Lab 2

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. The physical length of the marks on the scale depends on the degree of magnification. 

Formula: Micrometer pitch length(under microscope)= 

Actual micrometer pitch length/Objective lens magnification

Objectives

To measure and count the cells using a microscope 

Materials and reagents

Microscope fitted with an ocular microscope

Slide micrometer

Stained preparation of yeast and bacteria

Procedure

(refer to lab manual)

Results

Ocular Micrometer

Stage Micrometer

The scale of Ocular Micrometer and Stage Micrometer

Calculation:

All division of stage micrometer = 1mm =1000 micrometer (µm).

10x objective lens : 9.5 ocular units = 1mm.

40x objective lens : 3.9 ocular units = 0.1 mm.

The length of the yeast.

10x objectives lens : 0.1 ocular units = 0.0105 mm=  10.5 µm.

The width of the yeast.

10x objectives lens : 0.1 ocular units = 0.0105 mm=  10.5 µm.

Discussion

The dimensions of cells may be determined using an ocular micrometer. However, several steps must be completed first before microorganisms can be measured. This is because, the ocular micrometer is not calibrated with the microscopic field and with the help of stage micrometer this can be corrected. When calibrating, the scale of ocular micrometer need to be adjusted and superimposed on a parallel line with the stage micrometer. A known distance of ocular unit on the stage micrometer will be used for calculations.

1. After calibrating and using 10x objective lens it is determined that 1mm of stage scale is equal to 9.5 ocular units.
2.  Next, using the 40x objective lens and after recalibrate the system it is determined that 3.9 ocular units is equal to 0.1 mm based on the stage scale.
3. Therefore, the length of yeast is 0.0105 mm or 10.5 µm as it is measured to be 0.1 ocular unit when using the 10x objectives lens.
4.  The width of the yeast is similar to its length as it is almost round in shape and also measured to be 0.1 ocular units.

Conclusion

From the experiment, the length and width of the yeast cells is 10.5 micrometer. By using ocular micrometer and stage micrometer, the size of the microorganism like yeast can be measured.

2.2 Neubauer Chamber 

Introduction

Neubauer Chamber which is called as hemocytometer is a device used to count cells. It was originally designed for the counting of blood cells.

The hemocytometer was invented by Loius-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 which produce 9 major large squares. 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.

Materials and reagents

Serial dilutions of bacteria cultures

Neubauer and coverslip

70% ethanol

Sterile Pasteur pipettes

Procedure

(refer lab manual)

Results

Yeast was observed using hemocytometer

Total cells in 8 small boxes =  116 cells.

Average cells for 1 small boxes : 116 cells  /8 = 14.5 cells.

Volume of one small box = 0.25 mm x 0.25 mm x 0.1 mm = 6.25 x 10^-3 mm³

                                                                                      =6.25 x 10^-3 x 0.001 cm ³

                                                                                       = 6.25 x 10^-6 cm³

                                                                                        = 6.25 x 10^-6 mL.

Dilution of yeast culture = 10 times.

Cell concentration = 14.5 cells / (6.25 x 10^-6mL).

                                  = 2320000 cells =2.32 x 10⁶ cells per mL
After dilution, cell concentration = 2.32 x 10⁷ cells per mL

Discussion

Diluted yeast culture of 10 times was used for this experiment as the culture is very concentrated and a drop of it is put between the coverslip and counting chamber of hemocytometer. This step are conducted using aseptic technique to ensure sterility

1. The chamber used contains many grids but only the middle large box is use for calculation.
2. The middle large box has a size of 1mm x 1mm with a depth of 0.1 mm and contains 16 smaller boxes.
3. This means that the volume of 1 small box is 0.25mm x 0.25mm x 0.1 mm which is equal to 6.25 x 10^-3 mm³ or 6.25 x 10^-6 mL when converted.
4.  8 small boxes are chosen out of 16 and the average cells in 1 small box are calculated.

 By using the formula :   

Cell concentration = Average cells for 1 small boxes                                                                                                       Volume of 1 small box

           with dilution aspects included the cell concentration is calculated. 

Conclusion

The cell concentration of yeast is 2.32 x 10⁷ cells per mL. By using the Neubauer chamber, we can count the number of cells and hence determine the cell concentration.

References

https://en.m.wikipedia.org/wiki/Ocular_micrometer

https://en.m.wikipedia.org/wiki/Hemocytometer