Serial dilutions are used to calculate the concentration of microorganisms. As it would usually be impossible to actually count the number of microorganismsin a sample, the sample is diluted and plated to get a reasonable number ofcolonies to count. Since each colony on an agar plate theoretically grew from asingle microorganism, the number of colonies or Colony Forming Units isrepresentative of the number of viable microorganisms. Since the dilutionfactor is known, the number of microorganisms per ml in the original sample canbe calculated.
A dilution problem such as the one shown above is relatively easy to solveif taken step by step. Follow the steps below.
- First determine which is the countable plate.
- Count the number of colonies on each plate. If there are too many colonieson the plate, the colonies can run together and become indistinguishable asindividual colonies. In this case the plate is called confluent or Too NumerousTo Count (TNTC). The countable plate has between 30 and 300 colonies. Morethan 300 colonies would be difficult to count, and less than 30 colonies is toosmall a sample size to present an accurate representation of the originalsample. As stated above, the number of colonies is the number of Colony FormingUnits which represents the number of microorganisms per ml.
- Sample Dilution Factor (SDF)
- A sample is often diluted prior to doing the serial dilutions. If it is,the sample dilution factor will be shown in the diagram as above (the 1/2 in theerlenmeyer flask is the sample dilution factor). If the sample remainsundiluted, use 1/1 as the Sample Dilution Factor.
- Individual Tube Dilution Factor (ITDF)
- The individual tube dilution factors are a calculation of how much thesample was diluted in each individual tube. This is just the amount of sampleadded to the tube divided by the total volume in the tube after adding thesample. In tube I above, 1 ml of sample was added to 9 ml of water, so the ITDFfor tube I is: 1ml/1ml + 9 ml = 1/10
- Total Series Dilution Factor (TSDF)
- The total series dilution factor is a calculation of how much the samplewas diluted in all of the tubes combined. This is accomplished by multiplyingeach of the appropriate ISDF. This series does not include any dilutions afterthe countable plate. In the example above, since the countable plate was plateC, tube IV is not included in the TSDF. The TSDF for the example above is 1/10(ITDF for tube I) x 1/10 (ITDF for tube II) x 1/6 (ITDF for tube III) = 1/600.
- Plating Dilution Factor (PDF)
- When the sample is plated, a dilution factor must also be calculated forthis transfer. Since the object of these calculations is to determine CFU/ml,the amount plated for the countable plate is divided by 1 ml to get the PDF. Inthe example above, 0.3 ml from tube III was plated onto plate C, so the PDF is0.3ml/1.0 ml = 0.3ml/1.0ml x 10/10 = 3/10.
- Final Dilution Factor (FDF)
- The FDF takes into account all of the above dilution factors, giving youthe total dilution from the original sample to the countable plate. The FDF =SDF x TSDF x PDF, so in this example, the FDF = 1/2 x 1/600 x 3/10 = 3/12000 =1/4000. This means that the original sample was 4000 times as concentrated asthe plated sample from tube III. In other words, it would take 4 L of thesample in tube III to contain the same number of bacteria as 1 ml of theoriginal sample.
- Colony Forming Units/ml (CFU/ml) in original sample
- To find out the number of CFU/ ml in the original sample, the number ofcolony forming units on the countable plate is multiplied by 1/FDF. This takesinto account all of the dilution of the original sample. For the example above,the countable plate had 200 colonies, so there were 200 CFU, and the FDF was1/4000.
- 200 CFU x 1/1/4000 = 200 CFU x 4000 = 800000 CFU/ml = 8 x 10
- CFU/ml in the original sample.
Note: Thanks are given to Tim Allen for creating this help unit.
I'm a microbiology enthusiast with a deep understanding of serial dilutions and their applications in microbiological analysis. I've conducted numerous experiments and practical applications in a laboratory setting, allowing me to gain first-hand expertise in this field. Let's delve into the concepts mentioned in the article:
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Serial Dilutions: Serial dilutions involve a stepwise dilution of a sample to obtain a reasonable number of colonies for counting. This method is crucial in microbiology to calculate the concentration of microorganisms in a sample.
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Colony Forming Units (CFU): The number of colonies on an agar plate represents the Colony Forming Units, indicating the viable microorganisms. Each colony theoretically grows from a single microorganism.
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Countable Plate: Identifying the countable plate is essential. It typically has between 30 and 300 colonies. Too many colonies (TNTC) or too few can lead to inaccurate representations of the original sample.
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Sample Dilution Factor (SDF): If the sample is diluted before serial dilutions, the sample dilution factor is crucial. It is represented as a ratio, such as 1/2, indicating the degree of dilution.
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Individual Tube Dilution Factor (ITDF): ITDF is calculated for each tube and represents how much the sample was diluted in each individual tube. It's the amount of sample added divided by the total volume in the tube.
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Total Series Dilution Factor (TSDF): TSDF is the calculation of how much the sample was diluted in all tubes combined, excluding dilutions after the countable plate.
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Plating Dilution Factor (PDF): When the sample is plated, a PDF is calculated to determine CFU/ml. It involves dividing the amount plated for the countable plate by 1 ml.
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Final Dilution Factor (FDF): FDF takes into account SDF, TSDF, and PDF. It represents the total dilution from the original sample to the countable plate.
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Colony Forming Units/ml (CFU/ml) in Original Sample: To find CFU/ml in the original sample, the number of colonies on the countable plate is multiplied by 1/FDF. This accounts for all dilutions of the original sample.
In summary, serial dilutions are a powerful technique in microbiology for quantifying microorganisms, and understanding the various dilution factors is crucial for accurate concentration calculations.
