Fall 2021
Sampling, Pipetting and Cell Counting
Pipettes: Introduction
Pipetters (also known as micropipetters or pipettes) are without a doubt the most common and
fundamental tool in a molecular biologist’s toolkit. Pipettes can be used to transfer a wide range of
volumes with precision, care, and sterility, making the difference between experimental failure and
success. Mastery of the pipetter is thus the first and most important skill you will learn when beginning
laboratory manipulations.
Today, we will learn the very simple “pipetting cycle” by conducting a volumetric exercise and apply this
technique to transferring volumes of cell cultures to survey the densities of these cultures with a few
easy calculations. This will open the doors to all manner of molecular and cell biology applications! First,
we must learn about the basic parts (Figure 1), functions, and volumes of micropipetters at our disposal.
As with any laboratory equipment, please wait for Riley to give the go-ahead before manipulating the
pipetters.
Figure 1: Basic parts of a
micropipetter. Note: the “push
button” is also commonly referred to
as the “plunger”. Image credit:
https://micropipette.info/
What are the ranges of the pipettes at your table?
1. P 10
2. P 20
3. P 1000
4. P 200
Select the correct pipette for desired volume:
1.25 uL : P 1-10 165 uL : P 200 200 uL : P 1000 20 uL : P 200
9.8 uL : P 20 2.00 uL : P 1-10 12.8 uL : P 20 465 uL : P 1000
, Fall 2021
Pipetting Exercise:
1. Zero your scale with an empty 1.5 mL tube on it.
2. Pipette 500 uL of water using a P1000 pipettor into the same 1.5 mL tube.
3. Check the mass of the water. One mL has a mass of one gram, so 500 uL or 0.500 mL should
have a mass of 0.500 grams.
How accurate was your pipetting? Our pipetting was very accurate, we got the same exact
answer.
4. Repeat this process for 200 uL (P1000 pipettor) and 100 ul (P200 pipettor). You can use the
same tube if you zero the scale each time.
5. Repeat this protocol with the other member of your group doing the pipetting so that each lab
partner does this at least once. (If you have time, repeat to improve your accuracy.)
Cell Counting
The ability to quantify cell number is critical in many applications across many scientific disciplines. In
human medicine, abnormal red blood cell count can be an indicator of anemia, bone marrow failure, or
even leukemia. Pharmacologists must correctly estimate cell number in order to set up experiments and
appraise the efficacy and safety of drugs.
The most straightforward method to quantify cells is through the use of a hemocytometer. A
hemocytometer is a red blood cell, so hemocytometer is a device to measure red blood cells. Despite its
name, the hemocytometer can be used to count many other cell types.
A hemocytometer is a specialized microscope slide and matching cover slip that hold an exact volume of
liquid in a counting chamber mounted on the slide. These devices are precisely manufactured so that
the surface are and the height between the bottom of the chamber and the cover slip is exactly known.
Why is this important?
estimate cell number in order to set up experiments and appraise the efficacy and safety of drugs. Since
they are precise with can count cells.
Left: what a hemocytometer looks like under
a microscope. The top-left area highlighted
in red has a volume of 0.1 μL(real
hemocytometers aren’t highlighted like this)
Below: A microscope photograph ¼ of a
loaded hemocytometer square. Notice the
multiple cell types and debris. Red indicates
actual symbiont cells, which should be
counted. In purple are cells of a different
type and should not be counted. Green
circles surround cells of cell debris which
appear to be symbionts, but are far too
small. These should not be counted.
Sampling, Pipetting and Cell Counting
Pipettes: Introduction
Pipetters (also known as micropipetters or pipettes) are without a doubt the most common and
fundamental tool in a molecular biologist’s toolkit. Pipettes can be used to transfer a wide range of
volumes with precision, care, and sterility, making the difference between experimental failure and
success. Mastery of the pipetter is thus the first and most important skill you will learn when beginning
laboratory manipulations.
Today, we will learn the very simple “pipetting cycle” by conducting a volumetric exercise and apply this
technique to transferring volumes of cell cultures to survey the densities of these cultures with a few
easy calculations. This will open the doors to all manner of molecular and cell biology applications! First,
we must learn about the basic parts (Figure 1), functions, and volumes of micropipetters at our disposal.
As with any laboratory equipment, please wait for Riley to give the go-ahead before manipulating the
pipetters.
Figure 1: Basic parts of a
micropipetter. Note: the “push
button” is also commonly referred to
as the “plunger”. Image credit:
https://micropipette.info/
What are the ranges of the pipettes at your table?
1. P 10
2. P 20
3. P 1000
4. P 200
Select the correct pipette for desired volume:
1.25 uL : P 1-10 165 uL : P 200 200 uL : P 1000 20 uL : P 200
9.8 uL : P 20 2.00 uL : P 1-10 12.8 uL : P 20 465 uL : P 1000
, Fall 2021
Pipetting Exercise:
1. Zero your scale with an empty 1.5 mL tube on it.
2. Pipette 500 uL of water using a P1000 pipettor into the same 1.5 mL tube.
3. Check the mass of the water. One mL has a mass of one gram, so 500 uL or 0.500 mL should
have a mass of 0.500 grams.
How accurate was your pipetting? Our pipetting was very accurate, we got the same exact
answer.
4. Repeat this process for 200 uL (P1000 pipettor) and 100 ul (P200 pipettor). You can use the
same tube if you zero the scale each time.
5. Repeat this protocol with the other member of your group doing the pipetting so that each lab
partner does this at least once. (If you have time, repeat to improve your accuracy.)
Cell Counting
The ability to quantify cell number is critical in many applications across many scientific disciplines. In
human medicine, abnormal red blood cell count can be an indicator of anemia, bone marrow failure, or
even leukemia. Pharmacologists must correctly estimate cell number in order to set up experiments and
appraise the efficacy and safety of drugs.
The most straightforward method to quantify cells is through the use of a hemocytometer. A
hemocytometer is a red blood cell, so hemocytometer is a device to measure red blood cells. Despite its
name, the hemocytometer can be used to count many other cell types.
A hemocytometer is a specialized microscope slide and matching cover slip that hold an exact volume of
liquid in a counting chamber mounted on the slide. These devices are precisely manufactured so that
the surface are and the height between the bottom of the chamber and the cover slip is exactly known.
Why is this important?
estimate cell number in order to set up experiments and appraise the efficacy and safety of drugs. Since
they are precise with can count cells.
Left: what a hemocytometer looks like under
a microscope. The top-left area highlighted
in red has a volume of 0.1 μL(real
hemocytometers aren’t highlighted like this)
Below: A microscope photograph ¼ of a
loaded hemocytometer square. Notice the
multiple cell types and debris. Red indicates
actual symbiont cells, which should be
counted. In purple are cells of a different
type and should not be counted. Green
circles surround cells of cell debris which
appear to be symbionts, but are far too
small. These should not be counted.
