Bio 152 Principles of Molecular Biology and Biotechnology
LABORATORY COMPONENT
Week 2 February 21 – 26, 2022
BIO 152 LABORATORY
Exercise 2
CELL COUNTING METHOD
I. Introduction
Various cell counting techniques have been widely used as a key step in experimental workflows
that require an accurate and consistent number of cells. These downstream applications include cell culture,
transfection experiments, cell proliferation or viability studies, cytotoxicity assays, and quantitative PCR. The
method of cell counting can be performed either manually through using a hemocytometer or via an
automated cell counter. In this exercise we will focus on manual cell counting technique with the aid of
hemocytometer.
The hemacytometer has been an essential tool for hematologists, medical practitioners, and
biologists for over a century. The device was initially used by medical practitioners to analyze patient blood
samples, which was the spark that created the field of hematology. At the present, the type of cells counted
on a hemacytometer have expanded to algae, yeast, cancer cells, stem cells, cultured cells, blood-based cells,
parasites, spores and more. Although a variety of automated cell counting instruments have been developed,
many researchers still fall back on manual counting with hemacytometer.
II. Expected Learning Outcomes
After accomplishing the exercise, you should be able to:
1. discuss the principle and applications of cell counting methods;
2. examine and identify parts of hemocytometer, and;
3. develop cell counting skills.
III. Study Guide
Read resource 1 providing you with quick and easy steps on hemocytometer cell density calculations.
An additional link is also provided for you to review on how to get dilution factor in link a and an
explanation in hemocytometer square size in link b. Resource 2 is an overview of the different cell
counting techniques aside from using the hemocytometer. For you to have a grasp of how to count cells
in a laboratory setting, watch the demo video in resource 3. Refer to the image in Figure 1 illustrating the
hemocytometer.
1. Hemocytometer Calculation available at:
https://www.hemocytometer.org/hemocytometer-calculation/
a. Dilution Factor
https://www.hemocytometer.org/dilution-factor/
b. Hemocytometer Square Size
https://www.hemocytometer.org/hemocytometer-square-size/
2. How to count cells: An overview of cell counting methods
https://www.linkedin.com/pulse/20140929030437-16207202-how-to-count-cells-an-overview-ofcell-counting-methods/
3. Counting Cells with a Hemocytometer
https://www.youtube.com/watch?v=pP0xERLUhyc
Bio 152 Principles of Molecular Biology and Biotechnology
LABORATORY COMPONENT
Week 2 February 21 – 26, 2022
Figure 1: Hemocytometer overview. (A) View of Hemocytometer with glass coverslip. Black arrow denotes “V”
notch where samples are loaded. (B) Illustration of Hemocytometer counting area. Boxes 1-4 (red) are usually
used to count cells.
IV.
Activity: Practice Cell Counting Skills
Practice a virtual simulation to demonstrate the techniques on how to properly use a hemocytometer in cell
counting in a laboratory setting by following the instructions. Before you start clicking the link, read first the
materials and procedures used to prepare the samples for the counting method for you to have an initial
background.
Materials:
Binocular Light Microscope
Hemacytometer and cover slip
Micropipettes P20 and P1000
0.4% Trypan Blue
Ethanol
Hand counter
Sample Preparation Procedures
a.
b.
c.
d.
From your 1mL algal cell suspension, pipette out 100uL of cells and transfer to a new tube.
Add 400uL of 0.4% Trypan Blue.
Mix gently. Take note to perform this using aseptic technique to prevent contamination.
Your sample is then prepared in the hemocytometer. Next, you are going to view and count under the
microscope. Follow the procedures in the virtual lab simulation.
Preparing the hemocytometer
a. Prepare a hemocytometer by cleaning the chamber surface with 70% ethanol. Wipe dry and position
the coverslip over the chambers.
Bio 152 Principles of Molecular Biology and Biotechnology
LABORATORY COMPONENT
Week 2 February 21 – 26, 2022
b. Pipette out 100uL of Trypan Blue-treated algal cell suspension and apply to the hemocytometer. Very
gently fill both chambers underneath the coverslip, allowing the cell suspension to be drawn out by
capillary action.
c. Place the hemocytometer on the stage of a binocular light microscope. Adjust the microscope to 10X
magnification and focus on the cells. You can switch to 40x to view a clearer picture of the number of
cells in each square.
d. Reminder: When counting, employ a system whereby cells are only counted when they are set within
a square or on the right-hand or bottom boundary line. See Figure 2 below illustrating the direction of
counting cells. Dilution factor is computed as final volume over initial volume.
Figure 2. Cell counting direction (green arrows).
Source:
https://ph.images.search.yahoo.com/images/view;_ylt=AwrwJRhuZ4tg9U0AsxO1Rwx.;_ylu=c2VjA3NyBHNsawNpbWcEb2lkAzM3ZTU2YzU2OWJkNGY xODI0ODVmMjdkMWZiZjkzNmQ5BGdwb3MDMzAEaXQDYmluZw-
-?back=https%3A%2F%2Fph.images.search.yahoo.com%2Fsearch%2Fimages%3Fp%3Dcell%2Bcounting%26fr%3Dmcafee%26fr2%3Dpiv-web%26tab%3Dorganic%26ri%3D30&w=350&h=369&imgurl=braukaiser.com%2Fwiki%2Fimages%2F3%2F3e%2FMicroscopecell_counting.gif&rurl=http%3A%2F%2Fbraukaiser.com%2Fwiki%2Findex.php%2FMicroscope_use_in_brewing&size=23.2KB&p=cell+counting&oid=37e56c569bd4f182485f27d1fbf936d9&fr2=piv-web&fr=mcafee&tt=Microscope+use+in+brewing++German+brewing+and+more&b=0&ni=21&no=30&ts=&tab=organic&sigr=6udkgPaaLrwa&sigb=ORPKlZEQ0Nov&sigi=MtOKn.JRQ2lY&sigt=moEsRGms6gPv&.crumb=XAW0IvDx.OP&fr=mcafee&fr2=piv-web
Virtual Simulation Instructions on what to CLICK.
1. Cell Biology Virtual Laboratory link:
Hemocytometer (Counting of Cells) (Theory): Cell biology Virtual
https://vlab.amrita.edu/?sub=3&brch=188&sim=336&cnt=1
Bio 152 Principles of Molecular Biology and Biotechnology
LABORATORY COMPONENT
Week 2 February 21 – 26, 2022
2. “Theory” button and read on the underlying principle behind the use of hemocytometer. Other than
the field of molecular biology, hemocytometer is usually used in microbiology.
3. “Procedure” button. Reflect on the laboratory procedures of how cells are counted in the given
enlarged grid line of the hemocytometer. Take note also of how the cell samples are being prepared
according to the viability of cells especially when the researcher is performing cell culture, the dye
used to determine nonviable cells, how to count cells in the grid lines, what indices are to be
calculated, and what could be the real lab scenarios.
4. “Self Evaluation” button. Answer the 8-item quiz to check your cell counting skills. Take photo of the
result of the evaluation.
5. “Simulator” button. It will require you to sign up for free to proceed with the cell counting simulation.
Now you have a microscope with a computer screen in your view. Click the left topmost 3-bars button
and it will give you a detailed section at the left to see the points that need to be answered. Try the
binocular view to see the cell samples in the microscope itself. There will be buttons below that will
allow you to navigate your microscope just like in a real lab setting.
6. Count the number of viable and nonviable cells in the given sample using 4 and 5 squares. Fill in the
table of your answers in the worksheet. Show your calculations.
Student Output:
Step-by-step screenshot photos as proof that you have done the simulation practice, fill in the
table, show your calculations and answer the questions.
V. Submission Details
1. Save the laboratory worksheet in .pdf using your last name as file name. (e.g.
Bio152LAB_Ex2_lastnames)
2. Submit only the laboratory worksheet on March 4, 2022 at 11pm.
VI. References
1.
2.
3.
4.
https://www.hemocytometer.org/hemocytometer-calculation/
https://vlab.amrita.edu/?sub=3&brch=188&sim=336&cnt=5
https://www.abcam.com/protocols/counting-cells-using-a-haemocytometer
https://www.stemcell.com/how-to-count-cells-with-a-hemocytometer.html
Bio 152 Principles of Molecular Biology and Biotechnology
LABORATORY COMPONENT
Week 2 February 21 – 26, 2022
VII. Laboratory Worksheet
Student Names:
Date of Submission:
Bio 152 Laboratory Exercise 2 WORKSHEET
CELL COUNTING METHOD
I.
Complete the table by filling in screenshot photos of each step.
Stepwise Instructions
Theory
Evidence Photos
Procedure
Result of Self Evaluation of each Group Member
Simulator Binocular View
Simulator Trinocular View
II. Fill in the table of your answers in the simulation and show calculations of the cell counting indices.
Square Number
Number of Viable Cells
Number of Nonviable Cells
1
2
3
4
a. Percent Cell Viability
b. Average Viable Cell Count per Square
c. Viable Cell per mL
d. Dilution Factor
e. Total Viable Cells per Sample
III. Answer the following questions briefly and directly in space provided. Maximum of 3 sentences only.
a. Explain very briefly how do you count cells using the grids of hemocytometer?
b. List down 3 probable errors and its causes in cell counting via hemacytometer.
Bio 152 Principles of Molecular Biology and Biotechnology
LABORATORY COMPONENT
Week 2 February 21 – 26, 2022
c. Why do you need to calculate for volume of media needed after computing the cells?
d. There is a total of 56 cells including 20 unstained cells altogether in the five counting squares
of hemocytometer. Given volume of media is 5ml. The average viable cell count per square
is 4 and the dilution factor is 2. Then calculate the percentage of cell viability, viable cells/ml
and the volume of media needed, if we need 5000 cells. (Show calculations)
e. What is the principle behind Trypan Blue Exclusion Test of cell viability? What could be other
dyes used to differentiate between viable and nonviable cells?
f. If you are provided with a cell sample, how will you be able to find out its concentration by
using a hemocytometer?
g. What are the other methods of cell counting? List them down and briefly describe the
principle behind each.