Name: Kong
Siao Thung
Matric Number: 111372
LAB 2
MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE
Introduction
2.1 Ocular
Micrometer
Ocular
micrometer is use in order to measure and compare the size of prokaryotic and
eukaryotic microorganisms. Microorganisms are measured with an ocular
micrometer which is inserted into the one of the microscope eyepieces. The
micrometer, which serves as a scale or rule, is a flat circle of glass upon
which are etched equally spaced divisions. This is not calibrated, and may be
used at several magnifications. When placed in the eyepiece, the line
superimposed may be calibrated using a stage micrometer on which parallel lines
exactly 10µm apart etched. By determining how
many units of the ocular micrometer superimpose a known distance on the stage
micrometer, you can calculate the exact distance each ocular division measures
on the microscopic field. When u change objectives you must recalibrate the
system. After calibration of the ocular micrometer, the stage micrometer is
replaced with a slide containing microorganisms. The dimensions of the cells
may then be determined.
2.2
Neubauer Chamber
Neubauer
chambers are more convenient for counting microbes. The Neubauer is a heavy
glass slide with two counting areas separated by a H-shaped trough (see Figure
2.1). A special coverslip is placed over the counting areas and sits a precise
distance above them.
Objective
To measure
and count cells using a microscope
Materials
and Reagents
2.1 Ocular
micrometer
Microscope
fitted with an ocular micrometer
Slide
micrometer
Stained
preparation of yeast and bacteria
2.2
Neubauer Chamber
Serial
dilutions of bacteria cultures
Neubauer
and coverslip
70% ethanol
Sterile
Pasteur pipettes
2.1 Ocular
Micrometer
Results:
Superimposed
image of ocular and stage micrometer (400X magnification)
10 division
on stage scale = 10x0.01mm
=0.1mm
10 division
on stage scale coincide with 40 division on ocular,
one ocular
=0.1mm/40
=0.0025mm
=2.5µm
Superimposed
image of ocular and stage micrometer (1000X magnification)
5 division
on stage scale = 5x0.01mm
=0.05mm
5 division
on stage scale coincide with 50 division on ocular,
one ocular
=0.05mm/50
=0.001mm
=1.0µm
Lactobacillus (1000x magnification)
Size of a Lactobacillus =2 x 1.0µm
= 2µm
Yeast
(1000x magnification)
Size of a
yeast = 14 x 1.0µm
=14µm
Discussions:
An ocular micrometer is a glass disk with a ruled scale that
fits into a microscope eyepiece. It is used to measure the size of objects. It use
together with a stage micrometer for measurement with greater accuracy. The
standard eyepiece reticle, when combined with a precision stage micrometer,
provides a rapid, convenient, and accurate means of conducting measurements in
the microscope.
We
use a stage micrometer to calibrate the ocular micrometer. A stage micrometer is essentially a ruler
that is mounted on a microscope slide that does have units (millimeters (mm) or
micrometers (mm)).
When calibrating, we line up the stage micrometer with the ocular micrometer and
count the number of divisions on the ocular micrometer per millimeter or
micrometer on the staged micrometer. The
number of divisions will change as the magnification changes.
2.2
Neubauer Chamber
Results:
Cells as observed on a Neubaur chamber (100x magnification)
Cells as
observed on a Neubaur chamber (400x magnification)
Average number of the cells per
square box
= (34+39+36+34+43+44+40+43+33+37)/10
=38.3
Volume of the square:
0.2mm x 0.2mm x 0.1mm = 0.004mm3
0.004mm³/1000 =
0.000004cm³
38.3 cells in 0.000004mL ,
thus the concentration of the cells
= 38.3cells/ 0.000004cm³
=9575000 cells/mL
Discussions:
The hemocytometer consists of a thick glass microscope slide with a
rectangular indentation that creates a chamber. This chamber is engraved with a
laser-etched grid of
perpendicular lines. The device is carefully crafted so that the area bounded
by the lines is known, and the depth of the chamber is also known.Therefore it
is possible to count the number of cells or particles in a specific volume of
fluid, and thereby calculate the concentration of cells in the fluid overall.
The
ruled area of the hemocytometer consists of several, large, 1 x 1 mm
(1 mm2) squares. These are subdivided in 3 ways; 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 central, 0.20 x
0.20 mm marked, 1 x 1 mm square 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. This gives
each square a defined volume.
The cell-sized structures counted lie between the middle of the
three lines on the top and right of the square and the inner of the three lines
on the bottom and left of the square.
In an improved Neubauer hemocytometer, the total number of cells
per ml can be discovered by simply multiplying the total number of cells found
in the hemocytometer grid by 104 .
Conclusions:
2.1 Ocular
Micrometer
The ocular
micrometer allows us to measure the size of Lactobacillus and yeast cells. We
can measure the exact size of these microorganisms. The size of a Lactobacillus
is 2µm and the size of yeast is 14µm under 1000x magnification.
2.2
Neubauer Chamber
By using the Neubauer chamber, we
can calculate the concentration of yeast cells of the sample we used. According
to the result, the concentration of cells is 9575000 cells/mL.
References:
academic.evergreen.edu/curricular/fcb/wk2calibration.doc
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