LAB 2 (NOR SHAQIRA BT AZLAN)


LAB 2 : MEASUREMENT & COUNTING OF CELLS USING MICROSCOPE

INTRODUCTION

               An ocular micrometer is a glass disk that fits in a microscope eyepiece that has a ruled scale, which is used to measure the size of magnified objects. The physical length of the marks on the scale depends on the degree of magnification. The ruler on a typical ocular micrometer has between 50 to 100 individual marks, is 2 mm long and has a distance of 0.01 mm between marks.
The procedure to use ocular micrometer is firstly, measure the actual size of the letter on the microscope slide using the millimeter ruler. This measurement will help you calibrate the ocular micrometer to determine if it is giving you accurate measurements.
Then, attach the ocular micrometer to the microscope eyepiece by unscrewing the eyepiece cap, placing the ocular micrometer over the lens and screwing the eyepiece cap back into place. Some microscopes may have an ocular micrometer pre-installed, allowing you to skip this step.



Figure : ocular scale (above) and stage micrometer scale (below)

                   The procedure in using Neubauer Chamber is firstly, Ensure that the special cover slip provided with the counting chamber (thicker than standard cover slips and with a certified flatness) is properly positioned on the surface of the counting chamber. When the two glass surfaces are in proper contact Newton's rings can be observed. If so, the cell suspension is applied to the edge of the cover slip to be sucked into the void by capillary action which completely fills the chamber with the sample. Looking at the chamber through a microscope, the number of cells in the chamber can be determined by counting. Different kinds of cells can be counted separately as long as they are visually distinguishable. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture the sample comes from. It is the number of cells in the chamber divided by the chamber's volume (the chamber's volume is known from the start).


RESULT

2.1 Ocular Micrometer

Yeast

400x magnification :
5 X 0.0025mm = 0.0125mm

figure : yeast 400x magnification
 1000x magnification :
 7 x 0.0011mm = 0.0077mm

figure : yeast 1000x magnification


Lactobacillus sp.

400x magnification : 
2 x 0.0025mm = 0.005mm

figure : Lactobacillus sp. 400x magnification
    
 1000x magnification : 
 5 x 0.0011mm = 0.0055mm
figure : Lactobacillus sp. 1000x magnification

2.2 Neubauer Chamber

sum of the cell 10 box = 642
average = 642/10 = 64.2

volume box (16 box) = 0.2 mm x 0.2 mmx 0.1 mm
                                 = 4 x 10^-3 mm
                                 = 4 x 10^-6 cm^3
64.2 cell in 4 x 10 ^-6 ml
1 ml = 642/ 4 x 10 ^-6 = 1.61 x 10^8 cell/ml


DISCUSSION

2.1 Ocular Micrometer

 magnification X400.

5 X 0.01 = 0.05
0.05/20 = 2.5 micrometer

Suppose the full length of the reticle scale covered 25 divisions of the stage micrometer.
Then the full length of the reticle scale is equivalent to (25 x 0.1mm) = 2.5mm long.
For an eyepiece reticle with 100 divisions, each division will measure 25µm at the stage for this
magnification.

magnification X1000.

5 X 0.01 = 0.05
0.05/47 = 1 micrometer

Selecting the X100 objective and repeating the exercise above would show that the reticle scale
now covers 10 divisions of the stage scale.
Then the full length of the reticle scale is equivalent to (10 x 0.1mm) = 1mm long.
For an eyepiece reticle with 100 divisions, each division will measure 10µm at the stage for this
magnification.

In summary you can apply these conversion factors to state what each division of the eyepiece
reticle is measuring for a selected magnification.
X400  :  1 division = 25µm
X1000 :  1 division = 10µm

2.2 Neubauer Chamber


Suppose that we  conduct a count on 10 box, and count 642 particles in all 10  small squares described. Each square has an area of 0.04 mm-squared and depth of 0.1 mm. The total volume in each square is (0.04)x(0.1) = 0.004 mm-cubed. We  have 10 squares with combined volume of 10x(0.004) = 0.04 mm-cubed. Thus we  counted 642 particles in a volume of 0.004 mm-cubed, giving you 642/0.04 = 1.6 x 10^4 particles per mm-cubed. There are 1000 cubic millimeters in one cubic centimeter (same as a milliliter), so your particle count is 16,050,000 per ml.

CONCLUSION

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.

Cell culture and many applications that require use of suspensions of cells it is necessary to determine cell concentration. One can often determine cell density of a suspension spectrophotometrically, however that form of determination does not allow an assessment of cell viability, nor can one distinguish cell types.




 REFERENCES


http://en.wikipedia.org/wiki/Ocular_micrometer
academic.evergreen.edu/curricular/fcb/wk2calibration.doc
academic.evergreen.edu/curricular/fcb/wk2calibration.doc
en.wikipedia.org/wiki/Hemocytometer
sfiles.crg.es/protocols/cellculture/img/neubauer.jpg/view







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