial dilutions (as described above) to produce a standard curve showing the relationship between culture density and OD as shown in Figure 2.
Bacterial cell counting – Optical density (OD) measurement
General method:

  • Produce a standard curve of OD plotted against cell count (CFU / mL):
a. Make a cell suspension of your bacterial strain.
b. Set your spectrophotometer to the correct wavelength, usually OD600 for bacteria.
c. Zero your spectrophotometer with appropriate media by pipetting the diluent you use later for your bacteria suspension into the appropriate cuvette or microtiter plate and recording a blank measurement at OD600
d. Make several dilutions of your bacterial strain and use a spectrophotometer to record their OD600 values. Be sure to use ice and be quick to stop bacterial growth!
e. Use the CFU method above to count the cells in each dilution and make a standard curve against the OD measurements by adding a linear trendline

Advantages: Once a standard curve is established, this technique is extremely fast, inexpensive, simple, relatively non-disruptive, high-throughput, and readily automated.
Disadvantage: Since the spectrophotometer is measuring light absorbance, it does not provide a direct measure of cell count. A calibration protocol is therefore needed to relate OD measurements to cell count.

Direct microscopic count

The hemocytometer is a counting-chamber instrument that was initially created to manually count blood cells1. Nowadays, hemocytometers are used for a variety of cell types and in different applications to count the total number of cells of many distinct cell types, including bacterial cells. This technique involves directly counting the number of cells in a given volume of liquid culture in various microscope fields.There are several types of hemocytometer, which all have different cell counting grids. The most well-known grid is the ‘Improved Neubauer’ chamber that has a 3×3 mm counting grid subdivided into nine 1 mm2 squares. Each square is then further divided into 16, 100, or 400 smaller squares to produce different grids for counting cells of different sizes (Figure 3). The chamber’s edges are designed to hold a special glass coverslip with a defined distance above the marked grid, thereby creating demarcated areas of known volume. The standard ‘all purpose’ depth is 0.1 mm. This is well suited for mammalian cells but it can be more difficult to focus on smaller cells like bacteria.
Bacterial cell counting – Neubauer chamber
For bacterial cells, an alternative countingchamber known as the ‘Petroff-Hausser chamber’ is available with a smaller depth of 0.02 mm. It has the grid of the Improved Neubauer chamber. Additionally, the Petroff-Hausser chamber has a 1.5 mm thick glass slide, allowing for the use of dark field microscopy.
General method:
  • Dilute the sample (as necessary) to achieve a countable cell concentration with a hemocytometer
  • Clean the hemocytometer grid with 70% ethanol and lens paper and gently place the coverslip on the counting chamber
  • Make sure your sample is representative by pipetting up and down a few times then pipette a small volume (e.g., 10 µl) of cell suspension near the edge of the grid chamber to allow the cells to enter by capillary action. Wait 1-2 min for the cells to settle
  • Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective and count the cells with a 40x objective in the central 1mm2 square as shown in Figure 3 For accurate count, total count should be ≥ 100 cells. For each smaller square (25 subdivided squares), the goal should be to have ≥ 3 cells.
  • Work out the concentration (cells / mL)
    Concentration = Number of cells x Chamber-Specific Multiplication Factor x Dilution Factor / Number of squares used
    For example, if you diluted your sample by 1:10 and counted 500 cells in the central square (1 mm2) using the Petroff chamber (0.02 mm depth):
    500 x 50,000* x 0.1 /1 = 100,000 CFU / mL
    *The Petroff chamber has a depth of 0.02 mm and covers a 1 mm2 area, making a volume of one square 0.02 mm3. To convert the count from cells per 0.02 mm3 to cells per ml, we must do the following calculation: 1,000/0.02 = 50,000

Advantages: Cheap, relatively simple and only uses a small amount of sample

Disadvantages: Time-consuming and tedious with a lot of scope for human error
The method you choose will likely depend on the context of your experiment. Are you checking the number of bacteria present in a water sample? Then, CFU is the method for you. Are you producing a batch culture of E. coli cells? Using OD measurements, you can track the growth phase of your culture. Or do you need something precise for downstream analysis? In this case, you may need to take the time to sit down and count cells using a Neubauer chamber. No matter what method you choose when counting cells, consistency is key! Make sure to plan your dilutions, pipette carefully, and be meticulous about what you count as a cell.
1. Vembadi, A., Menachery, A. and Qasaimeh, M.A. (2019) ‘Cell Cytometry: Review and Perspective on Biotechnological Advances’, Frontiers in Bioengineering and Biotechnology, 7, p. 147. Available at: https://doi.org/10.3389/fbioe.2019.00147

2. Stoddart, M.J. (2011) ‘Cell Viability Assays: Introduction’, in M.J. Stoddart (ed.) Mammalian Cell Viability: Methods and Protocols. Totowa, NJ: Humana Press (Methods in Molecular Biology), pp. 1–6. Available at: https://doi.org/10.1007/978-1-61779-108-6_1.