DILUTIONS IN MICROBIOLOGY LABORATORY

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In the microbiology laboratory, the dilution of fluids, solutions, test samples, antimicrobial agents, microbial cultures or test reagents is amongst the important techniques that are routinely performed. When a test sample is insufficient in volume or too concentrated to perform a particular test, the dilution of the original sample or solution is paramount to achieve a desired or expected result. Also, when a sample contains an excessive amount of cells that makes the individual counting of the cells either microscopically or visually difficult in counting chamber or microscope, dilution technique is used to obtain a workable sample solution.

Dilution is the laboratory technique of reducing the original concentration of a sample or solution either by half or to a more practical (workable) concentration. It involves a reduction in the concentration of the original solution. To dilute a solution simply means to reduce its concentration. After dilution, the concentration of the resulting solution is usually related to that of the undiluted original solution. Working with a concentrated solution containing microbial cells for example in the microbiology laboratory can result to too many colonies of cells on a culture plate. Culture plate becomes overcrowded after incubation, and this makes counting and other visual examination to be a little bit impossible.

Dilution is therefore used in this scenario to reduce the amount of viable cells in the inoculum. For example, if a body fluid, test reagent or culture sample is too concentrated and requires a dilution, a diluent that is osmotically balanced with the sample to be diluted should be used. The rationale for using an osmotically balanced diluent for a solution (or cell) to be diluted is to ensure that the substance being diluted does not shrink or lyse but instead remains osmotically intact in the process of dilution. Below is a table showing different dilutions and their corresponding diluent parts (Table 1). Some tips in dilution are as follows:

  • To make a given volume of a fluid from a particular dilution, the following simple calculation applies: For example, to make 8 ml of a 1:20 dilution of a given solution, divide the amount to prepare (i.e. 8 ml) by the dilution given (i.e. 20). That is, 8/20 = 0.4 ml. Thus, to prepare 8 ml of a 1:20 dilution: add 0.4 ml of the solution to 7.6 ml of its corresponding diluent.  Remember: 0.4 ml + 7.6 ml = 8 ml.
  • When calculating the dilution of a given fluid, use the following formular:

For example: calculate the dilution of sample X when using 50 µl of sample X and 950 µl of diluent. Using the above formular:     50+950 / 50  = 1000 / 20 = 20

Thus, the dilution of sample X is 1:20

  • If you add 1 ml of diluent to 1 ml of a solution, the total volume becomes 2 ml (1 ml + 1 ml = 2 ml). This is called a 1:2 (1 in 2) dilution (i.e. 1 ml of solution in a total volume of 2 ml). The dilution factor here is 2.
  • If you add 27 mls of diluent to 3 mls of solution, the total volume becomes 30 mls (3 ml + 27 ml =30 ml). This shows a dilution of 3/30 or 3 in 30. Because it is impractical to work with 3 in 30, the dilution ratio of 3:30 must be changed to a practical figure by dividing both sides with 3. This gives us a 1 in 10 (1:10) dilution. The dilution factor in this case is 10.

Table 1: Dilution table

DilutionSample partsDiluent parts
1:101.09.0
1:100.54.5
1:100.10.9
1:501.049.0
1:500.524.5
1:500.14.9
1:1001.099.0
1:1000.549.5
1:1000.19.9

METHODS OF DILUTION

There are several dilution methods that are routinely employed in the microbiology laboratory to get a desired concentration of a concentrated solution.

  • Ten fold dilutions: The tenfold dilution is a decimal type of dilution (e.g. 10-1 or 1:10, 10-2 or 1:100) in which each concentration of the diluted solution is one tenth that of the original concentration. That is, each of the diluted solutions in the different tubes (say tubes 1-5) is 10 times more dilute than the concentrated stock solution from which the dilution was performed. A 10 fold dilution is usually performed by first measuring out 9 mls each of a suitable diluent into a series of test tubes (e.g. tubes 1-5). Then add 1 ml of the concentrated stock solution into the first tube containing 9 mls of the diluent. This first tube into which both the diluent (9 mls) and the concentrated solution (1 ml) were both mixed gives us a 10 fold dilution. This procedure can be repeated up to five or more times depending on the task at hand by taking 1 ml from the first diluted tube into the next tube containing 9 mls of the diluent. When performed in six (6) different tubes, the resulting dilutions will be: 1:10, 1:100, 1:1000, 1:10000, 1:1000000, and 1:1000000. It is noteworthy that 1 ml is discarded when you get to the last tube in the series.
  • Two fold dilutions: A 2 fold dilution which can also be called a doubling dilution is a type of dilution series in which the concentration of the resultant solution or mixture is half the concentration of the initial solution. Two fold serial dilutions is the laboratory technique of making a concentrated solution to be less concentrated prior to further analysis with same solution by adding equal volumes of both the diluent and concentrated solution in the same tube. As previously explained, serial means repeated while dilution means to make less concentrated. In a twofold dilution, a doubling dilution of the concentrated solution can be obtained by measuring out equal volumes of the diluent and the concentrated solution into a tube. For example, if you measure out 2 mls of a concentrated solution into a tube containing 2 mls of diluent, the resulting dilution becomes a 2 fold dilution (i.e. 1:2). If this is repeated three more times, you get a 1:4, 1:8 and 1:32 dilutions respectively. Table 2 show some common conversions in dilution.

Table 2: Some useful conversions in dilution

1 g1000 mg
1 g1 000 000 µg
0.1 g100 mg
0.01 g10 mg
1000 µg1 mg
1 liter1000 ml
1 ml1000 µl

Note: The conversion list in this table is not exhaustive but important for some of the basic dilution calculations undertaken in the microbiology laboratory.

FURTHER DILUTION CALCULATIONS

The formular for basic dilution calculations in the microbiology laboratory and other related biological disciplines is usually stipulated as: M1V1 = M2V2. This formular is very convenient to use for the majority of dilution problems. The formular implies that the concentration and volume of one particular solution (in this case the stock or concentrated solution) is equal to the concentration and volume of the other solution (in this case the diluted solution). The diluted solution is expected to have the same amount of active constituents (as obtained in the stock solution) after the dilution must have taken place. The term concentration can also mean molarity for this purpose.

M1 = Starting molarity of the highly concentrated solution. It is the concentration of the more concentrated solution (usually known as the stock solution), and its unit is designated as Mole (M).

V1 = Volume of the concentrated solution (usually known as the stock solution) that will be diluted. The unit of the volume here is ml.

M2 = Final molarity of the diluted solution, and its unit is designated as Mole (M). 

V2 = Total volume of the new solution i.e. the diluted solution. Its unit is designated as ml.

Note: It is important that you confirm that the units of the solutions (e.g. the volume) are always of the same components before proceeding with any calculations. Volume can assume any of the following units: ml, L or µl. When units are not the same, you have to convert to get them to be of the same components before proceeding with the calculations.    

Links for dilution calculation in biological sciences and other fields of sciences:

These links will help you do your dilution calculations without stress.

SOLUTION DILUTION CALCULATOR: https://www.sigmaaldrich.com/DE/de/support/calculators-and-apps/solution-dilution-calculator

SERIAL DILUTION CALCULATOR: https://www.omnicalculator.com/chemistry/serial-dilution

DILUTION CALCULATOR OF MASS CONCENTRATION: http://www.endmemo.com/bio/dilution.php


SERIAL DILUTION CALCULATOR AND PLANNER: https://www.aatbio.com/tools/serial-dilution

FURTHER READING

Ashutosh Kar (2008). Pharmaceutical Microbiology, 1st edition. New Age International Publishers: New Delhi, India. 

Block S.S (2001). Disinfection, sterilization and preservation. 5th edition. Lippincott Williams & Wilkins, Philadelphia and London.

Courvalin P, Leclercq R and Rice L.B (2010). Antibiogram. ESKA Publishing, ASM Press, Canada.

Denyer S.P., Hodges N.A and Gorman S.P (2004). Hugo & Russell’s Pharmaceutical Microbiology. 7th ed. Blackwell Publishing Company, USA. Pp.152-172.

Ejikeugwu Chika, Iroha Ifeanyichukwu, Adikwu Michael and Esimone Charles (2013). Susceptibility and Detection of Extended Spectrum β-Lactamase Enzymes from Otitis Media Pathogens. American Journal of Infectious Diseases. 9(1):24-29.

Finch R.G, Greenwood D, Norrby R and Whitley R (2002). Antibiotic and chemotherapy, 8th edition. Churchill Livingstone, London and Edinburg.

Russell A.D and Chopra I (1996). Understanding antibacterial action and resistance. 2nd edition. Ellis Horwood Publishers, New York, USA.

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