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Cell Counting Using the

Dye Exclusion Principle


Timothy Xavier Tan
Angelo Tolentino
Eloisa Francesca Umali
Janine Pearl Villaflor
Kim Nicole Yap

DYE EXCLUSION TEST


Used to determine the number of viable cells present in a
cell suspension
Based on the principle that live cells possess an intact
cellular membrane which can actively exclude certain
dyes
DYES: trypan blue, eosin, or propidium iodine
Dead cells do not have such integrity and are stained
BLUE

HEMATOCYTOMETER or NEUBABUER CHAMBER


Used in cell counting
It includes
a.) Neubauers slide
b.) cover slip
c.) cover glass

HEMATOCYTOMETER or NEUBABUER
CHAMBER
NEUBAUERS SLIDE
- Thick slide with two ruling
area on center
- Ruled area are separated by
H- shaped
gutter/trough/depression
- Beyond the two vertical arm
of trough there are two raised
shoulder (ridge) which
support cover glass
- Lining is coated by shining
metal or rhodium

HEMATOCYTOMETER or NEUBABUER
CHAMBER
Consists of TWO CHAMBERS (COUNTING CHAMBER)
One chamber is divided into 9 large squares with
dimensions of 1x1mm
A cover glass (0.1mm) is supported over these squares
TOTAL VOLUME OF EACH SQUARES
1.00 mm x 0.1mm or
0.1mm3 or
10-4 cm3

Each large square has a volume of 0.0001mL

COUNTING CHAMBER GRID

Dos and donts


Clean hematocytometer and cover slip with 70% ethanol
Bacteria used: _____
Mixture sample = bacteria cell suspension + 0.3% Trypan blue in 3%
Formalin
Trypan blue - stains dead or dormant cell
Formalin fixation of cell
Viable cells transparent
Nonviable cells color blue
Make sure the mixture covers the entire grid in one continuous
motion with no bubbles and fluid does not overflow into the gutter
More than 50 cells per square, DILUTE the cell suspension with the twice
amount of Phosphate Buffer Saline
Phosphate Buffer Saline used for diluting cells, balanced salt solution,
pH is 7.4

Dos and Donts


Ordinary glass coverslip CANNOT be used with
hematocytometer
Using the wrong coverslip can:
1. Distort the cell due to the capillary force of the
liquid under the coverslip unless it is measured to the
precise amount tot fill the space under the coverslip
2. Lean to an inaccurate count of cells

COUNTING RULES
DO NOT COUNT CELLS
TOUCHING:
Bottom line
Right line
This is to avoid double counting

Count in the CORNER


SQUARES
4 corner squares for
WBC
1 central square for
RBC/platelet

Guide Questions:
2.

5.

REFERENCES
http://www.slideshare.net/GovardhanJoshi/hemocytomet
er?qid=5ddd9dcc-48cb-4d15-b930c3a4188658e5&v=&b=&from_search=2
https://en.wikipedia.org/wiki/Hemocytometer#/media/Fil
e:Haemocytometer_Grid.png
Laboratory Manual in Cell Molecular Biology