To find the CFU / mL, insert the OD measurement into the standard curve equation.Īdvantages: Once a standard curve is established, this technique is extremely fast, inexpensive, simple, relatively non-disruptive, high-throughput, and readily automated.ĭisadvantage: Since the spectrophotometer is measuring light absorbance, it does not provide a direct measure of cell count.Pipette the sample solution into a cuvette or microtiter plate and use the spectrophotometer to measure absorbance at OD 600.Zero your spectrophotometer with appropriate media at OD 600.Once a standard curve has been produced then you can use it to estimate the number of cells present in your sample by using the linear regression equation y = mx + c (m=slope and c=intercept).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.Be sure to use ice and be quick to stop bacterial growth! Make several dilutions of your bacterial strain and use a spectrophotometer to record their OD 600 values.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 OD 600.Set your spectrophotometer to the correct wavelength, usually OD 600 for bacteria.Make a cell suspension of your bacterial strain.Produce a standard curve of OD plotted against cell count (CFU / mL):. 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. Therefore, to accurately estimate cell concentration from observed OD values, a spectrophotometer must be calibrated separately for each strain. 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. The sample is placed in a transparent cuvette or microtiter plate and light scattering, or turbidity, is measured in relation to a media blank. Measuring the OD of your sample is the easiest way to determine which phase of growth your culture is in. death phase, when cells lose viability.stationary phase, when conditions become unfavourable for growth and bacteria stop replicating.exponential or log phase, where cells divide at a constant rate.lag phase, the delay before the start of exponential growth.During batch culture, a typical bacterial growth curve shows four distinct phases of growth: Cell growth isn’t linear and instead has different phases of growth that reflect the cells condition and environment. This technique is widely used to estimate measurements of microbial growth, and is a valuable method for monitoring cell growth during fermentation. Optical density is determined by a spectrophotometer and provides a quick estimate of the number of cells present. Total CFU = 150 / 0.001 = 150,000 CFU / mLĪdvantage: Simple method that doesn’t require specialist equipment.ĭisadvantage: It is very slow due to the time required for colony formation. Number of colonies counted (CFU) / Total Dilution Factor = Total CFU / mLįor example, if you counted 150 colonies on the plate with the dilution factor of 1:100. Volume plated (mL) x DF of the plate = Total Dilution Factor Once you have the CFU for each plate, perform the following calculations:.Time to do some cell counting! Count how many colonies you can see on each plate.Choose a plate with a reasonable number of colonies (between 30 and 300 colonies).(DF = total volume of dilution/sample volume) Label plates with the Dilution Factor (DF) and incubate overnight (or as necessary).Pipette and spread 100 µL onto agar plates of the correct growth medium using a cell spreader.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.
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