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Haemoctymeter Instructions. Haemocytv3.doc Dr Neville A. Punchard

HAEMOCYTOMETER INSTRUCTION SHEET

(for improved Neubauer Haemocytometer)

PLEASE NOTE: The haemocytometer is made of glass, so do not drop it! Only the cover slip provided with theHaemocytometer, which requires a special one, should be used with it

DESCRIPTIONThe haemocytometer has a central area, slightly lower (" mm) than the rest of surface of the slide This depression formsthe counting area# chamber ($igure "a% below)

&oo'ing at the surface of the slide% finely ruled areas that form grids can be seen ($igure "b% above) These define an area

on the slide of a 'nown sie The area measured from the grid and the height between the slide and the base of thedepression of the counting area defines a 'nown volume y counting the cells or other ob*ects in this volume the no of cells etc per unit volume can be calculated and this value used to give the number in the original volume

INSTRUCTIONS FOR USE

(1). +nsure both the haemocytometer and its cover slip are clean -f they are dirty wash them in distilled water and thenwipe them over with alcohol .hen dry breath on the surface of the haemocytometer and quic'ly place the cover slip in

position so that it is centred over the counting chamber

(2). /ently, but firmly press down on the cover slip, using a thumb placed either side, holding the haemocytometer firmly between both hands, until Newton0s rings (refraction rings of light of a rainbow#oil slic' coloration) are seen -f no ringsare seen try again -f problems are e1perienced obtaining rings, eg on a hot day, then cool the haemocytometer under running cold water for few minutes before drying and then trying to the mount the cover slip again

(3). Ta'e (suc') up a small amount of the cell suspension#solution to be counted in a fine tipped 2asteur pipette /entlytouch the pipette against the side of the cover slip where it touches the base of the depression ($igure 3, below) 4 drop of

liquid should be drawn out of the pipette and into the chamber -f it is not then gently and slowly squeee out one dropThis will fill one counting chamber -f both chambers are to be used then repeat for the other chamber

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(4). 2lace the haemocytometer under a microscope Turn the microscope on and under low power (56#5") focus on thegrid area of the counting chamber PLEASE NOTE7 do not ta'e too long to count as the liquid will start to evaporate fromthe sides of the chamber, and the chamber dry out, with the heat of the lamp

(5). 8nder the microscope the grids area can be seen to be formed of 9 large squares in a 353 grid ($igure :% below) +achof these squares covers an area of " mm; which, with the depth of the chamber being " mm, means that the volume ofeach large square is " mm3

The central area of the grid is made up of triple ruled lines that in the centre form a 656 grid of ;6 squares with a totalvolume of " mm3 ($igure :, above, and $igure 6a, below ) +ach of these ;6 squares has an area of : mm; and avolume of : mm3 +ach one of these ;6 squares is further divided up into "< smaller ones in a :5: grid ($igure 6b, below) +ach of these "< smaller squares has a surface area of "#: mm; (;: mm;) and a volume of ;6 mm3

COUNTING A HIGH-DENSITY CELL POPULATION.

.hen counting high densities of cells squares use the :1 ob*ective and count the four corner and one centre : mm;square ($igure

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=ells falling on the bordering triple lines should only be counted if they are on either the top or on the left lines only+1clude those touching the bottom and the right hand side lines .hen performing cell counting, the most accurate count

is achieved when ";>; cells can be counted -f you have more than this in the five squares then you should dilute your sample and re>count -f you have less than this you should use either the low density cell population method as described below or centrifuge and resuspend your cells in a smaller volume

Cal!la"# "$# %&#&"'a"%& % #ll* a* +#l%,:The centre triple ruled grid has a volume of " mm3 ?ou have counted the cells in 6#;6 of this grid, ie 6 out of the ;6squares Thus to convert to the number of cells in " mm3 you have to divide by 6 and then multiply by ;6 To convert thisanswer from cell#" mm3 to cells# ml you have to multiply by ": ( " mm3 5 ": @" mm3@ " mm3)

&et no of cell counted in 6 squares @ A

no of cells# ml suspension @ 25 1/4

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NOTE: T$* 0%#* &%" "a# &"% a%!&" a& 0l!"%&* a0# & '#a'a"%& % "$# *"a& %' a& %"$#' 0l!"%&* '%' "%

*"a&&. Y%! ,ll $a6# "% al!la"# "$# ##"* % "$#*# %' %!'*#l.

COUNTING A LO7-DENSITY CELL POPULATION.

-n this case use "1 ob*ective lens and count the squares (" mm3

) in each corner of the 353 grid, ie outside the tripleruled area, ( labelled 4, , = and B in $igure : above) Bivide the number of cells counted by : and multiply by " : to getthe number of cells per ml

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