Microbiology Techniques

Serial Dilution Method Definition, Procedure, Application

THIS BLOG INCLUDES: hide 1 Definition of Serial dilution 2 Purpose of Serial dilution Technique 3 Serial dilution calculator 4 Formula/calculations for...

Serial dilution Procedure
Serial Dilution Method Definition, Procedure, Application

Definition of Serial dilution

Hey there, you might be thinking, what is serial dilution?

As the term indicates, it is a series of succeeding dilutions that performed to create a less dense or less concentrated solution from a high dense or concentrated solution.

In a single and very simple word, Serial dilution is a laboratory technique, in which a stepwise dilution process is performed on a solution with an associated dilution factor. In the laboratory, this method is used to decrease the counts of cells within a culture to simplify the operation.

In serial dilution, the cell count or density gradually decreases as the serial number increases in each step. This makes it easier to calculate the cell numbers in the primary solution by calculating the total dilution over the whole series.

Purpose of Serial dilution Technique

The main purpose of serial dilution technique is to find out the concentration or the cell counts of an anonymous sample by counting the number of colonies that are cultured from the serial dilutions of the sample.

It also used to avoid having to pipette very small volumes (1-10 µl) to make a dilution of a solution. The incubated plates from the serial dilution generate an easily countable number of colonies, hence we can easily enumerate the number of cells present within the sample.

Serial dilution calculator

Formula/calculations for serial dilution technique

In serial dilution, the selected sample is diluted through a set of standard volumes of sterile diluents, such as be distilled water or 0.9 % saline. After that, a small amount of sample from each dilution is used to prepare a series of pour or spread plates.

The extension of dilution factor or the number of serial dilution is increased based on the concentration cells present within the unknown sample. For example, if a sample is collected from a highly polluted source, then the dilution factor will be increased. In contrast, if the source of the sample is less polluted then a low dilution factor should be sufficient.

In laboratories two-fold and ten-fold dilution is used to titer antibodies or prepare diluted analytes. In serial dilution technique, 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:

Note: After the first tube, each tube is the dilution of the previous dilution tube.

Now calculate the total dilution factor;

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

Example:

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 will be = previous dilution × dilution of next tube = total dilution of 10-1 × 10-1 = 10-2

Importance/Application of Serial dilution Method

Serial dilution is a widely employed laboratory technique for experimental sciences such as pharmacology, biochemistry, homeopathy, and physics.

  1. In a microbiology laboratory, it is used to determine the density or counts of cells/organisms in an unknown sample to achieve an incubated plate with a countable number of colonies.
  2. It is also used to achieve the desire concentration of the reagents and chemicals from a higher density oi biochemistry lab.
  3. Serial dilution is also used to get the required concentration of chemicals and compounds in pharmaceutical laboratories as this technique is more efficient than individual dilutions.
  4. In homeopathy, homeopathic dilutions are employed where a substance is diluted within distilled water or alcohol. It is assumed that dilution enhances the strength of the diluted substance by stimulating its vital energy.

Serial dilution Procedure

The following is the procedure for a ten-fold dilution of a sample to a dilution factor of 10-6:

  1. Take 7 sterile and clean test tubes.
  2. The selected sample is taken into a test tube and the remaining 6 test tubes are filled with 9 ml of sterile diluent such as distilled water or 0.9% saline.
  3. Take a sterile pipette.
  4. Drawn 1ml of sample into the sterile pipette. The sample must be properly mixed, if necessary use a vortex meter.
  5. 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.
  6. Mix the sample properly with the diluent by shaking the tube.
  7. Now discard the pipette tip and add a new pipette tip to the pipette.
  8. 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.
  9. Repeat step 8 for the remaining tubes, transfer  1 ml from the previous tube to the next 9 ml diluents.
  10. The dilution for the bacteria/cells in the last test tube will be 10-6 (1 in 1,000,000).
Serial dilution Procedure
Serial Dilution Method Definition, Procedure, Application | This image is modified from https://chromoscience.com/2020/03/14/the-serial-dilution/

Limitation of serial dilution technique

Serial dilution faces some challenges such as;

  1. A mistake might occur throughout the distribution of the sample, and the transfer errors result in a less reliable and less accurate transfer. This leads to the highest dilution having the most errors and the least efficiency.
  2. It is performed in a stepwise manner, therefore it needs a more long period of time which restricts the capability of the method.
  3. This technique only reduces the counts of bacteria/cells but not separate them like in other methods such as flow cytometry.
  4. Required trained experts to perform this technique.

Reference

  • Cullen, J. J., & MacIntyre, H. L. (2016). On the use of the serial dilution culture method to enumerate viable phytoplankton in natural communities of plankton subjected to ballast water treatment. Journal of applied phycology, 28(1), 279–298. https://doi.org/10.1007/s10811-015-0601-x
  • https://www.biomol.com/dateien/Bethyl–Serial-Dilutions.pdf
  • Basic Practical Microbiology-Manual. The society of General Microbiology. Retrieved from https://microbiologyonline.org/file/7926d7789d8a2f7b2075109f68c3175e.pdf

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Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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