Serial 1 10 Dilutions

Dilutions A dilution is a common laboratory technique used to obtain the desired concentration. A dilution will always reduce the concentration of the sample.

V1C1 V2C2 Serial Dilutions Making Solutions For example, to make a dilution of a 1M NaCl solution, you would mix.

Serial Dilutions MLAB 1335 Immunology/Serology Terry Kotrla Introduction Many of the laboratory procedures involve the use of dilutions. It is important to understand.

Each tube contains 9 ml of media. To complete the Serial Dilution, Tube 1 will be inoculated with 1 ml of culture to give a dilution.

The dilution factor DF can be used alone or as the denominator of the fraction, for example a DF of 10 means a dilution, Serial Dilutions.

There are many ways of expressing concentrations and dilution. The following is a brief explanation of some ways of calculating dilutions that are common in biological science and often used at Quansys Biosciences.

To make a fixed amount of a dilute solution from a stock solution, you can use the formula: C1V1 C2V2 where:

V1 Volume of stock solution needed to make the new solution

C1 Concentration of stock solution

V2 Final volume of new solution

C2 Final concentration of new solution

Example: Make 5 mL of a 0.25 M solution from a 1 M solution.

Plug values in: V1 1 M 5 mL 0.25 M

Rearrange: V1 5 mL 0.25 M / 1 M V1 1.25 mL

Answer: Place 1.25 mL of the 1 M solution into V1-V2 5 mL – 1.25 mL 3.75 mL of diluent

To make a dilute solution without calculating concentrations, you can rely on a derivation of the above formula:

Final Volume / Solute Volume Dilution Factor can also be used with mass

This way of expressing a dilution as a ratio of the parts of solute to the total number or parts is common in biology. The dilution factor DF can be used alone or as the denominator of the fraction, for example a DF of 10 means a dilution, or 1 part solute 9 parts diluent, for a total of 10 parts. This is different than a dilution ratio, which typically refers to a ratio of the parts of solute to the parts of solvent, for example a 1:9 using the previous example. Dilution factors are related to dilution ratios in that the DF equals the parts of solvent 1 part.

Example: Make 300 uL of a 0 dilution

Formula: Final Volume / Solute Volume DF

Plug values in: 300 uL / Solute Volume 250

Rearrange: Solute Volume 300 uL / 250 1.2 uL

Answer: Place 1.2 uL of the stock solution into 300 uL – 1.2 uL 298.8 uL diluent.

If the dilution factor is larger than the final volume needed, or the amount of stock is too small to be pipetted, one or more intermediary dilutions may be required. Use the formula: Final DF DF1 DF2 DF3 etc., to choose your step dilutions such that their product is the final dilution.

Example: Make only 300 uL of a 00 dilution, assuming the smallest volume you can pipette is 2 uL

Choose step DFs: Need a total dilution factor of 1000. Let s do a followed by a 0 10 100 1000

Plug values in: 300 uL / Solute Volume 10

Rearrange: Solute Volume 300 uL / 10 30 uL.

Answer: Perform a dilution that makes at least 30 uL e.g. 4 uL solute into 36 uL diluent, then move 30 uL of the mixed into 300 uL – 3 uL 297 uL diluent to perform the 0 dilution.

A dilution series is a succession of step dilutions, each with the same dilution factor, where the diluted material of the previous step is used to make the subsequent dilution. This is how standard curves for ELISA can be made. To make a dilution series, use the following formulas:

Move Volume Final Volume / DF -1

Diluent Volume Final Volume – Move Volume

Total Mixing Volume Diluent Volume Move Volume

Example 1: Make a 7-point 1:3 standard curve, starting Neat, such that you can pipette duplicates of 50 uL per well.

Calculate the minimum diluent volume per step: 50 uL per well 2 for duplicates 100 uL minimum. Add extra volume to compensate for pipetting error, for example, 20 uL, which brings our desired Diluent Volume to 120 uL.

Calculate Move Volume: Move Volume 120 uL / 3-1 60 uL

Calculate Total Mixing Volume: Total Mixing Volume 120 uL 60 uL 180 uL

Prepare the first point of the standard curve, which is 180 uL of Neat standard.

Prepare the diluent for the rest of the points, or six aliquots of 120 uL of diluent.

Move 60 uL of the first point into the second and mix thoroughly, move 60uL of that into the next, and so on.

Example 2: Make a 7-point 1:2 standard curve, starting at a 1:5, such that you can pipette duplicates of 50 uL per well.

Calculate minimum diluent volume per step: 50 uL per well 2 for duplicates 100 uL minimum. Add extra volume to compensate for pipetting error, for example, 20 uL, which brings our desired Diluent Volume to 120 uL.

Calculate Move Volume: Move Volume 120 uL / 2-1 120 uL

Calculate Total Mixing volume: Total Mixing Volume 120 uL 120 uL 240 uL

Calculate first point dilution volumes: you need 240 uL of a 1:5

Prepare the first point of the standard curve, which is a 1:5, so pipette 240uL /5 48 uL solute into 192 uL diluent

Move 120uL of the first point into the second and mix thoroughly, move 60uL of that into the next, and so on.

This section is not a recipe for your experiment. It explains some principles for designing dilutions that give optimal results. Once you understand these principles.

Determine the proper dilution liquid. The liquid that you will be diluting your substance in is very important. Many solutions will be diluted in distilled water, but this is not always the case. If you are diluting bacteria or other cells, you will likely want to dilute in culture media. 2 The liquid you choose will be used for every serial dilution.

If you re unsure what liquid to use, ask for help or check online to see if other people have performed a similar dilution.

Prepare several test tubes with 9 mL of dilution liquid. These tubes will serve as your dilution blanks. 3 You will be adding your undiluted sample to the first tube and then serially diluting into the following tubes.

It s helpful to label all of your tubes before you begin so you don t get confused once you begin with the dilutions.

Each tube will be a 10-fold dilution starting from the undiluted tube. 4 The first tube will be a dilution, the second a 0, the third a 00, etc. Determine the number of dilutions you need to do beforehand so you don t waste tubes or diluting liquid.

Prepare a test tube with at least 2 mL of your undiluted solution. The minimum amount needed to perform this serial dilution is 1 mL of undiluted solution. If you only have 1 mL you will not have any remaining undiluted solution. Label this tube US for undiluted solution.

Thoroughly mix your solution before starting any dilutions. 5

Perform the first dilution. Draw 1 mL of undiluted solution from test tube US with a pipette and transfer it to the test tube labeled containing 9 mL of the dilution liquid and mix thoroughly. There is now 1mL of the undiluted solution in 9 mL of the dilution liquid. The solution, therefore, has been diluted by a factor of 10.

Perform the second dilution. For the second serial dilution, you will take 1 mL of solution from tube and add it to the 9 mL of dilution liquid in the tube 0. Thoroughly mix tube before adding to the next tube. Again, mix the tube 0 following dilution. The solution from test tube has been diluted 10-fold into test tube 0.

Extend this procedure to perform longer serial dilutions. This process may be repeated as many times as necessary to achieve the desired solution. In an experiment involving concentration curves, you can use a serial dilution to create a series of solutions with dilutions of 1, , 0, 1:1,000.

Method 2 of 2: Calculating Final Dilution Factor and Concentration

Calculate the final dilution ratio in a serial dilution. The total dilution ratio can be determined by multiplying the dilution factor of each step leading up to the final step. This can be mathematically illustrated with the equation Dt D1 x D2 x D3 x x Dn where Dt is the total dilution factor and Dn is the dilution ratio. 6

For example, let s say you did a dilution of your liquid 4 times. Plug your dilution factor into the equation: Dt 10 x 10 x 10 x 10 10,000

The final dilution factor of the fourth tube in your serial dilution is ,000. The concentration of your substance is now 10,000 times less than the original undiluted solution.

Determine the concentration of the solution following dilution. To determine the final concentration of your solution following serial dilution you will need to know your starting concentration. The equation is Cfinal Cinitial/D where Cfinal is the ending concentration of the diluted solution, Cinitial is the starting concentration of the original solution and D is the dilution ratio previously determined. 7

For example: If you started with a solution of cells with a concentration of 1,000,000 cells per mL and your dilution ratio is 1,000, what is the final concentration of your diluted sample.

Confirm that all units match. When performing any calculation, you want to make sure that your units always match at the end. 8 If you started with cells per mL make sure you are ending with cells per mL. If your starting concentration is parts per million ppm, then your final concentration must be ppm.

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