LAB 2: MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE

SCHOOL OF TECHNOLOGY INDUSTRY

UNIVERSITY SCIENCES MALAYSIA

DEGREE IN BIOPROCESS

IBG 102

BIOLOGY FOR TECHNOLOGIST

Name	1.     LOH SHI WEI (137602) 2.     LAI CHONG SING (137592) 3.     SITI NORASYIKIN BINTI SALMI (137673) 4. SITI NUR SUHAILI AFIQAH BINTI SARIMAN (137674) 5.     NUR LIYANA ATHILAH BINTI MOHD AFFANDI (137636)
Title	LAB 2: MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE
Date of practical	19/09/17
Date of Report Submitted	25/09/17
Lecturer	DR. TYE

LAB 2: 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. The physical length of the microorganism on the scale depends on the degree of the magnification. Ocular micrometer is used in order to measure and compare the size of prokaryotes and eukaryotes microorganism. The micrometer which serves as a scale rule is a flat glass upon which are etched equally spaced division. The size of microorganism is determined by how many units of the ocular micrometer superimposed a known distance on the stage micrometer, we can calculate the exact distance of each ocular division measure on the microscopic field. Then the stage micrometer is replaced with the slide containing microorganism. The dimension of cell may be determined. The distance between the lines of an ocular micrometer is an arbitrary measurement that only has meaning if the ocular micrometer is calibrated for the objective being used.  A stage micrometer, also known as an objective micrometer, has scribed lines on it that are exactly 0.01mm (10 micrometres) apart. The exact distance between each ocular division measures on the microscopic field can be calculated by determining how many units of the ocular micrometer superimpose a certain distance on the stage micrometer. The calibration is important in order to obtain the measurement with more accurate and precise. In addition, it is important to know that the system should be recalibrated when the objective lens is changed. After calibration of the ocular micrometer, the stage micrometer is replaced with a slide containing microorganism

OBJECTIVE

To measure and count cells using a microscope

PROCEDURE

1.      The stage micrometer was placed on the stage
2.The lowest power objective was used to focus the microscope until the image on the stage micrometer was observed superimposed on the eyepiece scale.

3.      The division of the eyepiece scale on the sage scale was determined.

4.      The measurement of an eyepiece division was calculated in micrometer.

5.      The step 1 to 4 were repeat by using the high-power and oil immersion objective.

6.      The diameter of the field was calculated and was recorded for each objective.

7.      The average dimensions of a sample of yeast cells was determined

MATERIALS AND REAGENTS

Light microscope

Ocular micrometer

Stage micrometer

Stained preparation of yeast 

RESULT

Objective lens	Ocular micrometer	Stage scale (mm)
4x			0.025
10x			0.010
40x	39	0.1	0.0025
100x	10	0.01	0.001

                                                                                                                         

Diameter of single yeast cell

cell	Diameter reading at 100x magnification (division)
1	8
2	6
3	7
Average

Diameter of single yeast = Reading at 100x magnification (division) x 1 division (mm)

                                        = 7 x 0.001

    = 0.007 mm

DISCUSSION

1.      The ocular micrometer has no unit. In order to measure the diameter and the length of the yeast cell, the ocular micrometer should calibrate with the stage micrometer.

2.      The change of magnification of the objective lens will change the scale of the stage micrometer but not for the ocular micrometer.

3.      For example, through the experiment, for 4X magnification, there would be 38 divisions found on the ocular micrometer corresponded to 1 mm of the stage micrometer. Hence, for 1 division of ocular microscope would be approximately to 0.0263mm on the stage micrometer.

4.      When going to 10X magnification, there would be 96 divisions found on the ocular microscope corresponded to 1mm of the stage micrometer. Hence, 1 division on the ocular micrometer would be 0.01mm of stage micrometer.

5.      When keep going to 40X magnification, there would be 39 divisions found on the ocular micrometer corresponded to 0.1mm of stage micrometer. Thus, 1 division of ocular micrometer will be 0.0025mm of stage micrometer.

6.      Similarly for 100X magnification, there would be 100 divisions found on the ocular micrometer corresponded to 0.1mm of stage micrometer. Hence, 1 division of the ocular micrometer would equivalent to 0.001mm of stage micrometer.

7.      By comparing the stage micrometer for each magnification, we could know that the magnification from 4X to 10X is almost 2.5 times, from 10X to 40X is 4 times while from 40X to 100X is 10 times. Hence, we can also use this concept to get the diameter of the cell alternatively.

8.      When carrying out the experiment, the parallax error should be avoided when calibrating the ocular micrometer with the stage micrometer to get the accuracy length and width of the cell.

CONCLUSION

            Through this experiment, can learn usage of the ocular micrometer together with the stage micrometer in a correct pathway. The diameter of the single yeast cell is 7um.

NEUBAUER CHAMBER

INRODUCTION

The neubauer chamber is a thick crystal slide with two counting areas separated by H-shaped through. Neubauer chamber remains the most common method used for cell counting in the world. In a simple counting chamber, the central area is where cell counting are performed per unit volume. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts. The hemocytometer was invented by Louis-Charles Malassez and 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. The 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.

OBJECTIVE

1.      To improve our knowledge how to calculate the area of cell.

2.      To count areas of the microbes using Neubauer chambers.

MATERIAL AND REAGENTS

Serial dilutions of bacteria culture

Neubauer and coverslip

70% ethanol

Sterile Pasteur pipettes

PROCEDURES

1.      A drop of diluted yeast culture was added to the space between the coverslip and the counting chamber using a sterile Pasteur pipette.

2.      One minute was allowed for the to settle

3.      The cells in the four corner and center squares was counted

4.      The Neubauer and the coverslip was cleaned with the 70% ethanol.

RESULT

Square	No. of cell
1	122
2	141
3	65
Average	109

Volume = (0.25mm x 0.25 mm x 0.1mm) = 6.25x10^-3 mm³

Cell concentration (cells/ml): = 6.25x10^-3 mm^{3 =} 6.25x10^-6 mL

                                                = 109 ÷ 6.25x10^-6 mL

= 1.744 x 10⁷ cells/mL

DISCUSSION

1.      The Neubauer chamber consists of 9 large squares with the size of 1mm x 1mm.

2.      In the middle large square, there are consists 25 medium squares with the size of 0.25mm x 0.25mm x 0.1mm. Each medium square is consists of 16 smaller squares.

3.      The middle square is used for calculating purpose.

4.      About 3 of the randomly choose medium squares, the number of cells were calculated by finding their average number.

5.      From the experiment, the average number of cells in each medium square was 109 while the cell concentration was 1.744x107 cells/mL.

CONCLUSION

Neubauer Chamber is used to count microbes and hence determine the cell concentration. Based on the result obtained, the yeast concentration is 1.744x10⁷ cells/mL.

REFERENCES

https://www.microscopeworld.com/t-microscope_reticle_measuring.aspx
http://www.abcam.com/protocols/counting-cells-using-a-haemocytometer
http://vlab.amrita.edu/?sub=3&brch=188&sim=336&cnt=2