LAB 2: MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE by NUR DIANA BT ABDUL JALIL (114120)

2.1 Ocular Micrometer

INTRODUCTION

Ocular Microscope

Microscopes are often used to measure small objects. For instance, forensic scientists use microscopy to measure the distance between microscopic tool marks or the diameter of fibers. Compound microscopes have ocular micrometers, or rulers that can help you measure items under the microscope. However, the scale on the ocular micrometer changes with total magnification, and thus has no absolute value. Therefore, the ocular micrometer does not have units and it needs to be calibrated prior to use. 

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 will 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.




  







OBJECTIVE

To measure and count cells using a microscope

Results

l.Lactobacillus sp.
                                                                  400x magnification

                                                                  1000x magnification

2. Yeast
                                                                400x magnification

                                                                 1000x magnification

calibration:

400x: 
5 x 0.01 = 0.05
0.05/ 20 = 0.0025 mm

1000x:
5 x 0.01 = 0.05mm
0.05/ 47 = 0.0011 mm

·         Lactobacillus sp.
    400x magnification : 
     2 division x 0.0025 mm = 0.005 mm
    
     1000x magnification : 
      5 division x 0.0011 mm = 0.0055 mm

·         Yeast
      400x magnfication :
      5 division x 0.0025 mm = 0.0125 mm
       
      1000x magnification :
       7 division x 0.0011 mm = 0.0077 mm



DISCUSSION

An ocular micrometer is a small glass disk with thin lines and numbering etched in the glass.
An ocular micrometer was placed into one ocular on your microscope so that the lines superimposed on the image will allow to measure the specimen.
For each magnification, must compare the lines on the ocular micrometer to the lines on a stage micrometer.
The stage micrometer is a glass slide with precisely space lines etched at known intervals.
The vertical distance of an object that is in focus.  When magnification is increased, less of the object is in focus (depth of field decreases) – but greater detail of the area in focus can be seen.
We can adjust the focus of our eyepiece in order to make the scale as sharp as possible. If we do that, also adjust the other eyepiece to match the focus. Any ocular scale must be calibrated, using a device called a stage micrometer
The ocular lenses usually magnify 10X.  Thus the total magnification observed is the multiplication of the power of magnification of the ocular times the objective.  For example an object magnified by the ocular and the 40X high-dry objective is viewed at 4002 times its real size.  Most ocular lenses can be moved back and forth to adjust to the interpupillary distance of the student.  When first using the microscope, adjust the ocular lenses back and forth until a circular field is viewed with both eyes open.  Additionally, many microscopes allow the ocular lenses to be adjusted up and down (mechanical tube length adjustment) and there is a scale alongside the tube.  After adjusting the interpupillary distance, read the distance off the scale and adjust the tube length of the ocular lens to the same value.  Now the ocular lenses are adjusted to our eyes.

Conclusion
Ocular micrometer a glass disk that fits in a microscope eyepiece and that has a ruled scale when calibrated with a slide micrometer, direct measurements size of a microscopic object can be made.

Reference


2.2 Neubauer Chamber


INTRODUCTION

Neubauer Chamber

Neubauer chamber or hemocytometer are more convenient for counting microbes. It is a heavy glass slide with two counting areas separated by a H-shaped through figure. To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid. The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the V-shaped wells with a pasteur or other type of pipet. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power.

It is essential to be extremely careful with higher power objectives, since the counting chamber is much thicker than a conventional slide. The chamber or an objective lens may be damaged if the user is not not careful. One entire grid on standard hemacytometers with Neubauer rulings can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed.







Objective
1.      To count cells using microscope.

RESULT


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
To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned.
Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid.
The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the H-shaped wells with a pasteur or other type of pipet.
The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered.
The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power.


Conclusion
Neubauer chamber is a device used for determining the number of cells per unit volume of a suspension. Hemocytometer was widely used since it was originally designed for performing blood cell counts.


Reference


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