Matric card no.:111431
Lab 2: Measurement and Counting of Cells using Microscope
2.1 Ocular Micrometer
Introduction:
Ocular micrometer is used 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
eyepieces. The micrometer, which serves as a scale or ruler, 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 certain distance markers on the micrometer
field. The actual distance 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 you 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.
Objective:
To measure the cells by using a microscope.
Results:
 |
| Photo that shows the size of yeast cell under 400x magnification. |
 |
| Photo that shows size of yeast cell under 1000x magnification. |
 |
| Photo that shows the size of Lactobacillus cells under 1000x magnification. |
1.)
For 400x magnification:
Calibration= stage micrometer ( mm ) /
eyepiece division ( no. of intervals )
= 0.01mm / 4
= 0.0025mm
=2.5μm
2.)
For 1000x magnification:
Calibration= stage micrometer ( mm ) /
eyepiece division ( no. of intervals )
= 0.001mm / 10
= 0.001mm
= 1μm
1.)
Therefore,
a.) The size of Yeast under 400x magnification= Intervals on the eyepiece graticule X
a.) The size of Yeast under 400x magnification= Intervals on the eyepiece graticule X
calibration under 400x magnification
= 2 X 2.5μm = 5μm
b.)The size of Yeast under 1000x magnification =
Interval on the eyepiece graticule X
calibration under 1000x magnification
=5 X 1= 5μm
c.) The size of Lactobacillus
under 1000x obj.magnification = Interval on the eyepiece graticule X
calibration under 1000x
magnification
= 3 X 1μm= 3μm
= 3 X 1μm= 3μm
Discussions:
1.)
An ocular micrometer is a glass disk that
attaches to a microscope’s eyepiece. I t has a ruler that allows the user to
measure the size of magnified objects.
2.)
The distance between the marks on the ruler
depends upon the degree of magnification. When we change magnifications, it
appears as though as the size oat the stage micrometer is changing while the
ocular micrometer remains fixed. Both micrometers actually stay fixed, but the
view of the stage microscope becomes distorted as the magnification changes.
3.)
Before we started to measure the cells, we took
some time to move the stage until the lines of the ocular micrometer
superimposed on the stage micrometer. This step is taken due to we could only
able to count the spaces of each micrometer to a point at which the lines of
the micrometers coincided.
Conclusion:
As a conclusion, the size of microorganisms can be measured by using ocular micrometer. Besides, i found that the size of Yeast is bigger than the Lactobacillus.
Reference:
2.2 Neubauer Chamber
Introduction
Neubauer chambers are more convenient for counting microbes.
The Neubaucer is a heavy glass slide with two counting areas which seperated by
a H-shaped trough. A special coverslip is placed over the counting areas and
sits a precise distance above them.
Objective:
To count cells by using microscope.
Results:
 |
| Photo that shows the no. of cells (Yeast) under 40x objective magnification. |
1.)
Volume of the 16 small boxes = 0.2mm x 0.2mm x 0.1mm
=
0.004mm³
=
0.000004 cm³
=
0.000004 mL
12
|
11
|
12
|
|
9
|
16
|
||
15
|
15
|
17
|
|
8
|
8
|
2.)
Total no. of cells counted in the 10 small boxes
= 123cells
3.)
Average no. of cells in 10 small boxes = 123cells/
10
= 12.3cells
4.)
The cell concentration = 12.3cells / 0.000004 mL
= 3075000
cells / mL
Discussions:
1.)
There are several precaution steps in this
experiment:
a.)
Clean chamber and coverslip with lens paper with
a little ethanol to remove any grease.
b.)
Hold the pipette with one hand and use the upper
surface of the index finger of the other hand to guide the tip; the pipette
should be held at 45 ̊ angle to the chamber. The chamber should fill very quickly with
liquid by capillary action. Not much pipette bulb pressure is required.
If there is any slow filling or if the liquid front is not even, for example
when a bubble is formed, we need to start over.
c.)
Place
the cover slip over the counting platform, pressing on the elevated ridges of
the hemocytometer, but not the center.
2.)
During calculation of cells, we do not need to
count the whole chamber. We just count in a section of the chamber and use the
grid to determine what proportion of the chamber that is. Then we continue our
calculation by estimating how many cells are in the chamber.
3.) Scan
square subdivisions from left to right, up to down and count the cells. Count
the cells if they are touching the left or top line of each square (doesn’t
matter which ones- it can be bottom and right line but be consistent!) too,
make sure that the cells that touching the lines are not counted twice.Do not
count the cell in the bottom row either.
Conclusion
From this experiment, I found that the cell concentration
for yeast is 3075000cells per mL. Besides, I have learned the method to calculate the
cells by using the Neubauer chamber.
References:
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