INSTRUMENTS
Tissue Cassette
• Cassettes keep specimen safely submerged in
liquid.
• Efficient flow-through slots.
• Snap-latch and hinge-lock design prevent early
separation of base and lid and allows for one
hand operation.
• Large labeling area on two sides of cassette.
Tissue holder
• To hold the tissue when embedded in L mold
• The wax is allowed to solidify
• Then the solid wax with the tissue along with
tissue holder is placed in the microtome for
trimming.
Wintrobe’s Tube
• The wintrobe’s tube is about 11cm long bore
diameter is 2.5 mm and the bottom 10 cm are
graduated.
• EDTA blood is used and the tube is filled till
zero mark on top with the help of a pipette.
• Set it vertically and read exactly after 1 hr.
• It can be used for ESR estimation and for PCV
estimation.
Measuring Cylinder or Mixing Cylinder
• Used to measure the volume of liquid.
• Generally more accurate and precise than
laboratory flask and beakers.
• Sometime used to measure the volume of a
solid indirectly by measuring the displacement
of a liquid.
Differences Between RBC AND
WBC Pipette.
RBC pipette WBC
pipette
1) It has a red It has a white
bead bead
2) It has It has
graduations graduations
upto mark 101 upto mark 11
3) Size of bulb is Size of bulb is
larger smaller
RBC PIPETTE
WBC PIPETTE
DILUTION FACTORS
For CELL counting
Blood is filled till mark 0.5 and fluid is
then filled till mark 101 or 11.
Both are thoroughly mixed.
Discussion
1. In certain conditions, such as polycythemia, the red
blood cell count may be extremely high, which makes it
difficult to obtain an accurate count. In this instance,
make a larger dilution of blood. For a 1:301 dilution,
add 20L of whole blood to 6.0mL of diluting fluid.
2. For a patient who has severe anemia and in whom the
RBC is low, make a 1:101 dilution by adding 20L of
whole blood to 2.0mL of diluting fluid.
Discussion
3. Make certain the pipettes, hemacytometer, and cover
glass are free from dirt, lint, and dried blood. Ensure
that the diluting fluid is free from blood and other
contamination.
4. RBC takes longer to perform than a WBC because of
the larger number of cells. Therefore, proceed as
quickly as possible once the cells have settled. Drying
of the dilution in the counting chamber causes
inaccuracies in the final cell count.
5. The range of error for a manual RBC is generally about
10 to 20%
Drabkins solution
The original (Drabkin's) reagent had a pH of 8.6. The following modified
solution, Drabkin-type reagent, as recommended by the
International Committee for Standardization in Haematology,[2] has a
pH of 7.0–7.4. It is less likely to cause turbidity from precipitation
of plasma proteins and requires a shorter conversion time (3–5 min)
than the original Drabkin's solution, but it has the disadvantage
that the detergent causes some frothing:
Potassium ferricyanide (0.607 mmol/l) 200 mg
Potassium cyanide (0.768 mmol/l) 50 mg
Potassium dihydrogen phosphate 140 mg
(1.029 mmol/l)
Nonionic detergent 1 ml
Distilled or deionized water To 1 litre
Uses
• Used for haemoglobin estimation.
HEMOCYTOMETER
Hemo: blood
Cyto: cell
Meter: measurement/counter
Thus, it is an instrument used to
count the blood cells.
*** Hemacytometers a precision-made slide for
performing
manual cell counts with the aid
of a
microscope.
*** Hemacytometers are used when……
1- automated cell counters and hematology analyzers are unavailable
2- blood cell counts are extremely low
3- to get a cell count for body fluids (spinal fluid, joint
fluid,commonly
The most semen counts,
used and other bodilyisfluids)
hemacytometer the Neubauer chamber.
It includes:
a) Neubauer’s slide
b) Cover slip
c) RBC pipette
d) WBC pipette
NEUBAUER’S SLIDE
** It is the name given to a
thick glass slide. In the centre
of the slide, there is an
H- shaped groove. On the two
sides of the central horizontal
bar, there are scales for
counting the blood cells.
** The depth of the scales is
1/10mm or 0.1mm.
** Each scale is 3mm
wide and 3mm
long.
** The whole scale is
divided into 9 big
squares.
** Each square is
1mm long and
1mm wide.
With the microscope, using a 4x objective, identify the nine main
squares of the counting chamber
delimited by three lines each as shown in the following image:
• The gridded area of the hemocytometer consists
of nine 1 x 1 mm (1 mm2) squares. These are
subdivided in 3 directions; 0.25 x 0.25 mm
(0.0625 mm2), 0.25 x 0.20 mm (0.05 mm2) and
0.20 x 0.20 mm (0.04 mm2). The centralsquare is
further subdivided into 0.05 x 0.05 mm
(0.0025 mm2) squares. The raised edges of the
hemocytometer hold the coverslip 0.1 mm off the
marked grid, giving each square a defined volume
.
Requirements
• Empty hemocytometer grid at 100x power.
• The original suspension must be mixed thoroughly before taking a
sample. This ensures the sample is representative, and not just an
artifact of the particular region of the original mixture it was drawn
from.
• An appropriate dilution of the mixture with regard to the number of
cells to be counted should be used. If the sample is not diluted
enough, the cells will be too crowded and difficult to count. If it is too
dilute, the sample size will not be enough to make strong inferences
about the concentration in the original mixture.
• By performing a redundant test on a second chamber, the results can
be compared. If they differ greatly, the method of taking the sample
may be unreliable (e.g., the original mixture is not mixed thoroughly).
Applications
• Blood counts: for patients with abnormal blood cells, where
automated counters don't perform well.
• Sperm counts
• Cell culture: when subculturing or recording cell growth over
time.
• Beer brewing: for the preparation of the yeast.
• Cell processing for downstream analysis: accurate cell numbers
are needed in many tests (PCR, flow cytometry), while some
others require high cell viability.
• Measurement of cell size: in a micrograph, the real cell size can
be inferred by scaling it to the width of a hemocytometer
square, which is known.