Simply described, serial dilution is the gradual dilution of a solution having an associated dilution factor.

• Serial dilution in biology is frequently used to reduce the number of cells in a culture in order to make the process simpler.
• Like the title indicates, a sequence of consecutive dilutions may be used to transform a thick solution into a concentration that is more usable.

Objectives of Serial Dilution

• The serial dilution approach seeks to determine the concentration (number of organisms, bacteria, viruses, or colonies) of an unknown sample by calculating the number of colonies that have been produced from subsequent dilutions of the sample.
• To make it simpler to determine the concentration of the cells in the original solution by determining the overall series’ total dilution, serial dilution reduces the density of cells at each phase.
• Serial dilutions are frequently used to dilute a solution instead of pipetting extremely small volumes (1–10 l).
• By using dilution to generate incubated culture plates containing a countable number of colonies, it is possible to calculate the quantity of microorganisms contained in a sample (between 30 and 100).

Serial Dilution Formula/Calculations

• A consistent quantity of sterile diluent, such as distilled water or 0.9 percent saline, is used to dilute a sample during the serial dilution procedure.
• Later, employing a modest calculated amount of each dilution, a sequence of pour or spread plates are made.
• Based on the anticipated quantity of cells or other organisms present in a sample, the degree of dilution is determined. For example, if a water sample is taken from a location with significant levels of contamination, the dilution factor is increased. On the other hand, a sample that is less contaminated may only need a little dilution factor.
• Sequential two-fold as well as ten-fold dilutions are frequently used in the lab to titer antibodies or manufacture diluted analytes.
• When doing a serial dilution, the dilution factor for either each test tube or the total dilution factor for the whole series may be approximated.
• Each tube in a set’s dilution factor is as follows:

A ten-fold dilution is produced by combining 1 ml of sample with 9 ml of diluent. The test tube’s dilution factor in this case will be:

• Every tube following the first dilutes the one before it. 

The overall dilution factor is now.

• The total dilution factor for the second tube is equal to the first tube’s dilution of the second tube’s dilution.

Example

For the first tube, dilution factor = 10-1 (1 ml added to 9 ml)

For the second tube, dilution factor = 10-1 (1ml added to 9 ml)

Total dilution factor = previous dilution × dilution of next tube

= total dilution of 10-1 × 10-1 = 10-2

Online serial dilution calculator

AAT Bioquest, Inc. (https://www.aatbio.com/tools/serial-dilution)

Merck (https://www.sigmaaldrich.com/chemistry/stockroom-reagents/learning-center/technical-library/solution-dilution-calculator.html)

Omni Calculator (https://www.omnicalculator.com/chemistry/serial-dilution)

Endmemo (http://www.endmemo.com/bio/dilution.php)

Handymath (https://handymath.com/cgi-bin/serdil6.cgi?submit=Entry)

Tocris Bioscience (https://www.tocris.com/resources/dilution-calculator)

Physiology Web (https://www.physiologyweb.com/calculators/dilution_calculator_mass_per_volume.html)

Selleck Chemicals (https://www.selleckchem.com/dilutioncalculator.jsp)

ApexBio Technology (http://www.apexbt.com/dilution-calculator)

CiteAb (https://www.citeab.com/toolbox/dilutions)

Fluffy Frog (http://fluffyfrog.net/calc/)

Functional Biosciences (https://functionalbio.com/web/calc.php)

CUSABIO (https://www.cusabio.com/m-298.html)

Procedure of Serial Dilution

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

1. The sample/culture is taken in a test tube and six test tubes, each with 9 mL of sterile diluent, which can either be distilled water or 0.9% saline, are taken.
2. A sterile pipette is taken.
3. 1 ml of properly mixed sample/culture is drawn into the pipette.
4. The sample is then added to the first tube to make a total volume of 10 ml. This provides an initial dilution of 10-1.
5. The dilution is thoroughly mixed by emptying and filling the pipette several times.
6. The pipette tip is discarded, and a new pipette tip is attached to the pipette.
7. Now, 1 ml of mixture is taken from the 10-1 dilution and emptied into the second tube. The second tube now has a total dilution factor of 10.2.
8. The same process is then repeated for the remaining tube, taking 1 ml from the previous tube and adding it to the next 9 ml of diluents.
9. As six tubes are used, the final dilution for the bacteria/cells will be 10-6 (1 in 1,000,000).

Serial Dilution Applications/Uses

Many experimental fields, including biochemistry, pharmacology, physics, as well as homoeopathy, use serial dilution.

1. To measure the concentration or amount of cells or organisms in a sample, serial dilution is performed in microbiology to create an incubated plate with an easily quantifiable number of colonies.
2. In biochemistry, serial dilution is used to dilute substances and reagents from a higher concentration to the required concentration.
3. Serial dilution is used in pharmaceutical labs because it is more efficient than individual dilutions for acquiring the precise amounts of substances and chemicals.
4. In homoeopathy, a chemical is diluted in distilled water or alcohol before being employed as a homoeopathic dilution. Diluting a material is considered to increase its potency through igniting its life energy.

Serial Dilution Limitation/Problems

Although serial dilution is a helpful method in labs, there are certain difficulties with it. among them are:

• It’s possible for a sample propagation error to occur, which causes transfer faults that make the transfer less precise and accurate. This causes the highest dilution to have the greatest number of errors and the lowest degree of precision.
• The method’s efficiency is constrained by the fact that serial dilution must be carried out in a stepwise fashion over a longer length of time.
• Serial dilution simply permits the decrease of bacteria or cells; unlike other methods like flow cytometry, it does not permit the separation of bacteria or cells. 
• Aseptic technique specialists and highly educated microbiologists are also needed for this procedure.

Serial Dilution Examples

• Tea or coffee are two common examples of serial dilution that occur in everyday life. To get the appropriate coffee concentration in coffee, we add some cold-pressed coffee and then pour water over it.
• The dilution of acids and bases in chemistry to achieve the desired concentration is another instance of serial dilution.
• The serial dilution of culture to count the quantity of bacteria in a sample using a plating technique is an important example of serial dilution.

References

Basic Practical Microbiology-Manual. The society of General Microbiology. Retrieved from https://microbiologyonline.org/file/7926d7789d8a2f7b2075109f68c3175e.pdf

Avishai Ben-David, Charles E. Davidson, Estimation method for serial dilution experiments, Journal of Microbiological Methods, Volume 107, 2014, Pages 214-221, ISSN 0167-7012,

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

https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_I/04%3A_Dilution_Worksheet_and_Problems

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