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 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. The scale allows the user to measure and compare the size of microbes observed. The lens need to be calibrated or adjusted. It is also need to be calibrated by using the stage micrometer. After calibration is done, the stage is changed with slide containing microorganisms. Hence, the dimension of the cells is identified. 

Materials

• Microscope fitted with an ocular micrometer
• Slide micrometer
• Stained preparation of yeast and bacteria

Objectives

• To master the usage of micrometer
• To measure the dimension of the microbes
• To identified size of different microbes

Methodology

1. The stage micrometer was placed on the stage.
2. The superimposed on the eyepiece scale on the micrometer stage is focused using the lowest power objective.
3. The divisions of the eyepiece scale correspond top a definite number of divisions on the stage scale are determined.
4. The measurement of an eyepiece division in micrometer (μm) is calculated. 
5. The steps are repeated using high-power objectives lens and oil-immersion objective.

Observation

For the first step calibration were performed to find it true value of measurement for the ocular micrometer then 10 random cell was selected for measurement of the size of a cell. The cell were measured then average size cell were calculated using formula from below:                                                                      

( average size cell= total size of all cell/number of cell)

The average size cell was calculated by using the measurement below :

1 eyepiece divission = 1μm line on eyepiece

40X magnification = 25μm

100X magnification = 10μm

400X magnification = 2.5μm

1000X magnification = 1μm 

Result

Pick random 10 cell than measure it size using ocular micrometer

Cell	Diameter (μm)
1	10
2	10
3	5
4	6
5	5
6	8
7	6
8	4
9	5
10	5

Average cell diameter = total diameter of cells / total number of cells

= (10+10+5+6+5+8+6+4+5+5)/10

= 6.31 μm

Discussion

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.

Conclusion

2.2 Neubauer Chamber

Introduction

Neubauer chambers are more convenient for counting microbes. The Neubauer is a heavy glass slide with two counting areas separated by a H-shaped trough. A special coverslip is placed over the counting areas and sits a precise distance above them.

Neubauer chamber remains the most common method used for cell counting around the world. This technical data sheet has been composed in order to help experienced, as well as non-experienced, researchers perform a proper cell counting using a Neubauer chamber or Hemocytometer. The principles described in this technical data sheet apply to any cell counting chamber, although the dimensions and volumes of each chamber may differ. The technical data sheet describes the best practices, applications, and recommendations when performing a cell count.

Materials

• Serial dilutions of bacteria cultures(yeast)
• Neubauer and coverslip
• 70% ethanol
• Sterile Pasteur pipettes

Objectives

• To calculate the concentration of cells per square
• To calculate the amount of cell in 1 square
• To find the average amount of cell per square

Methodology

1. A drop of diluted yeast culture is added to the space between the coverslip and the counting chamber using sterile Pasteur pipette.
2. The cells was rested for 1 minute.
3. The cells are counted in the four corner and the center squares. The Neubauer and coverslip are cleaned using 70% ethanol.

Counting

1. The chamber contains many grids, producing nine (9) major large squares
2. For calculation purposes, only the middle large square is used.
3. The middle large square has a size of 1 mm x 1 mm and a depth of 0.1 mm
4. Inside the middle large squares, there are 25 smaller squares, each with the size of 0.2 mm x 0.2 mm
5. 10 of these 25 smaller squares are randomly chose and  the number of yeast cells in each of the squares are calculated.
6. Average the number of cells per square
7. Assuming the average number of cells = Z; 1 mm3 = 0.001 cm3; 1 cm3 = 1 mL
8. Cell concentration = (250,000 x Z) cells/mL

Observation

A drop of yeast is put onto the hemocytometer and it is covered with a slip. The hemocytometer is then is put under the microscope. Lens is adjusted until a clear picture of cells on the hemocytometer is obtained. Cells then are counted per square in order to find the concentration of square by finding the total cell counts and the average amount. The average of concentration is obtained by using a formula stated below in the result.

Result

Square of 0.25mm × 0.25mm	Number of count cell present
1	125
2	118
3	125
4	115
5	102

Average number of cell = total cell count / volume

Volume= 0.25mm × 0.25mm × 0.1mm = 0.00625mm^3

1ml^3 = 1cm^3

0.00625mm^3 x (0.001cm^3 / 1mm^3) = 6.25x10^-6cm^3

6.25x10^-6cm^3 =  6.25x10^-6ml^3

Average number of cells = (125+118+125+115+102) / 6.25x10^-6ml^3

Discussion

1. Hemocytometer is a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is consisting of grid of perpendicular lines.
2. The area and the depth of the chamber is known so that the number of cells in a specific volume of fluid can be calculated.
3. The principles of the calculation is, choosing 10 smaller squares out of 25, inside the middle large square. Calculate the number of yeast cells in each of the 10 squares, and then the average number of cells per squares.

The middle large square is represented by the red square, the smaller square is represented by the yellow square.

Volume of small square = 0.2mm x 0.2mm x 0.1 mm

= 0.004 mm3

= 4 x 10-3 ml

Sum of cells in 10 small box = X cells

Average cells = total cells/10 = Y

Concentration of yeast cell = Y/ (4 x 10-6) ml

Conclusion

The cell populations can be measured by counting the number of cells in smaller squares and the volume of suspension is equal to the area of smaller square times the depth of film. By using Neubauer Chamber, we can easily calculate the concentration of microorganisms or cells which have an average number of cells in 10 boxes divided by volume of suspension.

LAB 1: PRINCIPLES AND USE OF MICROSCOPE AND EXAMINATION OF CELLS

INTRODUCTION

A microscope is an instrument used to see objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using such an instrument. Microscopic means invisible to the eye unless aided by a microscope.

There are many types of microscopes, and they may be grouped in different ways. One way is to describe the way the instruments interact with a sample to create images, either by sending a beam of light or electrons to a sample in its optical path, or by scanning across, and a short distance from the surface of a sample using a probe. The most common microscope is the optical microscope, which uses light to pass through a sample to produce an image.

The Parts of the Microscope

The figure shows the crucial parts of the microscope in order the microscope to function

The light sources bulb is located in the base at the instrument. It projects light upwards through the diaphragm, slide and lenses. The light sources in control by the on-off switch and the brightness of the bulb is controlled by the voltage control. The condenser helps to focus the light onto the sample analyzed. They are particularly helpful when coupled with the highest objective lens. The condenser should be adjust within 1cm of the slide when using the 40x objective and as close as possible when using 100x objective.

The condenser diaphragm controls the intensity and size of the cone light projected on the specimen. This will make sure that the correct amount of light enters the objective lens. The stage is the flat platform that support the slide being observed. The stage may be moved backwards or forwards and from left to right by using coaxial knobs.

When the coarse-focus is turned, the stage moves up or down, in order to adjust the focus. The fine-focus adjustment knobs is used fine to ajust the focus. The light microscope usually has three or four objective lenses on a microscope. They consist of 4x, 10x, 40x and 100x magnification powers. The objective lens focuses the light passing through the specimen to form a magnified primary images.

The eyepiece tube connect the eyepiece to the objective lens. It receives and redirect the light through the objective lens. The eyepiece contains the ocular lens, which provides a magnification power of 10x to 15x, usually. This is where you look through.

Magnification and resolution

The total magnification of the specimen or sample observed are calculated by multiplying the objective lens multiplication power and the eyepiece lens multiplication power. The calculations are as shown below:

4x objective X 10x eyepiece = 40x magnification

10x objective X 10x eyepiece = 100x magnification

40x objective X 10x eyepiece = 400x magnification

100x objective X 10x eyepiece = 1000x magnification

Magnification and resolution are two important things for microscope. Resolution is not magnification. Magnification is a microscope’s ability to increase size. When you magnify an images probability you may get blur images because of the useless resolution. Microscope resolution is the shortest distance between two separate points in a microscope’s field of view that can still be distinguished entities. Use the resolving power of the lens on the microscope to adjust the resolution.

OBJECTIVE

• To improve the skills in order to use a simple bright-field microscope correctly.
• To learn the importance of magnification and resolution of microscope.
• To learn the ways to take care of the microscope.

MATERIALS

• Microscope slide and cover slip

PROCEDURES

Setting up:

1. Sit on stool with knees under the bench and microscope is moved so that straining of eyes can be avoided and a comfortable environment is achieved.
2. After the power that lead to the microscope is plugged in and the power in turned on, the microscope light is turned on using main on-off switch.
3. The light intensity is adjusted using the brightness control.
4. The revolving nosepiece is rotated to bring the 4x objective lens into the light path.
5. A clean slide is taken and marked with a line on it using a marker pen. The slide is then placed on the stage and secured by the spring slip. The slide is moved into the light path using coaxial stage control knob.
6. After looking through the both eyepieces and adjusting them until a single circle of light is seen, a note in the manual of the setting is made about the interpupillary distance scale.
7. The tube length adjustment (diopter) ring on the right eyepiece is rotated and matched the interpupillar with a distance setting 1.6.
8. By using the right eye only, the marker-pen mark is focused and adjustment is made using the coarse and fine adjustment knobs.
9. By using the left eye only, focus the left eyepiece is focused by using the tube lens adjustment (diopter) ring. Then, a note in class manual of the diopter ring setting is made. A perfect binocular vision is now obtained.

Low power (10x) objective viewing:

1. The marker pen marked slide is replaced with a specimen slide.
2. Fine adjustment knob is used to focus and the stage is moved to obtain a view of the specimen. While watching from the side of the microscope, the lens is changed to the 10x objective lens.
3. An object is placed in the center of the glass above the light source for the condenser to be focused. The condenser light is adjusted until the the object is in focus.
4. The condenser is then lowered just sufficiently to throw the object out of focus.
5. To optimize image definition and contrast, one eyepiece is removed, empty tube is observed through and the condenser diaphragm is adjusted so that its edge can just be seen inside the circle of light. For specimen of poor contrast, the aperture further is reduced for a better image. The eyepiece is replaced and fine adjustment is re-focused.

High power (40x) objective viewing:

1. The specimen is focused with the 10x objective. While watching from the side of the microscope, the lens is changed to the 40x objective.
2. The condenser is raised to within 1 cm of the slide.
3. The fine focus is used for a better view and the light intensity is increased using the brightness control if necessary.
4. The condenser diaphragm is adjusted for optimum contrast.

Oil immersion (100x) objective viewing:

1. The specimen is focused with the 40x objective. The 100x (oil immersion) objective is selected while watching the microscope from the side. The objective is aware from touching the slide.
2. The objective is carefully turned to one side of the light path then one or two drops of oil are placed onto the slide. The objective is rotated until it is against the light path.
3. The condenser is raised as close to the slide as possible.
4. The fine focus is used for a better view and the light intensity is increased using the brightness control if necessary.
5. The condenser diaphragm is adjusted for optimum contrast.

After used:

1. The specimen slide was removed and discarded into the appropriate discarded container.
2. The light brightness control was reset to its lower setting.
3. The lowered power objective was reset to the working position.
4. The oil was cleaned from 100x objective using lens tissue.
5. The microscope was turned off at the on-off switch and the power was turned off at the power point. The cord was disconnected and wrapped it around the base of the microscope carefully.
6. The cover was replaced.

How do we care of the microscope:

1. The microscope is a delicate and expensive instrument. It is essential that you observe certain procedure for the care of the instrument.
2. Carried a microscope carefully, holded it firmly by the arm and supported it at the base. Kept the instrument upright.
3. The microscope should never be placed close to the edge of the bench.
4. Did not tamper with or removed any part. If the microscope did not seen to be functioned properly, asked for help.
5. Did not handle the lenses with your fingers. Used only specified lens tissues for cleaning lenses.
6. Did not allow liquids, particularly acid and alcohol, to come into contact with any part of the microscope.
7. A cover slip was used when examined objects or organisms mounted in water or other fluids.
8. Lowered the stage before placed or removed a slide.
9. Before put the microscope away, put the lowest power objective in worked position and replaced the cover.

EXAMINATION OF THE CELL

INTRODUCTION

INTRODUCTION

A cell is a small, membrane-bound compartment that contains all the chemicals and molecules that help support an organism's life. An understanding of the structure of cells is one of the first steps in comprehending the complex cellular interactions that direct and produce life.

Microscopes are required in examining cells in every sort of aspect including shape of structure , size and many others . In achieving the exact image of the particular microorganism being obseved , the observer must perform the right technique to obtain any visible image of the microorganism . Oil imersion objective is used to observe the microorganism closely after staining . While the condition of the microorganism can be determine and studied through wet mount methods .

OBJECTIVE

• To gain knowledge and proper techniques in preparing the cells .
• To visualize the image of the whole part of cells and microorganisms.
• To discover the scondition of the microorganisms.

MATERIALS

1. Culture
2. Immersion oil
3. Lens tissue
4. A microscope slide containing stained microorganisms
5. Inoculating loop
6. Bunsen burner
7. Slide and coverslip

PROCEDURE

Stained cells :

1. Microscope was set up based on the procedure given and then the slide was examined under the oil immersion lens .
2. The shape and size of each organisms including also the structures that was found through the observation had been taken using a camera.

The wet mount :

1. One drop of culture was transferred aseptically to the centre of a glass slide by using a sterile Pasteur pipette .
2. A mark was drawn on a coverslip by using a marker pen to help us focus on the microorganism better.
3. To ensure the marker pen faces down , the coverslip was taken and turned. Then, one edge of the coverslip was placed and lowered gently onto the slide to cover the drop of culture. The culture was spread in between the coverslip and the slide.
4. The slide was placed on the microorganism stage and the 4x objective lens was set to focus the culture. Result shown that about two groups of highly motile protozoa was found within the observation carried out.
5. The 10x and the 40x objective lens were used in observing the cell. Smaller moving objects were detected randomly within certain times when observed more closely. Even some of the bacterias observed were larger in size.
6. The cells was then observed using oil immersion lens ( 100x objective lens ). This time the condenser and diaphragm were reconfigured.
7. The procedure were repeated towards other cultures.

DISCUSSION

This experiment was conducted to learn how to use a simple bright-field microscope correctly. All part of the microscope has its own function and technique to be used. The image of the sample that are observed from the light microscope was actually the shadow of the object that are magnified from the sample. In this experiment, the glass slide was marked with “x” and was observed under the microscope under 40x, 100x and 400x magnification. In order to be able to see the X mark, the coaxial knobs and the lamp condenser are needed to be adjusted differently according to the magnification of the objective lens for a better result. X mark are observed from 4x objective lens up to 40x objective lens. The results obtained are as on the figures above and can be observed as the magnification increase, the image of X mark can be seen clearly as it was magnified and more detail of the X mark can be clearly observed.

Stained cells:

The light microscope is the apparatus for viewing microorganism. Each group need to learn a simple bright-field microscope correctly by using at least three specimen slide with four objectives wit magnifications 0f 4x, 10x, 40x, 100x.

The wet mount:

The wet mount method was used to determine size and shape of living microorganism. There are some special precautions that being used while applying this method, especially when we want to take one drop of the sample culture. The bottle cover needs to open 45 degree to avoid contamination. When the image is able to see under the microscope, the image is being draw. As stated before, use of oil immersion, make more powerful and high power magnification have short focus length, facilitating the use of oil.

RESULT

Images show the Clostridium Perfingens under 40x lens and the drawing of the image 

Images show the Clostridium Perfingens under 100x lens and the drawing of the image

Images show the yeast under 40x lens and the drawing of the images

Images show the yeast under 100x lens and the drawing of the images

CONCLUSION

Based on the experiment conducted, we can conclude that the 10x objective X 10x eyepiece = 100x magnification is potential to produce a firm, clear and detail of images of the microorganisms that being observed. Furthermore, the bright microscope also shows a wonderful coloured imaged from every edge and angle of the microorganisms.Based on the experiment above, we can learn on how to use microscope with the right technique and effectively so we can get a clear image of the microorganisms. We can observe that the Lactobacillus bacteria is in rod-shaped and Cocci bacteria is in spherical or generally round shape. However, we still able to found some of them are in spiral-shape.

Microscope is an instrument that are used to observe an image or microorganism clearer. Based on the experiment that had been conducted, we can conclude that ‘X’ mark is clearer with 400x magnification compared to 40x & 100x magnification. The higher the magnification, the clearer the image. Therefore, the objective of this experiment is achieved.

Stained cells:

In our experiment we use four objectives with magnifications of 4x, 10x, 40x, 100x.We start at 4x objective, followed by low power 10x objective viewing following by 40x and 100x.The specimen looked clearer at 40x magnification.We can see the shape of the specimen and the colour of the specimen.From microscope’s view we manage to know wether the specimen is gram-negative or gram-positive based on their colour.

Some specimen cannot seen clearly by using magnification at 4x, 10x, and 40x.So we use oil immersion(100x). Function of oil immersion in light microscope is used to increase the resolution of a microscope and in order to avoid the loss of clarity associated with refraction.From this method,we can see specimen clearly.

The wet mount:

From wet mount methods,we enable to study the sizes and shape of living microorganism.Besides that we enables to determine if the cells are motile.We manage to see the moving specimen by using oil immersion(100x) by using correct method and follow some precautions.