LAB 2 : MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE

Laboratory's Information :
Laboratory's Number - LAB A203
Laboratory Title : MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE
Lecturer's Name : Associate Professor Dr. Liong Min Tze
Laboratory Assistant : Madam Najmah
Group Members : Charles Ng Wai Chun, Siti Hawa Binti Ramli, Nuramirah Binti Ramlan
Date of Laboratory : 29 September 2015

LAB 2 : MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE

2.1 OCULAR MICROMETER 

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 under the microscope especially in measuring and comparing the size of prokaryotic and eukaryotic microorganisms. Suitable scales for their measurements should be somewhere in the microscope itself and the physical length of the marks on the scale depends on the degree of magnification. We must calibrate the ocular micrometer with the stage micrometer first before we want to calculate the size of a microorganism. We can use different types of magnifications to observe the samples we prepared but the calibration factors for the ocular micrometer is specific for each ocular objective combination because the objectives have different values of magnification. Therefore, each objective lens must be calibrated separately.

Steps in inserting a ocular micrometer into the eyepiece.

The ocular micrometer.

Objective

1) To measure and count cells using a microscope.

Materials and Reagents

1) Microscope fitted with an ocular micrometer.

2) Slide micrometer.

3) Stained preparation of yeast and bacteria.

Procedure

(refer to the laboratory manual)

Results

When the ocular micrometer is superimposed with stage micrometer under 40x magnification,

the calculation of one division\

50 division of stage scales superimpose with 20 divisions of ocular micrometer

1 division on stage scale = 50 x 0.01mm

                                        = 0.5mm

Therefore, one ocular division = 0.5mm / 20

                                                 = 0.025mm/25μm

When the ocular micrometer is superimposed with stage micrometer under 100x magnification,

the calculation of one division

10 divisions on stage scales superimposed with 10 divisions on ocular micrometer

1 division on stage scale = 10 x 0.01mm

                                        = 0.1mm

Therefore, one ocular division = 0.1mm / 10

                                                  = 0.01mm/10μm

For the size of one of the the yeast cells,
it covered half of an ocular division under 100x magnification,
so, its size will be

1/2 division x 0.01 mm = 0.005 mm 

                                      = 5μm

Discussion

1) Based on the experiment we conducted, we found that in order to measure the sizes of certain microorganisms, the ocular micrometer has to be calibrated with the stage scale to count the exactly length of each division in the eye piece.

2) The size of the scales of the ocular micrometer will not change as of what we can see, but the stage micrometer will change when different magnifications were applied. Therefore, there will be different values of one division under different magnifications.

3) Basically, ocular micrometer does not have units on them, so, to make use of the ocular micrometer , it has to be assigned or calibrated with the stage micrometer in order to get the the correct unit and scale

4) Then, the size of  yeast can be measured using the calibrated ocular micrometer.

Conclusion

            Ocular micrometer is a glass disk are oftenly use to measure the size of the cells easily. The exact size of a microorganism can only be determined by utilizing a calibrated ocular micrometer  It has a ruled scale and it fits in a microscope eyepiece. We can calculate the exact distance each ocular division measures on the microscopic field by determine how many units of the ocular micrometer superimpose a known distance on the stage micrometer.

Reference

1) OCULAR and STAGE MICROMETERS. Retrieved from http://fire.biol.wwu.edu/cmoyer/zztemp_fire/biol346_W06/labman_week2.pdf 

2) Wikipedia, the free encyclopedia, 8 June 2015. Retrieved from https://en.wikipedia.org/wiki/Ocular_micrometer

2.2 NEUBAUER CHAMBER

Introduction

            The neubauer chamber or hemocytometer is a device used to count cells. It is originally designed for the counting of bloods cells. The hemocytometer consists 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. This device is carefully crafted so that the area bounded by the lines is known and the depth of the chamber is also known. It is therefore possible to count the number of cells or particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

The hemocytometers.

Materials and Reagents

1) Serial dilutions of bacteria cultures.

2) Neubauer and coverslip.

3) 70% ethanol.

4) Sterile Pasteur Pipettes.

Procedure

(refer to the laboratory manual)

Results

Yeast cells on the hemocytometer.

10 results from randomly picked square boxes : 20, 19, 28, 27, 34, 29, 30, 33, 36, 26

The average number of 10 boxes = 282 / 10

                                                      = 28.2 cells

Volume of the square: 0.25 mm X 0.25 mm X 0.1 mm = 0.00625 mm³

0.00625mm³ / 1000 = 0.00000625cm³ = 0.00000625 mL 

There are 28.2 cells in 0.00000625 mL, 

thus concentration of the cells

 = 28.2 cells / 0.00000625 mL

 = 4512000 cells/mL

= 4.512 x 10⁶ cells/mL 

 Discussion

1) To count the number of cells using the Neubauer chamber, we must ensure that the coverslip is properly positioned on the surface of the counting chamber.
2) The yeast cells must be allowed to settle down before we observe and count the cells with the help of a microscope so that the yeast cells will not flow into the trough of the Neubauer chamber when we are in the process of counting the cells.
3) The randomly picked 10 square boxes from 25 square boxes has to be really random and avoid count the cells outside the square box.
4) If there are cells located between 2 square boxes, it should be counted once only and counting of the same cell twice has to be avoided so that the calculation be precise.

Conclusion

           Neubauer chamber is a thick crystal glass slide with two counting area separated by a H-shaped trough, by using the method of calculating the average of cells in a sample on the small boxes of chamber that we chosed randomly and we divided it to the volume of each box, we can calculate the concentration of the cells.

Reference

1) Celeromics. Cell Counting with Neubauder Chamber, Basic Hemocytometer Usage.  Retrieved from http://www.celeromics.com/en/resources/Technical%20Notes/cover-cell-glass.php

2) Rasayanika Biotechnology. Hemocytometer. Retrieved from http://www.rasayanika.com/product/lab-instruments/sigma_aldrich/hemocytometer/

3) Wikipedia, the free Encyclopedia. 2 February 2015. Retrieved from https://en.wikipedia.org/wiki/Hemocytometer