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Serial Dilution Method of Counting Bacteria

    What is a Serial Dilution?

    What is serial dilution definition? serial dilution is a sequence of repeated dilutions used to produce a less dense or concentrated solution from a dense or concentrated one. Serial dilution is a laboratory technique that involves performing a stepwise dilution process on a solution with an associated dilution factor. 

    This procedure is used in the laboratory to reduce the number of cells in a culture to make the process easier. The cell count or density gradually falls as the serial number increases in each step of serial dilution. Calculating the overall dilution over the entire series makes it easier to calculate the cell numbers in the primary solution.

    Objectives of Serial Dilution

    The main goal of the serial dilution in microbiology is to determine the concentration or cell counts of an anonymous sample by counting the number of colonies cultivated from the sample’s serial dilutions.

    The serial dilution technique in microbiology is intended to estimate the concentration (number of organisms, bacteria, viruses, or colonies) of an unknown sample by counting the number of colonies cultured from serial dilutions of the sample.

    It was also used to avoid pipetting very small amounts (1-10 µl) of a solution to dilute it. We can readily count the amount of cells present in the sample since the incubated plates from the serial dilution yield an easily countable number of colonies.

    Serial Dilution Calculations

    The selected sample is diluted through a series of standard quantities of sterile diluents, such as distilled water or 0.9 percent saline, in serial dilution in microbiology. Following that, a small portion of each dilution’s sample is used to make a series of pour or spread plates.

    Serial dilution technique in microbiology based on the concentration cells contained within the unknown sample, the dilution factor or the number of repeated dilutions is raised. If a sample is taken from a heavily polluted source, for example, the dilution factor will be raised. If the sample source is less polluted, on the other hand, a low dilution factor should suffice.

    In laboratories two-fold and ten-fold dilution is used to titer antibodies or prepares diluted analytes. In serial dilution in microbiology, the dilution factor can be calculated either for a single test tube or for the entire series (total dilution factor).

    The dilution factor of single tubes within a series; In the case of ten-fold dilution, where 1ml of sample is transferred to 9 ml of diluent, the dilution factor for that test tube will be:

    Now calculate the total dilution factor;

    Total dilution factor for the second tube = dilution of first tube × dilution of the second tube


    If the dilution factor of the first tube, r = 10-1 (1 ml added to 9 ml) and the dilution factor of the second tube= 10-1 (1ml added to 9 ml), then,

    Total dilution factor = previous dilution × dilution of next tube = total dilution of 10-1 × 10-1 = 10-2

    Serial Dilution Method Procedure

    Serial Dilution Method of Counting Bacteria

    1. The serial dilution technique in microbiology for ten-fold dilution of a sample to a dilution factor of 10-6 is as follows.

    2. Take 7 clean and sterile test tubes. The sample/culture is placed in a test tube, and six test tubes are filled with 9 mL of sterile diluents, which can be distilled water or 0.9 percent saline.

    3. Take a clean pipette. 1ml of sample was drawn into the sterile pipette. The sample must be thoroughly mixed; if necessary, vortex equipment should be used.

    4. Then transfer this 1ml sample within the first test tube to make the total volume of 10 ml. It provides an initial dilution of 10-1. Make sure during the transfer, the tip of pipette doesn’t touch the wall of test tube or no amount of sample remains at the tube wall.

    5. Mix the sample properly with the diluent by shaking the tube. Now discard the pipette tip and add a new pipette tip to the pipette.

    6. Transfer 1 ml of mixture sample from the 10-1 dilution to the second tube by using pipette. The 2nd tube now has a total dilution factor of 10-2.

    7. Repeat step 8 for the remaining tubes, transfer 1 ml from the previous tube to the next 9 ml diluents. The dilution for the bacteria/cells in the last test tube will be 10-6 (1 in 1,000,000).

    Applications of Serial Dilution

    1. Serial dilution technique in microbiology is used in a microbiology laboratory to determine the density or counts of cells/organisms in an unknown sample in order to produce an incubated plate with a countable number of colonies.

    2. Serial dilution method in microbiology also used to get the desired concentration of reagents and chemicals from a higher density in biochemistry lab.

    3. Serial dilution is used in pharmaceutical laboratories to obtain the required concentration of chemicals and compounds because it is more effective than individual dilutions.

    4. Homeopathic dilutions are used in homoeopathy when a substance is diluted in distilled water or alcohol. It is assumed that dilution enhances the strength of the diluted substance by stimulating its vital energy.

    Limitations of Serial Dilution

    1. An error may occur during the sample’s propagation, and transfer inaccuracies result in less accurate and precise transfer. As a result, the highest dilution has the most inaccuracies and the lowest accuracy.

    2. Because serial dilution is done step by step, it takes a longer time, limiting the method’s efficiency.

    3. Serial dilution in microbiology only allows for the reduction of bacteria/cells, not their separation as in other techniques such as flow cytometry.

    4. Serial dilution in microbiology also necessitates highly trained microbiologists and aseptic technique experts.

    Examples of Serial Dilution

    1. In chemistry, example of serial dilution method is the dilution of acids and bases to achieve the desired concentration.

    2. Serial dilution method in microbiology of culture to determine the number of bacteria in a given sample via plating is another important example of serial dilution.

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