EXPERIMENT 3: MICROSCOPY Objective: 1. To demonstrate the correct use of a compound light microscope. 2.

To identify the basic morphologies of bacterial and fungi. Introduction: Compound light microscope is an instrument to view samples or objects that be seen by naked eyes. It is using the visible light to transmit image to our eyes. This type of microscope has more than one lens, so it is called as compound microscope. The magnification of compound microscope is the result of two lenses which are ocular lens and objective lenses. Ocular lens located at the upper part of microscope which is closest to our eyes. It will magnify images ten times (10X) than its actual size. Usually there are three or four objective lenses. Scanning objective lens is the shortest objective, which will magnify power four times (4X) than actual size. The low power objective is the next shortest objective but longer than scanning objective lens. It magnifies objects ten times (10X) their actual size. Oil immersion objective is longer than high-dry objective. High-dry objective can magnify objects fourty times (40X) than their actual size. While, oil immersion objective will magnifies objects 100X from their actual size. A drop of oil must be applied on the specimen while this objective. It will be fill space between slide and lens. Oil immersion is a technique used to increase the resolution of microscope. Microscope resolution is the ability of the lens to separate or distinguish small objects that are close together. Without immersion oil, many lights did not enter the objective due to the reflection and refraction. Immersion oil will reduce the reflection and refraction of light then focuses them into the lens. This will increase the resolution and numerical aperture.

Methods: 1. Region of a smeared and stained specimen which is well-spread and stained was focused at low power. 2. Turret was rotated to 4 and0x objective. Desired portion of specimen was located in the center of field. Refocus carefully so that specimen is focused as sharply as possible. 3. Turret was partially rotated so that 40x and 100x objectives straddle the specimen. A small drop of oil was placed on the slide in the center of the lighted area. The small drop of oil was noted directly over the area of the specimen to be examined. 4. Turret was rotated so that the 100x oil immersion objective touches the oil and clicks into place. Focus with only fine focus. Hopefully, the specimen will come into focus easily. Do not change focus dramatically. If you still have trouble, move the slide slightly left and right, looking for movement in the visual field, and focus on the object which moved. 5. Do not alter focusing with more then one specimen. A drop of oil was placed on the second specimen, and slide was slide laterally until it is in place. 6. Never go back to the 10x or 40x objectives after you have applied oil to the specimen since oil can ruin the lower power objectives. 7. The 100x oil immersion objective was wipe carefully with lens paper to remove all oil when had finished for the day. Oil from the slide was wiped thoroughly with a Kimwipe. Cleanse stage should any oil have spilled on it. The immersion oil container was recapped securely and was replaced in drawer.

3.1

Observation of microbes using light microscope

Materials: Prepared slides: E.coli, Bacillus sp., Staphylococcus aureus, Streptococcus sp., Vibrio sp., Salmonella sp., Methods: 1. Illumination system was switched on. 2. Slide was put on the stage and the lowest objective lens (10x) was positioned above the slide. 3. The stage was raised to its highest limit and was adjusted for optimal illumination. 4. The stage was lowered slowly until images were observed. Proper zone to be focused at higher magnification was identified by adjusting position of the slide using the knobs at the side of the stage. 5. 40x objective lens was moved halfway and a drop of immersion oil was applied on the slide for positioning of the immersion objective lens. The lens would be immersed completely in oil. 6. Images obtained were observed, draw and labeled.

3.2

Observation of fungi microscopic traits.

Materials: Petri dish with fungi cultures: Penicillium, Saccharomyces, Lactophenal Cotton Blue, glass slide Methods: 1. A little of the fungal culture was transferred onto a clean slide using a hooked or a looped wire. The culture that is transferred should be obtained from the peripheral region of the fungal cultures. For microscopic observation, fungal should be taken from the area around the centre of the culture as this region will contain spores that will enable identification. Do not transfer too much of the agar onto the slide. 2. The mycelium that has been transferred onto the slide is then spread finely on the slide to enable clear visualization of fungal structures. 3. A drop of Lactophenol Cotton Blue was placed on the cultures and cover slip was placed above the culture. Press gently to spread the culture evenly. 4. Structure of the fungi was observed microscopically using the microscope technique.

Results: 3.1 Observation of microbes using light microscope

E.coli

Magnification Power: 10 x 10

Magnification Power: 10 x 40

Magnification Power: 10 x 100

Bacillus sp.

Magnification Power: 10 x 10

Magnification Power: 10 x 40

Magnification Power: 10 x 100

Staphylococcus aureus:

Magnification Power: 10 x 10

Magnification Power: 10 x 40

Magnification Power: 10 x 100

Streptoccoccus pyogenes

Magnification Power: 10 x10

Magnification Power: 10 x 40

Magnification Power: 10 x100

Salmonella sp

Magnification Power: 10 x10

Magnification Power: 10 x 40

Magnification Power: 10 x 100

3.2

Observation of fungi microscopic traits.

Saccharomyces

Magnification Power: 10 x 10

Magnification Power: 10 x 40

Magnification Power: 10 x 100

Penicillium

Magnification Power: 10 x 10

Magnification Power: 10 x 40

Magnification Power: 10 x 100

Discussion: Objective lens on compound light microscope will collect lights emerging from samples and focus them into the objective turret. Magnification from this lens will combined with magnification from ocular lens when viewing images. Usually there are three or four types of objective lens. They are scanning objective lens, low power objective lens, high-dry lens and oil immersion lens. All of this objective lens has different magnification. Scanning objective lens is the shortest lens therefore its magnification power is the lowest. Its function is to locate specimen on slide since it will magnify images to 40x of their actual size. Thats why we have to start with the lowest lens when observing a slide. Furthermore, this lens also will help in observing the entire specimen in a few minutes. The low power objective lens has 100x magnification power. It will allow us to quickly scan large area of the specimen. So that, we can spot area which need to be observe and studied under high-dry objective lens. There are two types of high power objective lens, which are high-dry objective lens and oil immersion objective lens. These objective lenses will magnify specimens to provide detailed images. High-dry objective lens will magnify images to 400x of their actual size while oil immersion lens will magnify until 1000x of actual size. Oil immersion objective lens has the longest lens, so it has the shortest wavelength of light. Actually, only a little light ray can enter into objective because of the reflection and refraction of light at the surface of the objective lens and slide. In order to increase the resolution, immersion oil will be applied on the space between slide and lens. This is because the reflection and refraction of light ray will be reducing. Thus, many light rays will be entering into the objective lens and slide.

Figure 1: Reflection and refraction of light ray with and without immersion oil

Due to different environment and habitat, most of bacteria have different shape and structure of cell wall. Actually, we can observe the basic morphology of bacteria under compound light microscope. But in this experiment, most of the images from prepared slides not really clear under 40x, 100x and 400x objective lens, so it is hard to observe for their shape and arrangement in colonies. This problem occurs maybe because of some problem happens during preparation of slide. There are some techniques applied when preparing slide. When bacteria are heat-fixed and stain, they tend to clumps together. So, it is difficult to detect the individual cell except under the highest resolution microscopy. It is necessity to used immersion oil to view better images. Beside that, when slide is staining, the percentage of the bacteria to die is higher and then will disrupt their actual shape. Furthermore, technique while handling microscope also important while observing specimens. The reason why we cannot see the shape and arrangement of specimens observed maybe because we cannot focus to the specimen and cant control light entering the lens. This will result in blur images of specimens observed. Most bacterial present in rod-shaped known as bacillus or round-shape known as cocci. But there are some species with curved, spiral-shaped, or irregularly shaped. Bacterial cells form clumps which have specific arrangement which are paired, in chains, in clusters or random. E.coli, Bacillus sp. and Salmonella sp. are rod-shape bacteria while Staphylococcus aureus and Streptoccoccus pyogenes are round-shape bacteria. Staphylococcus aureus is arranged into grape-like cluster while Streptoccoccus pyogenes arranged into chains. For experiment observing the microscopic fungi, we can clearly observe thallus include sporangium of Penicillium and Saccharomyces. We can observe microscopic fungi better than bacteria because microscopic fungi are much bigger than bacteria. But we cannot clearly observed and differentiate their mycelium. This is maybe because of too much of agar was transfer onto the slide while slide preparation. This will result in too thick section of fungi on our slide. Conclusion: The correct use and handling compound light microscope will help us to view clear and better images of the specimens observe. Some mistake done while using this microscope will result in the blur and unclear images. Under compound light microscope actually we can clearly seen the basic morphologies of bacteria and fungi, which is we can differentiate their shape and arrangement in a colony.

References: 1. Light Microscope, retrieved on 15th October 2011 from http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html 2. What is A Light Compound Microscope?, retrieved on 15th October 2011 from http://tami-port.suite101.com/what-is-a-compound-light-microscope-a68284 3. Oil immersion, retrieved on 15th October 2011 from http://en.wikipedia.org/wiki/Oil_immersion 4. What is the function of a scanning objective on the microscope?, retrieved on 16th October 2011 from http://answers.yahoo.com/question/index? qid=20100902161535AAley1K 5. What is the function of the low-power objective on a microscope?, retrieved on 16th October 2011 from http://wiki.answers.com/Q/What_is_the_function_of_the_lowpower_objective_on_a_microscope#ixzz1avjXXUe6 6. Fungi Under the Microscope, retrieved on 16th October 2011 from http://sites.google.com/site/scottishfungi/identification/microscopic