COLLEGE OF ARTS AND SCIENCES

CMBI 6617 Cell Culture Techniques with laboratory

Pipetting and Dilutions

A. Practice with equipment

It is critical for cell culture that you work comfortably and cleanly to carry out basic skills, such as
pipetting and opening tubes and bottles efficiently. Practice the following techniques until you are
comfortable enough to do them quickly and easily. When working with cells, these techniques will be
performed in a laminar flow hood, but can be practiced on your bench. Be careful not to cross
contaminate and to not touch pipettes to other surfaces.
Before getting started, you should always prepare your work area:

• wipe down work surfaces with 70% ethanol

• make sure all necessary equipment and reagents are close at hand

Equipment required:

• 1, 5, and 10 ml pipettes.

• Pipet Aid

• 1.5 ml and 15 mL tube with buffer for pipetting practice

• Tubes, bottles and flasks as provided.

• Micropipettes

• 20, 200 and 1000 uL pipet tips

Protocol:
1. Using a selection of tubes, bottles and flasks, practice removing and replacing the caps one handed.
You must be able to do this without drawing the lid across the opening. It is also important to avoid
touching anywhere near the opening, or palming the lids.

2. Starting with the 10 mL pipet, mix the contents of the tube. Mixing involves drawing the solution
into and out of the pipet 20 times in a minute. Be careful not to spill the solution, blow bubbles or
draw the fluid into the cotton plug in the pipet.
3. Repeat the process with the 1ml pipet and the 5 ml pipet.
4. Starting with the 1000 uL micropipette practice drawing up liquid. Be slow and careful to avoid
causing bubbles or retention of liquid into the shaft of the micropipette.
Pipetting and Dilutions CMBI-6617

5. Repeat the process with the 200 and 20 uL micropipettes

B. Units of scale in cell biology

Cells and their parts can be characterized by properties of length, mass, volume and concentration. A
major difficulty for students in understanding these properties is the fact that the units used are much
smaller than they are typically used to dealing with. A second problem is often confusion over the
differences between these properties. Table 1 lists the units we will most commonly need.

Table 1: Units of scale commonly used in the laboratory.

Length Mass Volume Concentration

1m 1g 1L 1M

milli 1 mm 10-3 m 1 mg 10-3 g 1 ml 10-3 l 1 mM 10-3 M

micro- 1 μm 10-6 m 1 μg 10-6 g 1 μl 10-6 l 1 μM 10-6 M

nano- 1 nm 10-9 m 1 ng 10-9 g 1 nl 10-9 l 1 nM 10-9 M

pico- 1 pg 10-12 g 1 pM 10-12 M

C. Dilutions and Stock solutions

Moles and Molarity
Moles (mol) and molarity (M) are both units. Moles is the SI unit for the number of molecules of a
chemical, while the concentration of molecules (per Liter of substance) is quantified as the molarity (M).
(M = mol/L) .
One mole of a chemical has a mass equal to its Molecular Weight (MW). (For chemicals, MW may be
listed as formula weight, FW). For example, MgCl2 has a FW of 203.3 . A one molar solution of MgCl2 (1M
MgCl2) would have = 203.3 g MgCl2 per Liter of water.

Making Solutions from Dry Chemicals

To determine how much of a compound you need to make a certain volume of that compound at a
particular Molarity, use the following equation:
M x MW x V = g

Where:
M = Final Molarity desired (mole Liter-1)
MW = Molecular weight of the compound (or FW) (g x mole-1)
V = Desired final volume (Liters)
g = grams of compound to add to solvent

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For example, if you want 500 mL of a 1M solution of MgCl2, you would make

1 mol/L x 203.3 g/mol x 0.5 L = 101.65 g

Note: Check the units! I converted mL to L for the Volume

To make the solution, you would add 101.65 grams of MgCl2 to 400 mL water; mix. If a specific
pH is desired, you would measure the pH and adjust as necessary, before bringing the total
volume up to 500 mL (or, Q.S to 500 mL).

Making solutions from a hydrated compound

Some chemicals come with water molecules attached. In that case, the FW of the compound listed on
the bottle includes the mass of the water. You can use the FW as above to determine Molarity, but you
need to remember that the attached water molecules will contribute water to the solution, potentially
diluting the final concentration. The easiest way to deal with this is just to use good basic lab practices:
start with less than the final desired volume, mix the compound, and then Q.S. to the desired final
volume.

Diluting Stock Solutions to a Particular Concentration

While we often have to work with minute quantities of compounds or suspensions the starting stocks
are often very concentrated, often more than 1000-fold higher than desired. Micropipettes allow us to
very accurately dispense volumes as small as 1 or 2 μl. Unfortunately, this is still not convenient or
accurate enough for many applications. The most reproducible method of working with stocks is to
make dilutions. There are different circumstances where a variety of dilutions are needed. The most
common form is the use of stock reagents. In later lab exercises we will use stocks of antibiotics that
have been prepared at 100x. This means the antibiotic stock is 100-fold higher than the desired final
concentration. 1 ml of 100x stock can be added to 99 ml of media to achieve the desired final product.
Stocks of this form are useful because they store well, can be used for a variety of final solutions, and
can be added without significantly altering the concentrations of the other media components.
If you know the concentration of the stock solution and want to determine how much to use to reach a
particular dilution, use the following rearrangement of the formula M1V1 = M2V2:

Concentration you want x Final Volume = Volume to add

Concentration you have
Then Q.S to the final volume.

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However, sometimes you want a range of concentration; or sometimes the volumes in question are too
small to accurately measure with micropipettes. In that case, you might choose to do a serial dilution.

Serial Dilutions
If you start with a concentrated solution, dilute it, then take some of that diluted stock and dilute it
some more, then take some of that diluted dilution and dilute it again, you are making a serial dilution.
A series of dilutions like this is an easy and accurate way to effectively dilute a concentrated stock, or in
some cases, a cell suspension.

Serial dilutions are used when the objective is to examine a range of concentrations of a compound or
reagent. This forms a series of samples that have the concentrations effectively divided over a
logarithmic scale. A simple example would be to have a series of 1:10 dilutions. The resulting samples
would all differ by one power of ten (1000, 100, 10, 1, 0.1, 0.01). This has significance for compounds
that have a physiological effect, because often the effect of changing a concentration from 1 μM to 5
μM is greater than changing from 100 μM to 200 μM. It is another example of the difference in fold
difference.

Dilutions are written as ratios (ex. 1:5), which represent the initial and final volumes. In other words,
take 1 mL of your stock and bring it up to a final volume of 5 mL with the appropriate diluent. If you
wanted 10 ml you would mix 2 ml of sample plus 8 mL of diluent.

General Protocol: Making a Serial Dilution

Let’s start with a simple 1:10 dilution series.

1. Make a note of the Initial Concentration (Ci).

2. Decide the final volume (Vf) you need for each dilution
3. Decide how many different concentration you need (N), and line up that many containers.
4. Decide what the dilution factor will be. In this case, the dilution factor, X = 10
a. Tube one will have a dilution factor of 1/X or 1/10.
b. Tube two will have a total dilution factor of 1/X2, or 1/100
c. Tube three will have a total dilution factor of 1/X3, or 1/1000
d. And so on.
5. Calculate what the final concentration will be in each container
a. The first will have a concentration :

1 x C
X i

b. The second will have a concentration:

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1 x C
2 i
X
c. And so on.
6. Label the containers with the final concentration they will hold.
7. Put the final volume (Vf) into each new container.
8. Now figure out how much of your stock to add to the first container to get the desired
concentration. Use the formula:

Vd = Vf
X-1
9. Set your pipette to Vd. This is the volume you will use for your dilutions.
10. Now, make your dilutions.
a. Put a new pipette tip on your micropipette or get a new pipette
b. Pipette Vd of the stock into the first container, X
c. Mix well.
d. Change the pipette tip or pipette.
e. Pipette Vd of the solution, X, into the next container, X2
f. Continue.
g. When you get to the final container, discard the volume, Vd, from the container so that
all containers will have the same volume.

Example

• We have a stock of 1M. (Ci = 1M)

• We want a 1:10 dilution series, (X = 10).
• We want 5 dilutions (N = 5)
• The final volume is 100 uL (1 x 10-4L) (Vf = 100 uL)

Dilution #1 = 1/X x 1M = 1/10 x 1M = 0.1M

Dilution #2 = 1/X2 x 1M = 1/100 x 1M = 0.01M

Dilution #3 = 1/X3 x 1M = 1/1000 x 1M = 0.001M

Dilution #4 = 1/X4 x 1M = 1/10,000 = 1/103 x 1M = 0.0001M = 0.1 mM

Dilution #5 = 1/X5 x 1M = 1/100,000 x 1M = 0.00001M = 0.01mM

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Note, that if we tried to dilute directly from our stock to 0.01 mM we would have to do the
following:

0.00001M/1M x 100 uL = 0.01 uL

*how did I calculate that?

It is not possible to accurately dispense 0.01 uL from a micropipetter! This is a good example of
when serial dilutions are easy and accurate.

The dilution volume, Vd = 100 uL/ 10-1 = 100/9 =11.1

So: I will take 5 tubes, labeling them #1 through #5 and with the concentrations I calculated above (and
with my initials and the date, too!)

I will put 100 uL of my solvent in each tube.

I will take 11.1 uL of the 1M stock and dispense that into Tube #1. After mixing, I will change pipette tips
and then take 11.1 uL out of Tube #1 into Tube #2. After mixing, I will change pipette tips and take 11.1
uL out of Tube #2 into Tube #3. I will continue until Tube #5. I will take 11.1 uL out of Tube #5 and
discard it. Now I have 6 tubes: the stock plus 5 dilutions, with 100 uL in each tube (unless my starting
volume in the stock tube varied, which is not important), and a range of concentrations from 1M to 0.01
mM.

Lab Exercise: Serial Dilutions

This exercise is designed to teach two skills and give you a chance to interpret basic data. We will be
practicing the use of micropipettes as you make serial dilutions. The dilution series will be quantitated
by a spectrophotometer, which will allow you to determine the reproducibility of your pipetting. In
addition, you will be given an unknown and determine its concentration by comparing its dilution series
to that of the known samples.

Equipment required:
• A 96 well plate (Figure 1)
• Standard Dye, 1%
• Unknown Dye
• Beaker of water
• Micropipettes and tips
• Plate Reader at 750 nm

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Protocol:
1. Orient the plate with the numbering across the top as shown in Figure 1.
2. Rows B, C, and D will be used for a 1:2 dilution. Rows F, G and H will be used for a 1:5 dilution.
3. We will be making three wells at 1:2 dilution; and three at 1:5

4. We want a final volume of 100 uL in each well.

5. We want to have triplicates of each dilution, so the dilution series will decrease as you move down
left to right but rows B,C,D will be triplicates of the known; rows F,G,H will be triplicates of the
unknown.
(a) In your lab notebook, calculate the dilution factor for each row (X, X2, and X3) for both 1:2 and
1:5 dilution series.
(b) Calculate the final concentration for your Known sample (Starting concentration is 1%) for each
concentration tested.

(c) Calculate the Dilution volume, Vd, to use for your dilution series.
ii. Make note of the Vd2 (dilution volume for the 1:2 series) and Vd5 (dilution
volume of the 1:5 series)
(a) Draw a table similar to Table 2 (or cut and paste it into your notebook). Fill out as much as you
can, now.
6. Using a micropipette transfer 100 μl of water into well A1 and into columns 2 to 6 of rows B, C and
D.
7. Pipette 100 uL of the stock dye into column 1 of rows B, C and D. This is our undiluted stock.
8. Pipette the Vd2 of the stock dye into column 2 of rows B, C and D. Note; this is a 1:2 dilution.

9. Starting with Row B: mix the contents of column 2 with the pipette and transfer the Vd2 to column 3
of the same row.
10. Repeat transferring the Vd2 from column 3 to 4 then 4 to 5 and 5 to 6. When you reach column six
after you mix in the 100 μl from column 5, remove the Vd2, so that all wells have 100 μl.
11. Repeat step 9 and 10 for Rows C and D.
12. In columns 7 through 12 you will be using the dye of unknown concentration, and setting it up
exactly as you did in columns 1-6. Using the unknown you were provided with; perform a 1:2
dilution series of the unknown.
13. Set up rows F, G, and H for a 1:5 dilution by repeating steps 7-12, but make sure to use the Vd5.

a. Remember to use the 1% Dye (the Known) for columns 1-6

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b. Use the Unknown Dye for columns 7-12.

14. Use a plate reader to determine the absorbance of your samples at 750 nm.

15. The data file will be sent to you. Print the data sheet and tape it into your notebook before you
come to the next lab. We will analyze it together in class.

Data Interpretation
Now we will see how well you did with your plating of the dilution series. I will demonstrate in class how
to do the following, and then you can each complete it on your own based on your own data.

1. Using Microsoft Excel, or similar software, calculate the average and standard deviation for each
dilution of your known and of your unknown.

2. Plot a graph of Absorbance vs. Concentration from your Known.

3. Determine the linear regression and trendline.

4. From the equation for your curve, determine the concentration of your unknown.

Figure 1: Setting up a 96 well plate for making serial dilutions.

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Table 2: Data collection for Dilution Series

Dilution Average Abs Standard Final

Sample Column Factor (750nm) Deviation Concentration

1 undiluted 1%

2 1:2

6
Known

7 1:5

10

11

12

1 Undiluted

2 1:2

5
Unknown

7 1:5

10

11

12

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References

Adams, DS. Lab Math: A Handbook of Measurement, Calculations, and Other Quantitative Skills for Use
at the Bench. CSHL Press, Cold Spring Harbor NY, 2003.

Freshney, RI. Culture of Animal Cells: A Manual of Basic Techniques and Specialized Applications, Sixth
Edition. Wiley Blackwell Publishers, Hoboken NJ, 2010.

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