pics for counting

THE NEUBAUER CHAMBER FOR COUNTING

THE NEUBAUER CHAMBER FOR COUNTING

1. INTRODUCTION

The Neubauer chamber (hemacytometer) is used for counting red blood cell, white blood cell, and blood plateletss. we use it in urine, csf, joint fluid. Also use it in fertility for sperm count.

(counting, densities, concentration.)

The improved Neubauer hemacytometer consists of a thick rectangular slide (plastic/glass) with an H-shaped trough forming two counting areas (Fig. 1). With the cover slip in place, each area forms a chamber with a depth of 0.1 mm.

The total ruled area of one chamber is 9 mm². The four corner squares (1 mm × 1 mm) are subdivided into 16 smaller squares and the centre square (1 mm × 1 mm) is subdivided into 25 smaller squares, each has an area of 0.04 mm² (.2 mm × .2 mm) (Fig. 2). All of the 25 smaller squares may then be divided into another 16 squares.

 

Fig. 1 Macroscopic view of the hemacytometer (Plastic)

Fig. 2 Ruling and dimensions of one chomber of the improved Neubauer hemacytometer

2. Cautions:

1) chose suitable dilution factor for easy read, even distribution. Avoid presence of air bubbles, overflowing, underfilling, cell clusters and uneven distribution of cells.

2) Allow cells to settle for 3–5 minutes before counting.

3) counting the upper and left boundary line, not the right and lower boundary line.

4) at least counting 200 cells.

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3 Counting

• For cells greater than 6 u and not too dense cultures, the total count is made in any one block (A, B, C, D, E) as shown in Fig. 2.
  
• For minute and dense populations, count the cells in the five small squares in the centre block (E Block).
  
  • Start at the top left square and count only those cells which lie within or touching the boundary line as shown in Fig. 5.
    
  • Make a duplicate count in the corresponding block in the second chamber.
    
  • Record the counts in individual 1 mm² blocks.
    

Fig. 3 Properly and improperly charged counting chamber

Fig. 4 Distribution of cells

Score board for each small square Total count is 48

Fig. 5 Method of counting cells

4. Calculation

Tips 1. one of nine blocks are 1x1x0.1 mm³

2. 1 cm³=1ml

3. SI unit is L, and 1000ml =1L

 

(a) If all the cells in the individual blocks are counted, the density (d) would be:

(b) If the cells were counted only in the five small squares in the centre block (E Block), the following formula may be used:

where 10 = the 10 squares of the 2 chambers and

4×10^-6 = the volume of sample over the small square area which is equivalent to .004 mm³ (.2 × .2 × .1) expressed in cm³ (ml).

5. REFERENCES

AO Bright line hemacytometer counting chamber, American Optical, Buffalo, N.Y., 1425, 19pp.

Guillard, R.L. 1973 Division rates. In Phycological Methods, Janet R. Stein (ed.), Cambridge University Press, Cambridge, 289–311pp.

Martinez, M.P., Chakroff, J.B. Pantastico. 1975 Direct Phytoplankton counting technique using the hemacytometer. In Phil. Agriculturist 59. 43–50

 

 

 

 

 

 

These triple lines are used to determine if cells lie within or outside the grid.

 
 

 
 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

Quiz

1. the diagram below shows the outline of an chamber. What are the dimensions for one of the nine large squares?
   

A	B	C
D	E	F
G	H	I

 

1. what is the total cell count , if the following counts were obtained from these squares:
   

   A=45
   

   C=42
   

   G=46
   

   I=39
   

2. If center E square's five small squars was conted ( as below), what the total cell count ?
   

   a=45
   

   b=42
   

   c=46
   

   d=39
   

   e=43
   

 

 

 

 

 
 

 
 

 
 

 
 

 

neuboaur chamber cell counting

neuboaur chamber cell counting