Microbiology Techniques

Serial Dilution Method Definition, Procedure, Application

Table of Contents show 1 What is a serial dilution? – serial dilution definition 2 What is the purpose of serial dilution?...

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This article writter by MN Editors on April 22, 2021

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Serial dilution Procedure
Serial Dilution Method Definition, Procedure, Application

What is a serial dilution? – serial dilution definition

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.

What is the purpose of serial dilution?

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

Serial dilution formula – serial dilution calculation

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 method

The following is the serial dilution 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.

Advantages of Serial Dilution

  • It can help reduce the size of cells to a lower concentration that is usable.
  • A certain amount of bacteria are eliminated with each dilution.
  • The number of colonies cultured from serial dilutions of the specimen is estimated to estimate the concentration of an unidentified sample. Then backtrack the measured enumerations to the unspecified concentration.

Serial dilution examples

  • One simple example of serial dilution is that in our daily life we every day make coffee or tea. In coffee, we pour some cold-press coffee and then add water to get the desired concentration of coffee.
  • Another instance of serial dilution is diluting of bases and acids in chemistry to achieve an appropriate concentration.
  • In laboratory the dilutions of cultures are used to determine the number of bacteria in a sample by plating is another important instance for serial dilution.

What is Ten-fold serial dilution?

A ten-fold dilution decreases an amount in a suspension or viral suspension by one ratio of ten, which is equal to one-tenth of the concentration at which it was originally found. A sequence of ten-fold Dilutions is known as ten-fold serial dilutes. In this manual, ten-fold serial dilutes are employed in titrations of the suspension of Newcastle disease virus to determine the amount of infection. The tests are conducted in tiny sterile test tubes. The tubes are generally constructed of glass, and it is recommended that they have lids that are fitted to limit the risk for contamination in the dissolution.

Steps of A 10 fold serial dilution

  1. Utilize a micropipette to disperse about 900mL of the dilute into the glass tube.
  2. Make use of a micropipette to transfer 100mL of test solution into the first well. Remove the end.
  3. Mix by shaking hand or by using a vortex mixer.
  4. The test well now has 100mL of the original test solution, which has been diluted by one-tenth to create an overall quantity of 1000mL.
Steps of A ten-fold dilution
Steps of A ten-fold dilution

Steps of 10 fold serial dilution

  1. Set up the glass test tubes that have been sterilized on a rack. Label each test tube clearly to mark the amount of dilute it has after the tenfold serial dilution process is completed.
  2. Utilize a micropipette to disperse an ounce of the dilute to the sterilized tubes.
  3. Utilize a micropipette for transfer of 100mL of test solution to the test tube. Mix. This is the first tenfold diluting.
  4. Make use of a micropipette equipped with a new sterile tip for carrying out a second 10-fold diluting.
  5. Continue the ten-fold diluting until you reach the final tube.
Steps of Ten-fold serial dilutions
Steps of Ten-fold serial dilutions

What is 2 fold serial dilution?

A twofold dilution decreases the concentration of the solution by a factor of two, which decreases the original concentration by one-half. Two-fold Dilutions is known as two-fold serial dilutes. In this manual, twofold serial dilutions take place in tiny volumes on microwell plates. They are employed in both haemagglutination and haemagglutination inhibition tests in order to determine the levels of test samples.

Steps of A 2 fold serial dilution

  1. Make use of the micropipette to disperse 25mL of PBS diluting fluid to the first well.
  2. Utilize the micropipette for transfer of 25mL of test solution into the first well.
  3. Make use of the micropipette for mixing by drawing the liquid and then expelling it. Repeat this procedure twice.
  4. The test solution now contains 25 mL from the original test solution, which has been diluted by one-half in the total volume of 50 milliliters.
Steps of A two-fold dilution
Steps of A two-fold dilution

Steps of Two-fold serial dilutions

  1. Make use of the micropipette to disperse 25mL of PBS diluting solution to all wells in a row on microwell plates.
  2. Make use of the micropipette for transfer 25mL of test solution to the initial hole and blend. This is the initial two-fold diluting.
  3. Use the micropipette’s same tip to perform the second two-fold dilation.
  4. Continue the twofold diluting until the final well on the microwell plate. The final well serves as a control well during the haemagglutination as well as haemagglutination inhibition tests.
Steps of Two-fold serial dilutions
Steps of Two-fold serial dilutions

FAQ

Q1. What is Log Dilutions?

The term “log dilution” refers to a 10-fold one which means that the concentration decreases by a multiplier of 10. For a tenfold dilution to be complete the ratio should be 1:10. The one represents the quantity of sample that is added. The 10 is the amount of the finished sample. For instance an amount of 1 milliliter is then added to 9 ml of diluent , to make 10 milliliters.

Example: 1:10 dilute If the concentration is 1,000 CFU One log dilution would reduce it by 100 CFU.

Log Dilutions
Log Dilutions

Q2. Decimal Numbers vs Scientific Notation

Decimal numbers may be transformed into scientific notations by shifting the decimal number by the same amount as the exponential number.

Decimal Numbers vs Scientific Notation
Decimal Numbers vs Scientific Notation

Q3. What is Multiple Dilutions?

Multiple dilutions are necessary to reduce the concentration of the sample in multiple ways. In the case of a concentration that is greater than three times that of 35,000 CFU/ml (104) with 35 CFU/ml being the desired concentration, the subsequent serial dilutions are possible.

Multiple Dilutions
Multiple Dilutions

Q4. What is Larger Dilutions?

The reduction in concentration by using less dilutions is achievable by using large-volume diluting. This is done by using the 1:100 dilution instead 1:10

 Larger Dilutions
Larger Dilutions

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
  • https://www.fao.org/3/ac802e/ac802e0r.htm#TopOfPage
  • https://www.fao.org/3/ac802e/ac802e0q.htm#TopOfPage
  • https://www.microbiologics.com/core/media/media.nl?id=1758034&c=915960&h=fc555e1fdf60b6138d07&_xt=.pdf
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