Counting Cells with a Hemocytometer

Summary

This video script demonstrates the process of using a hemocytometer to count cells, including the use of Trypan blue to differentiate between living and dead cells. It outlines the steps for preparing the cell suspension, loading the hemocytometer, and counting cells within a grid pattern under a microscope. The script also covers counting rules, handling artifacts, and calculating the percentage of viable cells and the concentration of cells in the culture, providing a clear guide for cell counting and viability assessment.

Takeaways

• 🔬 A hemocytometer is a specialized tool used for counting cells, featuring two identical wells on a microscope slide.
• 🧪 Cells are prepared for counting by diluting a cell suspension with Trypan blue, a dye that differentiates living from dead cells.
• 📏 Each well in the hemocytometer is divided into a grid pattern of 9 large squares, each with a volume of 10^-4 mL.
• 🔍 The counting procedure involves focusing on specific squares: the 4 corner squares and the center square.
• 📐 Cells touching the top and left boundaries of the grid are counted, while those on the right and bottom are ignored.
• 🔵 Dead cells, which have taken up Trypan blue, are identified by their blue color under the microscope.
• 🚫 Artifacts, such as blurry debris, are excluded from the cell count to ensure accuracy.
• 🔄 Proper handling and storage of the hemocytometer are crucial for minimizing artifacts.
• 🧮 The total viable cell count is determined by summing counts from the designated squares and calculating the percentage of viable cells.
• 🧪 The average number of cells per square is calculated by dividing the total viable cell count by the number of squares counted.
• 🔢 The concentration of viable cells is calculated by considering the dilution factor and the average count per square, resulting in cells/mL.

Q & A

What is a hemocytometer used for?

A hemocytometer is used for counting cells, specifically it is a device that helps in determining the concentration of cells in a suspension.

How is a hemocytometer different from a regular microscope slide?

A hemocytometer is a modified microscope slide that contains two identical wells or chambers for holding a small volume of cell suspension.

What is the purpose of using Trypan blue when counting cells with a hemocytometer?

Trypan blue is used to distinguish between living and dead cells. It passes through the membranes of dead cells, staining them blue, while living cells exclude the dye and appear mostly clear.

How should the cell suspension be loaded onto the hemocytometer?

The cell suspension should be loaded onto both chambers of the hemocytometer by pipetting it under the cover slip.

What is the volume of suspension contained in each square of the grid pattern on the hemocytometer?

Each square on the hemocytometer contains 10^-4 mL of suspension.

Which squares are counted for cell enumeration in the given lab's procedure?

In the given lab's procedure, cells are counted in the 4 large corner squares and the center square of the hemocytometer's grid pattern.

How are cells that touch the boundaries of the counting squares handled during counting?

Cells that touch the top and left boundaries of the counting squares are counted, while cells touching the right and bottom boundaries are ignored.

What is an artifact as mentioned in the script and why is it important to note it?

An artifact is an object or debris that appears blurry and lacks a well-defined shape, which can interfere with cell counting. It is important to note because artifacts are not included in the cell count to ensure accuracy.

How is the percentage of viable cells calculated from the counted cells?

The percentage of viable cells is calculated by dividing the number of viable cells by the total number of cells counted, and then multiplying by 100 to get the percentage.

What is the dilution factor and how is it calculated in this context?

The dilution factor is the ratio of the final volume of the solution to the initial volume of the cells. It is calculated by dividing the final volume by the initial volume of cells.

How is the concentration of viable cells determined from the hemocytometer counts?

The concentration of viable cells is determined by multiplying the average number of cells per square by the dilution factor and the volume of suspension per square (10^-4 mL).