The chamber is diamond etched and has a double improved Neubauer Ruling, which has a worldwide reputation in hospitals and laboratories for unmatched reliability, meeting the most demanding of standards. A hemocytometer is a laboratory instrument used for counting cells or particles in a sample of biological fluid, such as blood or cerebrospinal fluid. It consists of a glass microscope slide with a grid etched on its surface, which allows for precise counting of cells or particles within a defined area.
What is Neubauer Chamber or Hemocytometer?
The Neubauer chamber is a thick crystal slide along with the size of a glass slide. (30 x 70 mm and 4 mm thickness).
In a simple counting chamber, the central area is where cell counts are performed.
The chamber has three parts.
- The central part, where the counting grid has been set on the glass.
- Double chambers are most common than a simple chamber.
- In this case, the chamber has two counting areas that can be loaded independently.
Neubauer chamber’s counting grid is 3 mm x 3 mm in size. The grid has 9 square subdivisions of a width of 1mm.
In the case of blood cell counting, the squares placed at the corners are used for white cell counting.
Since their concentration is lower than red blood cells a larger area is required to perform the cell count. The central square is used for platelets and red cells.
This square is split into 25 squares of width 0,2 mm (200 μm). Each one of the 25 central squares is subdivided into 16 small squares.
Therefore, the central square is made of 400 small squares.
Glass Cover
- The glass cover is a squared glass of width 22 mm.
- The glass cover is placed on the top of the Neubauer chamber, covering the central area.
- The glass cover leaves room for the cell concentration between the bottom of the chamber and the cover itself.
- The chamber is designed so that the distance between the bottom of the chamber and the cover is 0.1 mm.
Procedure
Step 1. Sample preparation
Depending on the type of sample, preparation of a dilution with a suitable concentration should be prepared for cell counting.
Step 2. Counting
- Put the glass cover on the Neubauer chamber central area. Use a flat surface to place the chamber, like a table or a workbench.
- Put a disposable tip at the end of the micropipette.
- Adjust the micropipette to suck 10 μl. You can adjust it by turning the upper plunger roulette to select the required pipetting volume.
- Introduce the micropipette tip on the dilution previously prepared (STEP 1)
- Push the pipette plunger slowly until you feel it has arrived to the end of its travel.
- Remove the pipette tip from the dilution, and bring it to the Neubauer chamber. When the pipette is loaded, it must always be held in vertical position.
- Place pipette tip close to the glass cover edge, right at the centre of the Neubauer chamber.
- Release the plunger slowly watching how the liquid enters the chamber uniformly, being absorbed by capillarity.
- In case of the appearance of bubbles, or that the glass cover has moved, repeat the operation.
Step 3. Microscope set up and focus
- Place the Neubauer chamber on the microscope stage. If the microscope has a fixing clamp, fix the Neubauer chamber.
- Turn on the microscope light.
- Focus the microscope until you can see a sharpimage of the cells looking through the eyepiece and adjusting the stage.
- Look for the first counting grid square where the cell count will start. In this example, 5 big squares from a Neubauer-Improved chamber will be counted.
- Start counting the cells in the first square. Different laboratories have different counting protocols. In case of high cell concentration, a counting technique in zig-zag is used.
- Write down the amount of cells counted in the first square.
- Repeat the process for the remaining squares, writing down the counting results from all of them.
- The higher the number of cells counted, the higher the accuracy of the measurement.
Step 4. Concentration calculation
We apply the formula for the calculation of the concentration
The number of cells will be the sum of all the counted cells in all squares counted. The volume will be the total volume of all the squares counted.
Formula for cell concentration –
Total cells/ml = (Total cells counted x Dilution factor x 10,000 cells/ml)
Number of squares counted 1mm x 1mm = Area of square 0.1 cm x 0.1 cm = 0.01 cm² area of square Since the depth of the chamber is 0.1mm= 0.01cm
Volume of square = area x depth Volume = 0.01 cm² x 0.01 cm = 0.0001 cm3 = 0.0001 ml (10-4 ml) = 0.1 μl
What is a hemocytometer used for?
A device used for determining the number of cells per unit volume of a suspension is called a counting chamber.
Neubauer chamber is frequently used for counting the number of RBCs, WBCs, platelets, spores, bacteria, and yeast, and algal cells.
- Counting cells: A hemocytometer is primarily used to count cells or particles in a fluid sample. This is useful in medical research, clinical diagnostics, and veterinary medicine, as it allows for the quantification of cells in a sample, such as red blood cells or white blood cells.
- Measuring cell concentration: By counting the cells in a sample and knowing the volume of the sample, one can calculate the concentration of cells in the fluid. This information can be used to monitor disease progression, assess treatment efficacy, or determine the quality of a cell culture.
- Assessing cell viability: Hemocytometers can be used to assess the viability of cells by staining them with dyes that indicate live or dead cells. This can be useful in determining the effectiveness of a treatment or the toxicity of a substance.
- Environmental monitoring: Hemocytometers can also be used in environmental monitoring to count microorganisms in water samples, soil samples, or other environmental matrices.
- Industrial applications: Hemocytometers can be used in industrial quality control to measure the concentration of cells in a fermentation process, for example, to ensure the proper production of a specific product.
