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Cell Counting and Measurement Under Magnification Report

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Updated: May 3rd, 2022

Aims of exercise 1A

The aim of this exercise is in the measurement and recording of the size of cells and tissues. Another aim of this exercise is the calibration of the eyepiece graticule using a stage micrometer.

Aims of Exercise 1C

This practical is aimed at learning to harvest cells grown in a culture and to determine the number of viable cells in the sample. Once the results have been determined, they should be collated into a table. The final aim of this practical is to determine the effect the chemical has on cell viability.

Introduction

In the study of cells, one must be able to observe them under a microscope to determine their size, structure and density. Achievement of this objective requires the use of two microscopy tools; the eye piece graticule and the haemocytometer. The eye piece graticule, also known as the ocular micrometer, is used to determine the cell structure and cell sizes under a microscope. The haemocytometer is used to determine or calculate the density of cells under a microscope.

All living organisms possess a unique cell structure that varies in size and density. Determination of this two attributes is vital in the determination of the organism’s cell placement and how the cells interrelate to each other to form a functional living organism.

Materials and methods

Exercise 1A

Materials

The materials used were: Eye piece graticule, a calibration slide (1mm), slides of H & E stained mouse stomach and lens tissue.

Methods

The first step involves cleaning of the eyepiece graticule with a piece of lens tissue. Once this has been done, remove one of the eyepieces of the microscope and then unscrew the bottom of the eyepiece. Insert the eyepiece graticule into the eyepiece and then reattach the base of the eyepiece to the microscope. Confirm that the graticule is correctly inserted by checking if it is positioned in the right way. Once this has been done, find the minute scale bar on the calibration slide and place it on the microscope stage ensuring that it is centered directly over the beam of light. The next step involves the use of a scanning objective lens to focus the divisions of the stage micrometer until the two scales are parallel and lined one above the other. Calibrate the scales by locating two points along the scales where the lines coincide. Record the results obtained from the 4x objective lens and then swap it with the other lens, follow the above laid down procedure and record the subsequent results.

Exercise 1C

Materials

The materials used to conduct this experiment were: microscope slides, cover slips, light microscope (per pair), phase microscope (per bench), tip discard, culture of human cells, haemocytometer, haemocytometer cover slip, 1ml transfer pipettes, microfuge tubes, pipettes/tips, 0.4% trypan blue stain in PBS (fresh), 0.025% trypsin EDTA, paper towels and plain paper for diagrams.

Methods

Cell culture preparation for cell counting

Check and record the confluence of the flask area and then pour the growth medium into the liquid waste container. The next step is to use 1ml of room temperature phosphate buffered saline (PBS) to rinse the cell layer. The solution is then rocked back and forth gently over the cells and then the PBS is poured into the liquid waste. The cells are then rinsed with 1 ml of 0.025% Trypsin-EDTA, a further dose is added to the flask and the solution incubated for 3-5 min on a flat surface at 37o C. The flask is then rocked gently and its side tapped to loosen the cells and break the clumps (Freshne 229). The cells are then transferred to a clean, sterile microfuge tube with a disposable transfer pipette. The cells are then centrifuged at 1000 x g for 5 min and carefully pipette off the supernatant. The cells are then washed by re-suspending the cell pellet in 1ml of PBS and then centrifuged at 1000 x g for 3 min. a pipette is then used to carefully remove and discard the supernatant and then 500µL of ice-cold buffer PBS added and the cells sat at room temperature (Freshne 26).

Vital Staining With Trypan Blue

100µL of trypan blue stain is mixed with 100µL of cell suspension and then 10µL is pipette from the mixture at the end of the cover slip and then allowed to run under it. The haemocytometer grid is then visualized under the microscope using the scanning objective. Viable (colorless) and dead (blue) cells are then counted in one or more large corner squares and the results recorded (Ajit and Amit 135).

Results

Using the equation:

Number of divisions on stage micrometer x 10µm

Equivalent number of divisions on eyepiece

The following results can be deduced as depicted in tables 1.1 and 1.2. From the tables, it is clear that the greater the magnification lens used, the greater the accuracy achieved of the size of the cell structure.

To calculate the cell concentration per ml, the following equation was used:

Average no. of cells in a 1mm corner square * dilution factor * 104. This equation gave an approximate number of cells in the base solution of a large corner square. The viability of the cells was then calculated using the equation:

No. of viable cells counted x 100= % viable cells

Total cells counted (viable + dead)

Calculation of the number of cells present in a given sample is an intricate task that requires finesse and accuracy (Andersen 239). The stains used should act as a guide in the identification of the cells in a given sample.

Discussions

The use of microscopy to determine the cell size of a given microorganism is useful to determine the micro organisms’ cell structure. From this information, one can then be able to alienate the characteristics of a given micro organism based on its cells. Furthermore, use of different magnification for the undertaking of the same task increases the accuracy of the data obtained. Secondly the use of phase, dark field, bright field and DIC in the determination of the cell shape and structure allows for one to choose a result that best shows the cell structure. Finally, counting the number of cells in a given micro organism and the preceding process used to isolate the cells while at the same time preserving them is a crucial skill that can be used in the analysis of cells. A count of the number of cells is important to ascertain the number of cells that can be present in a given tissue sample and this information would be important to the scientist to determine how the cell functions (Andersen 245).

Tables

Table 1.1.

Magnification Number of eyepiece divisions Number of calibration slide divisions Measurement of one eyepiece division (µm)
40x 40 100 2.5 µm
100x 100 100 10 µm
400x 100 2.5 2.5 µm

Table 1.2.

Slide Magnification used Number of eyepiece divisions Calibrated measurement of one eyepiece division (µm) Calculated dimensions of the structure
Thickness of the gastric mucosal layer X40 2.5 2.5 6.25
Dimension of the gastric gland in the gastric mucosa X400 4 2.5 10
Size of the lumen (hole) in a gastric gland X400 4 2.5 10
Size of a cell nucleus X400 2 2.5 5

Table on cell counts

Concentration of copper sulphate Viable: dead replicate 1 Viable: dead replicate 2 Viable; dead replicate 3 Viable; dead replicate 4 Viable; dead replicate 5
0 274:5 64:6
50 263:21 23:1 376:16 580:44
100 51:11 46:11 31:3 242:49 82:3
200 83:46 64:3 148:128 124:14
300 186:11 109:17 15:2 75:7
500 281:13 164:10 20:2
1000 234:35 97:13 13:8 21:2

Answers to questions asked

Dark field microscopy (dark ground microscopy): this method describes electron and light microscopy which usually eliminates the un-scattered beam from the image. The resultant image usually has a dark surrounding around the place where there is no specimen (Murphy 142).

Differential interference contrast microscopy (DIC): This type is also known as Nomarski microscopy and is a method used in optical microscopy illumination to augment the contrast in unstained, crystal clear specimens. This technique utilizes interferometery to access data on the optical path length of the specimen so that areas that are invisible can be seen (Barry 68).

Viewing cells under the bright field technique presents somewhat different results in comparison to other methods. The bright field technique only shows the outline of the cell with the resultant image being highly illuminated. The phase contrast and the DIC microscopy produce somewhat detailed views of the cell outline and features with the exception that the latter has a bright diffraction halo which is almost similar to the bright field technique. Finally, the dark field microscopy produced a dark image with only the basic outline of the cell being brightly illuminated.

Calculation of cell concentration

Dilution factor=2, no. of cells=200

Cell concentration= 315 x 2 x104 = 630* 104/ml

Cell viability= 295/315 x 100= 93.65%

Analysis of the effects of the copper

At zero concentration, the viable to dead cell result is different as compared to when the copper has been added. In replicates 2 and 4, results can only be seen once the copper has been added. However, the results seem to differ marginally with different concentrations of the copper though the ratio seems to remain the same at a certain given range.

Works cited

Ajit, Varma and Amit, Kharkwal. Symbiotic Fungi: Principles and Practice. Noida, India: Springer, 2009. Print.

Andersen, Robert . Algal Culturing Techniques. London, UK: Academic Press, 2005. Print.

Barry, Masters. Confocal Microscopy and Multiphoton Excitation Microscopy: The Genesis of Live Cell Imaging. SPIE Press, 2006. Print.

Freshne, Ian. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. New Jersey, US: John Wiley & Sons, 2010. Print.

Murphy, Douglas B. Fundamentals of Light Microscopy and Electronic Imaging. New Jersey, US: John Wiley & Sons, 2001. Print.

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