MANUAL CELLS COUNTING WITH HEMOCYTOMETER/IMPROVED NEUBAUER CHAMBER
Wide variety and range of automated cell counting tools have been established, Neubauer Chamber/Hemocytometer left/remains the most widely technique used for cell counting around the globe. The most commonly used hemocytometer is the Improved Neubauer Chamber. Other hemocytometers contain the Thoma, Burker and Fuchs Rosenthal. Using these tool, the cells (e.g., RBCs (Red Blood Cells Erythrocytes), WBCs (White Blood Cells Leucocytes), and Platelets (Thrombocytes), Fungus spores, Bacteria and pollen) are observable below a microscope. Neubauer Chamber/Hemocytometer is a very thick glass plate/slab about a size of a glass slide having (30x70x4mm) in diameter. The counting area contains 2 square shaped ruled/lined zones. There are dejections or the channels on each side among the regions on which the squares are marked and identifiable therefore showing an “H” shape character.
The ruled/lined zone is 3mm2 allocated into Nine (9) big squares, every one with a 1mm 2 area/zones. The big central square is allocated into 25 medium squares having 2 or 3 lines. Every of these 25 squares is additionally distributed into 16 small squares having single lines and every of these smallest squares having area around of 1/400 mm2.
The coverslip is a square glass of thickness 22 mm. The coverslip is positioned on the upper portion of Neubauer Chamber/Hemocytometer, cover the central region. The ruled/lined portion is 0.1 mm inferior to the rest of the hemocytometer. So that when we count the cells the glass slip is already placed on the counting area, there is an opening of 0.1 mm (1/10mm) between the coverslip and the ruled/lined zone.
CELLS COUNTING AREAS IN NEUBAUER CHAMBER/HEMOCYTOMETER:
The counting may be finished either in the central big square or in the corner/curve squares, reliant on the size of the cells present in the sample under microscope.
- WHITE BLOOD CELLS (WBCs) COUNTING AREA:
The 4 big squares located at the corners are used for WBCs count. Subsequently their concentration is less than RBCs, a big area is necessary to complete the count.
- RBC COUNTING AREA:
The large center square is used for RBCs counts. As already stated, this area is split into 25 medium squares, which in turn are distributed into 16 squares. From these 25 medium squares, only the big corner squares and the center squares inside the big center square are used to do RBCs counts.
- PLATELET COUNTING AREA:
The big center square is used to count platelets. Platelets in all 25 squares inside the big center square are counted and calculated.
USED OF NEUBAUER CHAMBER
1. SAMPLE PREPARATION:
Depend on the form of specimen, preparation of dilution by a appropriate concentration must be ready for counting. Normally, the concentration scale for a counting with the hemocytometer is in between 250,000 cells/ml and 2.5 million cells/ml. The correct dilution of the mixture/blend with esteem to the no. of cells to be counted must be used. If the specimen is not diluted/mixed sufficient, the cells will be too crowded/jam-packed and challenging to count. If it is too diluted/insipid, the sample/ specimen size will not be adequate to mark durable implications approximately the concentration in the original mixture.
2. ARRANGING AND PREPARATION OF HEMOCYTOMETER/ NEUBAUER CHAMBER:
Clean and disinfect the chamber by placing the cover-slip with 70% ethanol. Take the slide, put the cover-slip on the chamber central part. Use a smooth surface to place the chamber, like a board or a worktop.
3. INTRODUCING THE SPECIMEN INTO THE HEMOCYTOMETER/ NEUBAUER CHAMBER:
By the help of micro-pipette, cautiously draw/pull up about 20ml of the cell dilution/mixture. Place the micro-pipette tip compared to the edge of the coverslip and slowly eject the fluid till the chamber is fully filled. By capillary act that will aid to confirm that the chamber is fully filled, but caution must be grasp not to over-fill the counting chamber. A volume of having capacity of 10 ml is enough to fill 1 chamber.
4. CELL COUNTING AND MICROSCOPIC FOCUSING ADJUSTMNT:
- Place/Put the chamber under the microscope. Focus it by using the 10X objective lens.
- As 10X objective lens is correct for white blood cells counting, count correctly the entire no. of cells present in 4 big corner squares.
- To count the red blood cells and Platelets, the microscope essential be transferred to a 40X objective lens. Count the cells in the separate zones as identified initially.
- All cells which are counted, write down on clean paper for calculation.
- If cells are touching/stirring the 4 perimeter/border sides of a corner square, only counted cells on 2 borders, whichever the 2 external margins or 2 internal borders.
CALCULATION OF COUNTING CELLS
Total no. of cells/ml of a specimen calculated from the no. of cells counted and area/portion counted. It is due to the ruled/lined zone of the hemocytometer comprise a precise capacity of the diluted/mixture of specimen. Subsequently a small capacity of the diluted mixture of specimen/sample is counted, a common formula needed used to convert the cells counts into the no. of cells/ml. The dilution factor (DF) which is used in the formula is determined/decided through the blood dilution/erosion used in the count. The deepness used in the formula is permanently remain 0.1. The region which is count will differ for every kind of cell which is count and is calculate by means of the proportions of the ruled/lined region.
EXAMPLE FOR COUNTING:
- Calculate the total white blood cells count by the help of Neubauer Chamber/Hemocytometer.
- of cells counted = N = 150 (Assumption)
- Region which is Counted = 1 mm2 x 4 = 4 mm2 (Region of 4 big corner squares)
- Deepness = 1/10 mm. Dilution Mixture = 1:20.
- Therefore White Blood Cells/Cubic mm of Whole Blood = N x 50 = 150 x 50 = 7,500