LAB 2 : MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE

2.1 Ocular micrometer

Introduction

Ocular micrometer is a glass disk that fits in a microscope eyepiece and that has a ruled scale; when calibrated with a slide of micrometer and hence the direct measurements of a microscopic object on prokaryotic and eukaryotic microorganism can be made. It will not change size when the objectives are changed. Therefore, each objective lens must be calibrated separately.

Ocular micrometers have no units on them - they are like a ruler with lines but no numbers. In order to use it to measure microorganism under a microscope, you must assign numbers to the lines. This is done by looking through your ocular micrometer at a stage micrometer mounted on a slide. The stage micrometer is a ruler with fixed known distances, so you can use it to measure the size of the microorganism on the ocular micrometer. The lines on the ocular micrometer are different distances apart depending on the magnification used on the microscope. It must be calibrated for each objective of the microscope. Stage micrometer is vary but most of them contain a line 2mm long that subdivided into 0.01mm (10 µm).

Figure 1 Ocular micrometer

                                          

Objective

-To measure the cells using a microscope

Materials and Reagents

-Microscope fitted with an ocular micrometer

-Slide micrometer

-Stained preparation of Penicillium

Procedure

1.   The stage micrometer is placed on the stage.

Figure 2 Stage micrometer

2.  The superimposed image of stage micrometer and eyepiece scale is focused using the objective lens with lowest power which is 4x.

Figure 3 Microscope

3.  The number of divisions of the eyepiece scale correspond to a definite number of divisions on the stage scale are determined.

Figure 4 Ocular scale and stage micrometer scale

                           
4.  The measurement of an eyepiece division in micrometer (µm) is calculated.
5.  The experiment is repeated by using the high power.

6.    Each division of the stage micrometer = 10 µm. If 100 eyepiece divisions = 11 stage divisions = 110µm, hence: 1 eyepiece division = 110/100 = 1.1µm
7.   The diameter of the field for each objective was calculated and recorded for future reference. 
8.  The sample cell’s average dimension in micrometer was determined.

  

Result

Total magnification = 40x objective X 10x eyepiece = 400x magnification  

Figure 5 Ocular micrometer and stage micrometer  under  400x magnification

                   

Discussion

1.   Ocular micrometer is a glass disc that fit in the microscope eyepiece that has ruled scale. It is used to measure the size of magnification object.
2.    Stage micrometer is a special glass slide with a known scale.
3.    Before we started to measure the size of microorganism, we spent some time to move the stage until the line of ocular micrometer is superimposed to stage micrometer. When the lines of micrometer are coincided, then we are able to measure the size of the microorganism.
4.    One division of stage micrometer = 0.01mm.
5.    For 400x magnification
     Stage scale = 0.01 mm
     Let x = 1division of the ocular micrometer
         3x =  8 division on stage micrometer x 0.01 mm
         3x =  0.08mm
           x =  0.0267mm

Therefore, one ocular division = 0.0267mm
Average dimension of sample Penicillium = 0.0267mm x 0.08 = 0.0213mm

Conclusion

Ocular micrometer let us measure the size of microorganism, Penicillium. Cells can be measured easily especially using oil immersion.

Reference
http://wiki.answers.com/Q/Why_is_it_necesary_to_calibrate_the_ocular_micrometer_with_each_objective&altQ=Why_it_is_necessary_to_calibrate_ocular_micrometer
http://www.ehow.com/how_5019336_use-ocular-micrometer.html#ixzz1rQeXORkR
http://www.medilexicon.com/medicaldictionary.php?t=55222
http://en.wikipedia.org/wiki/Ocular_micrometer

2.2 Neubauer Chamber

Introduction

To perform counting of cell accurately, Neubauer chamber is used which is analogous to the quadrat sampling. Neubauer chamber is a thick glass slide with two counting area separated by a H-shaped depression and it is 0.1mm deep(refer figure 1).A counting special cover slip which is having counting grid will set on the Neubauer chamber to allow calculation of the concentration of the cell.

Figure 1 Neubauer chamber

Materials and Reagents:

-Neubauer chamber and coverslip

-70% ethanol

-Pipette

Procedure
1.    Neubauer chamber was cleaned with 70% ethanol to remove any microorganism or unwanted debris and left it to dry.
2.    Neubauer chamber was placed and secured on the stage.
3.    The coverslip is placed carefully by 45 degree on the Neubauer chamber. 
4.    The microscope is focused until an image is formed.
5.    The diaphragm is set to smaller to increase image contrast. 

                        Figure 2 The grid layout observed through microscope of the Neubauer chamber

    

Discussion
Neubauer chamber consists of finely etched lines that crossing each other perpendicularly which creates a counting grid which aids us in counting the cell. The grid is been seen through 400x of magnification. The grid is divided into 9 large squares. Each square has a surface of  area of 1 mm² and the depth of the chamber is 0.1 mm. Therefore, the volume is 0.9 mm³.    There are 16 small squares in a large square. We also have to avoid the present of bubble in the slide of the specimen when we view the grid.

Conclusion   

In this experiment, we have learnt to setup the Neubauer chamber and the method of calculating the number of cells.

Reference
http://en.wikipedia.org/wiki/Hemocytometer

http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html