Introduction
Ocular micrometer is a glass disc with etched equally spaced divisions on surface that fits in a microscope eyepiece. It is used to measure and compare the size of prokaryotic and eukaryotic microorganisms. The ruled scale is not calibrated. When placed in the eyepiece, the line superimposed certain distance markers on the microscope field. The actual distance superimposed may be calibrated using a stage micrometer on which parallel lines exactly 0.01mm (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. Calibrating an ocular micrometer is only reliable for one objective on one scope. Changing objective lens may yield varying results, hence when you change objectives you must recalibrate the system. After the calibration of ocular micrometer, the stage micrometer is replaced with a slide containing microorganisms. The dimensions of the cells may be determined.
An Ocular Micrometer
Ruled scale of an Ocular Micrometer
Stage Micrometer
Ocular micrometer is a glass disc with etched equally spaced divisions on surface that fits in a microscope eyepiece. It is used to measure and compare the size of prokaryotic and eukaryotic microorganisms. The ruled scale is not calibrated. When placed in the eyepiece, the line superimposed certain distance markers on the microscope field. The actual distance superimposed may be calibrated using a stage micrometer on which parallel lines exactly 0.01mm (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. Calibrating an ocular micrometer is only reliable for one objective on one scope. Changing objective lens may yield varying results, hence when you change objectives you must recalibrate the system. After the calibration of ocular micrometer, the stage micrometer is replaced with a slide containing microorganisms. The dimensions of the cells may be determined.
An Ocular Micrometer
Ruled scale of an Ocular Micrometer
Stage Micrometer
Calibration of an ocular micrometer with a stage micrometer
Objective
To measure and count cells
using a microscope
Materials and Reagents
Light microscope
Ocular micrometer
Stage micrometer
Stained preparation of yeast
Procedure
1. The stage micrometer is placed on the
stage of the microscope.
2. The microscope is focused using
lowest power objective until the image on the stage micrometer is observed
superimposed on the eyepiece scale.
3. The number of divisions of the
eyepiece scale correspond top definite number of divisions on the stage scale
is determined.
4. The measurement of an eyepiece
division in micrometer (µm) is calculated.
5. The procedure is repeated using
high-power and oil immersion objective.
6. One example is shown below:
Each division of the stage micrometer =
10µm.
If 100 eyepiece divisions = 11 stage
divisions = 110µm, then:
1 eyepiece division =110/100 = 1.1µm
7. For future reference, the diameter of
the field is calculated and recorded for each objective.
8. The average dimensions (in µm) of a
sample of yeast cells is determined, followed by a sample of bacterial cells.
At least 10 observations should be included in the samples.
Result
The dimension of the yeast cell
For magnification of 10x eyepiece X 40x objective lens
= 400x magnification,
Stage scale = 0.01 mm
4 ocular unit= 0.01mm
1 ocular unit= 0.0025mm
= 2.5µm
Sample yeast
|
Ocular reading (unit)
|
Stage measurement (µm)
|
1
|
1.5
|
1.5x2.5=3.75
|
2
|
1.0
|
1.0x2.5=2.5
|
3
|
1.0
|
1.0x2.5=2.5
|
4
|
1.3
|
1.3x2.5=3.25
|
5
|
1.9
|
1.9x2.5=4.75
|
6
|
1.5
|
1.5x2.5=3.75
|
7
|
2.9
|
2.9x2.5=7.25
|
8
|
2.0
|
2.0x2.5=5.0
|
9
|
2.6
|
2.6x2.5=6.5
|
10
|
1.6
|
1.6x2.5=4.0
|
The average dimensions of yeast cell = (The total stage measurement) / (The total number of yeast) sample)
= (3.75+2.5+2.5+3.25+4.75+3.75+7.25+5.0+6.5+4.0) / 10
= 4.325 µm
Therefore, average dimension of sample yeast cell is 4.325 µm.
Discussion
To measure the size of the cell, an ocular micrometer
is required. Ocular micrometer is a glass disk that fits in a microscope
eyepiece and that has a ruled scale. When ocular micrometer is calibrated with
a stage micrometer, direct measurements or the correct scale of a cell can be
obtain. Stage micrometer is simply a microscope slide with a finely divided
scale marked on the surface. Before measure the dimension of the cell, the
stage has to be moved or adjusted until the line of ocular micrometer is
superimposed to stage micrometer. When the lines of micrometer are coincided,
then only the dimension of the cell can be measured. Besides, the appearance of image in ocular micrometer will not
change with the change of magnification but scale on stage micrometer will
change with the change in of magnification. In this experiment, with 400x
magnification, one stage scale with 0.01mm is equal to 4 ocular units which
mean one ocular unit equal to 0.0025mm or 2.5µm. Therefore,
average dimension of sample yeast is 4.325 µm.
Conclusion
With
the use of ocular micrometer together with the stage micrometer, the specific
size or dimension of microorganism, yeast cell can be measured easily.
2.2 Neubauer Chamber
Introduction
Neubauer chamber is the
most common method used to count microbes that are
in suspension . It is a heavy glass slide with two counting
areas which have specific depth.
The counting areas are separated by a H-shaped trough .
A special coverslip is placed over the counting areas and sits a precise
distance above them.
Neubauer Chamber
Counting Grid
Materials and Reagents
Serial dilutions of yeast culture
Neubauer and coverslip
70% ethanol
Sterile Pasteur pipettes
Procedure
1. A drop of diluted yeast culture is
added using a sterile Pasteur pipette to the space between the coverslip and
the counting chamber.
2. The cells are allowed to settle for
about one minute.
3. The cells in the four corner and
middle squares are counted and there should be more than 30 cells per area for
a reliable result.
4. The Neubauer chamber and the
coverslip are cleaned by using 70% ethanol.
Counting
1. The middle large square with size of
1 mm x 1 mm and depth of 0.1 mm is observed for calculation purposes.
2. There are 25 smaller squares, each
with size of 0.2 mm x 0.2 mm, inside the middle large square.
3. 10 out of 25 smaller squares are
randomly chosen to calculate the number of yeast cells in each of the squares.
4. The average number of cells per
square is then calculated.
5. The cell concentration is calculate
using the formula
Average
number of cells per square box/ Volume
of square in mL
Results:
Number of cells to be counted per square box
Average number of the cells per square box = (25 + 28 + 31 + 25 + 33 + 26 + 34 + 29 + 37 + 22) / 10
= 29 cells
Volume of the square: 0.25 mm x 0.25 mm x 0.1 mm = 0.00625 mm^3
= (0.00625 mm^3) / 1000
= 0.00000625 cm^3
= 0.00000625mL
29 cells in 0.00000625 mL,
Cell concentration = (29 cells) / (0.00000625 mL)
= 4640000 cells/mL
Discussions:
A
Neubauer Chamber is used to determine the concentration of cells by counting
the number of cells per unit volume of a suspension. A special coverslip called
haemocytometer is placed over the counting areas and sits a precise distance
above them. This haemocytometer contains 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, the number of
cells or particles in a specific volume of fluid is possible to be obtained,
and thereby calculate the concentration of cells in the fluid overall. Next,
the ruled area of the hemocytometer consists of several, large, 1 x 1 mm
(1 mm2) squares which 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.
There
are some precautions that need to be taken while determining the cell
concentration. First, always repeat the procedures to obtain average value.
Avoid improper filling of chambers which is too much or too little. We need to
ensure representative sample taken is counted with pipette and no air bubbles
are trapped. There are different types of
counting chambers available with different grid sizes, so we have to know the
grid height and size otherwise we will make calculation
errors. Next, while counting the number of cells, cells that touch the
top and right lines of a square should not be counted and cells on the bottom
and left side should be counted. Besides, we need to immobilize the moving
cells first before counting. Lastly, the objective of Neubauer Chamber is much
thicker than a regular slide, so try to avoid crashing of the objective into
the chamber when focusing.
Conclusion
Neubauer Chamber is used to count microbes and hence
determine the cell concentration. Based on the result obtained, the yeast
concentration is 4640000 cells/mL.
Reference:
There is a mistake in the calculations of the volume for heamacytometer and therefore the cell concentration is inaccurate
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