Lab 1: Principles And Use Of Microscope

Name of group member:
1. Chok Wen Xin (133291)
2. Leong Kah Yan (133310)
3. Yang Wen Huey(133376)
4.Joey Lew Hui Lin (133302)
Lab 1: Principles and use of microscope

1.1 Setting up and using the microscope

Introduction

Microorganism are living organism (such as a bacteria, fungi, viruses) too small to be seen with naked eye but visible under a microscope. Also called microbe. Microscopes are instruments designed to produce magnified visual or photographic images of objects too small to be seen with the naked eye. There are a lot of microscope such as Transmission electron microscopy (TEM), Scanning electron microscopy (SEM),Compound Microscope and so forth. The development of microscope started back in 14th century and until today. In this experiment, we use the light microscope to observe our specimen.

The part of the microscope

The following description is generalized to cover a typical microscope (see Figure 1.1).

Figure 1.1

  A light microscope is an instrument that uses visible light and magnifying lenses to examine small objects not visible to the naked eye, or in finer detail than the naked eye allows. Magnification, however, is not the most important issue in microscopy.The usefulness of any microscope is that it produces better resolution than the eye. Resolution is the ability to distinguish two objects as separate entities, rather than seeing them blurred together as a single smudge.

Eyepiece or ocular lens: Eyepiece is the lens, present at the top and is used to see the objects under study. Eyepiece lens contains a magnification of 10X.

Tube: Tube or the body tube, connects the eyepiece to the objective lenses.

Resolving nosepiece: It is also known as the Turret. Resolving nosepiece has holders for the different objective lenses. It allows the rotation of the lenses while viewing.

Objective lenses: There are four objective lenses found on the microscope, with ranges of 4X, 10X, 40X, 100X powers. Lenses are colour coded, the shortest lens is of the lowest power, and the longest lens is high power lenses.

Diaphragm: Diaphragm helps in controlling the amount of light that is passing through the opening of the stage. It is helpful in the adjustment of the control of light that enters.

Coarse adjustment knob: Used for focus on scanning. Usually the low power lens is used enabling the movement of the tube.

Fine adjustment knob: Used for focus on oil. Moves the body tube for focussing the high power lens.

Arm: It supports the tube of the microscope and connects to the base of the microscope.

Stage: The platform that is flat used for placing the slides under observation.

Stage clip: Stage clips hold the slides in proper place.

Base: Provides basal support for the microscope.

Power switch: The main power switch that turns the illumination on or off.

Magnification and resolution

Magnification is the ability to view an object as larger. A good image is obtained when the amount of specimen detail is also increased. Magnification alone will not achieve this.

In order to ascertain the total magnification when viewing an image with a compound light microscope, take the power of the objective lens which is at 4x, 10x or 40x and multiply it by the power of the eyepiece which is typically 10x.

Therefore, a 10x eyepiece used with a 40X objective lens, will produce a magnification of 400X. The naked eye can now view the specimen at a magnification 400 times greater and so microscopic details are revealed.

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

The resolving power or resolution of microscope is its ability to distinguish two very small and closely spaced objects as separate entities. A number of factors affect resolution- one of these is the condenser diaphragm. Closing the diaphragm image contrast but decrease resolution; opening the diaphragm decreases contrast but increase resolution.

        Magnification and resolution are equally important. You may greatly magnify an image, but unless the resolution is excellent you will see only fuzzy image. Likewise, you may obtain excellent resolution, but without magnification you will see no detail.

Objective

1. To learn the correct way to use a simple bright-field microscope.

     2.)    To understand the importance of magnification basic concept and microscope resolution

     3.)    To gain the skill to take care a microscope correctly after use

Materials and reagents

Microscope slide and cover-slip

Procedure

Setting up:

1. Sit on your stool with your knee under the bench and move the microscope so that you look through both eyepieces without straining. Make sure the sitting posture is comfortable enough to carry on the experiment.
2. Turn on the microscope light using the main on-off switch after plugging in the power lead of the microscope and turning on the power.
3. Adjust the light intensity using the brightness control. Position 5 is normally adequate.
4. Rotate the revolving nosepiece to bring the 4x objective lens into the light path.
5. Take a clean slide and mark a line on it with a marker pen. Place the slide on the stage, using the spring clip to secure it. By using the coaxial stage control knobs, move the slide into the light path.
6. Look through both eyepieces and adjust them until you see a single circle of light. Make note in your class manual of the setting on the interpupillary distance scale for future reference.
7. Rotate the tube length adjustment (diopter) ring on the right eyepiece to match your interpupillary distance setting obtained in 1.6.
8. Focus the marker-pen mark by adjusting the coarse and fine adjustment knobs by using the right eye only,
9. Using the left eye only, focus the left eyepiece using the tube lens adjustment (diopter) ring. Again for future, make a note in class manual of the diopter ring setting. You should now have perfect binocular vision.

Low power (10x) objective viewing:

1. Replace a marker pen marked slide with a specimen slide.
2. The specimen is focused using fine adjustment knob and the stage is moved in order to obtain a specimen view. Watching from the side of the microscope, changes to 10x objective.
3. Focus the condenser by placing an object, such as an inoculating loop or pencil tip, in the glass centre above the light source. Adjust the condenser light so that the object is in focus.
4. Lowered  the condenser just sufficiently to throw the object out of focus.
5. To optimize image definition and contrast, remove one eyepiece, look down the empty tube and adjust the condenser diaphragm so that its edge can just be seen inside the circle of light. For specimen of poor contrast, a better image may be obtained by reducing the aperture further. Replace the eyepiece and re-focus with fine adjustment.

High power (40x) objective viewing:

1. The specimen is focused with the 10x objective. Watching from the side of your microscope, change to the 40x objective.
2. The condenser is raised to within 1 cm of the slide.
3. Focus using fine focus and, if necessary, increase light intensity using the brightness control.
4. Adjust the condenser diaphragm for optimum contrast as above.

Oil immersion (100x) objective viewing:

1. The specimen is focused with the 40x objective. Watching from the side of your microscope, carefully select the 100x (oil immersion) objective. Do not allow the objective to touch the slide.
2. Carefully turn the objective to one side of the light path then place one or two drops of oil onto the slide. Rotate the objective so that I is again in the light path.
3. The condenser is raised as close to the slide as possible.
4. Fine control is used to focus and, if necessary, increase light intensity using the brightness control.
5. The condenser diaphragm is adjusted for optimum contrast as above.
6. Under a 100x objective lens,light will refract in different ways,causing the image to look very unfocused.
7. Oil have similar refractive index as glass,most of the light refracted through oil into lens.This increase in resolution due to a greater light-gathering ability.
8. It reduces light scatter as the light passes from the sample lens.

After use:

1. The specimen slide is removed and discarded into the appropriate discard container.
2. Light brightness control is reseted to its lowest setting.
3. Reset the lowest power objective to the working position.
4. By using lens tissue,clean the oil from the 100x objective..
5. Turn off the microscope light at the on-off switch and turn of the power at the power point. Disconnect cord and carefully wrap it around the base of the microscope.
6. The cover is replaced.

Care of the microscope:

Microscopes should be well maintained, and cared for right from the beginning. It is a delicate and expensive instrument. Proper care and maintenance of microscope will ensure this equipment will provide years of usage as well as pinpoint and accurate information.It is essential that you observe certain procedure for the care of the instrument.

1. Always carry a microscope carefully, holding it firmly by the arm and supporting it at the base. Keep the instrument upright.
2. The microscope should never be placed close to the edge of the bench.
3. If the microscope does not seem to be functioning properly, do not try to tamper with, remove any parts or fix it. Seek help from the lab assistant.
4. Do not handle the lenses with your fingers. Use only specified lens tissues for cleaning lenses.
5. Do not allow liquids, particularly acid and alcohol, to come into contact with any part of the microscope.
6. When examining objects or organisms mounted in water or other fluids, always use a cover slip .
7. The stage is always lowered before placing or removing a slide.
8. Always put the lowest power objective in working position and replace the cover,before putting the microscope away.
9. If using immersion oil, always ensure the objectives are cleaned immediately after use.  Objective, eyepieces and condenser may be removed for cleaning. Use only lens paper and lens cleaner.  Do not use solvents.
10. After using the microscope, turn off the illuminator and wait for it to cool for several minutes before putting it away. By allowing the bulb to cool you will extend its life.

Result:

Penicilum Conidia in 1000X magnification (Photo)

Penicilum Conidia in 1000X magnification (Hand Drawing)

1.2 Examination of cells

Introduction

       Because of the size of bacteria is too small, generally it does not studied with the low power objective lens or high power dry objective lens. Instead they are observed by using staining technique and with oil immersion objective.  

      The wet mount methods enable the sample to be viewed floats in a layer of water which is between the slide and the cover glass. The water performs an important optical function. Without it, the resolution power will be lower.

Objective

1. To gain the experience of using the microscope
2. To learn technique of preparing the cell properly
3. To illustrate the image of cells and microorganisms.

Materials and reagents

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. The microscope was set up based on the procedure above and the slide was examined under the oil immersion lens.
2. The shape and size including the structure of the organisms are observed and the image of organism was taken by a camera.

The wet mount:

1. A sterile Pasteur pipette was used to transfer one drop of culture to the centre of a glass slide aseptically.
2. A marker pen was used to make a mark on a coverslip. This mark will help us to focus on the microorganisms better later.
3. The mark was faced down by turning the coverslip. Then, one edge of the coverslip was placed onto the slide and lowed down gently so that it covers the drop of culture. The culture was spread between the coverslip and the slide.
4. The slide was placed on the microscope stage using the 4x objective to focus on the culture. About two groups of highly motile protozoa was observed .
5. Then, the cell was observed by using 10x and 40x objective lens. Some smaller moving objects were viewed randomly when observed more closely. Some of the larger types of bacteria were observed.
6. The cells were observed using oil immersion lens. The condenser and diaphragm were adjusted.
7. The procedure were repeated with other cultures.

Result:

Escherichia coli  in 1000X magnification

Discussion

Penicillium is a genus of ascomycetous fungi of major importance in the natural environment as well as food and drug production. When we under the 1000x magnification microscope we observed the colour Penicillium of was a part in blue rings and another part in red rings.The spores (conidia) are produced in dry chains from the tips of the phialides, with the youngest spore at the base of the chain, and are nearly always green.The common occurrence of Penicillium species in food is a particular problem。Some species produce toxins and may render food inedible or even dangerous. It is a good practice to discard foods showing the development of any mould. On the other hand some species of Penicillium are beneficial to humans. Cheeses such as Roquefort, Brie, Camembert, Stilton, etc. are ripened with species of Penicillium and are quite safe to eat. The drug penicillin is produced by Penicillium chrysogenum, a commonly occurring mould in most homes.

Escherichia coli (E. coli) bacteria normally live in the intestines of people and animals. Most E. coli are harmless and actually are an important part of a healthy human intestinal tract. However, some E. coli are pathogenic, meaning they can cause illness, either diarrhea or illness outside of the intestinal tract. The types of E. coli that can cause diarrhea can be transmitted through contaminated water or food, or through contact with animals or persons. E. coli infection causes serious problems with the blood and kidneys. We can prevent E. coli by only use pasteurized milk, dairy, and juice products and treated, or chlorinated, drinking water.

Conclusion

Based on this experiment, we can conclude that we can use the 10x objective X 10x eyepiece=100x magnification to give a clear and almost detail within the images of  microorganism to us. Bright field microscope also shows a colored images from every edge and angle of the microorganisms, that is more vitality compare to black and white colour.