LAB 2 REPORT BY YI LEE
Name: Choong Yi Lee
matrix number: 111359
LAB 2: MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE
2.1 Ocular Micrometer
Introduction:
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 certain distance markers on the
microscope field. The actual distance superimposed maybe 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 and count cells using a microscope
Results:
Measurement Skill
After Zoom
Measurement of smaller division under 1000x power of
magnification:
(0.01x10)/40 = 0.0025m
Lactobacillus under 1000x power of magnification
After zoom
It is shown that 18 smaller division, so the
dimension of this lactobacillus is
18 x 0.0025=0.045mm
Yeast under 1000x magnification power of
magnification
After zoom
There is 4 smaller division of Yeast, so the dimension
of yeast is (0.01x10)/4=0.025mm
Discussions:
- 1) The stage micrometer is placed on the stage.
- 2) The microscope is focused until the image is observed superimposed on the eyepiece scale.
- 3) The numbers of division of the eyepiece scale is determined.
- 4) The measurement of an eyepiece division is calculated in micrometer (μm)
- 5) The high power magnification and immersion objective is used.
- 6) The result is calculated and recorded.
Advantage
The advantage of using ocular micrometer is it
can be used to measure the size of various microbes in terms of diameter and
length. Some precautions steps should be taken and always repeat the procedure
so that can obtain the average value. Make sure that the image view from
eyepiece is focus accurately and
correctly. If the image is blur. The
measurement will be incorrect and the reading may wrong.
Principles
Ocular micrometers have no units on them - they are like a ruler with marks but no numbers. In order to use one to measure something under a microscope, you must assign numbers to the marks. This is done by looking through your OCULAR micrometer at a STAGE micrometer mounted on a slide. The stage micrometer is just a ruler with fixed known distances, so you can use it to tell how far apart marks are on the ocular micrometer.This has to be done because the marks on the ocular micrometer are different distances apart depending on the magnification used on the microscope. It must be calibrated for each objective.
Principles
Ocular micrometers have no units on them - they are like a ruler with marks but no numbers. In order to use one to measure something under a microscope, you must assign numbers to the marks. This is done by looking through your OCULAR micrometer at a STAGE micrometer mounted on a slide. The stage micrometer is just a ruler with fixed known distances, so you can use it to tell how far apart marks are on the ocular micrometer.This has to be done because the marks on the ocular micrometer are different distances apart depending on the magnification used on the microscope. It must be calibrated for each objective.
References:
Conclusion:
By using ocular microscope, the size of the
cell can be measure accurately.
Comparison of size between prokaryotes with eukaryotes can be made
clearly.
2.2 Neubauer Chamber
Introduction:
Neubauer chambers are more convenient for counting microbes. The Neubauer is a heavy glass slide with two counting areas by a H-shaped trough. A special cover slip is placed over the counting areas and sits a precise distance above them.
Neubauer chambers are more convenient for counting microbes. The Neubauer is a heavy glass slide with two counting areas by a H-shaped trough. A special cover slip is placed over the counting areas and sits a precise distance above them.
Results:
Yeast under 100x of magnification
.jpg)
Yeast under 400x of magnification
.jpg)
Results:
Box
|
Number of cells
|
1
|
34
|
2
|
39
|
3
|
36
|
4
|
34
|
5
|
43
|
6
|
44
|
7
|
40
|
8
|
43
|
9
|
33
|
10
|
37
|
Average= 38.3
Volume of the square:
0.2mm X 0.2mm X 0.1mm =0.004mm³
0.004mm³ / 1000 =0.000004cm³
0.2mm X 0.2mm X 0.1mm =0.004mm³
0.004mm³ / 1000 =0.000004cm³
38.3 cells in 0.000004mL, thus
Concentration of the cells = 38.3
cells / 0.000004 mL
= 9575000 cells/mL
= 9575000 cells/mL
Discussion:
Preparation:
1)
A drop of
diluted yeast culture is added using a sterile Pasteur pipette to the space
between the cover slip and the counting chamber.
2) Wait for one minute to allow the cells to
settle
3) Count the cells in the four corner and centre
squares. For a reasonably accurate count, it should have 30 cells per area.
4)
The
Neubauer and coverslip are cleaned with 70% ethanol.
Counting:
1)
The
chamber contains many grids, producing
nine major large squares.
2) For calculation purposes, only the middle large
square is used.
3) The middle large square has a size of 1mmx1mm
and a depth of 0.1m.
4) Inside the middle large squares, there are 25
smaller squares, each with the size of 0.2mmx0.2mm
5) Randomly choose 10 of these 25 smaller squares
and calculate the number of yeast cells in each of the squares
6) Avearge the number of cells per square
7) Assuming the average number of cells=Z ; 1mm^3=
0.001cm^3; 1cm^3=1mL
8)
Cell
concentration= (250000x Z) cells/mL
Requirement:
1)
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.
2) 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.
3)
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.
Precautions:
1)
During
preparation of bacterial dilutions amount of water and sample must be accurate.
2)
Avoid
the formation bubbles while preparing the sample as possible as you can.
Reference:
Conclusion:
With the use of neubauer chamber, we
are able to count the yeast in the sample.
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