Lab 1 : Principles and Use Of Microscope

INTRODUCTION

A microorganism is an organism that cannot seen by our naked eyes. It lives in a single unit or in a colony of cellular organism. The first microorganism discovered is by Anton van Leewenhoek’s in 1975 by using his own microscope.

Anton van Leewenhoek’s

There are many classes of bacteria such as bacteria, fungi, archaea, and protists.

Because of microorganism is in very small in size, so that in order to see it, a microscope must be use. Microscope is a kind of machine that used to magnified the view of small particle or things.

A microscope is an equipment used to see object that cannot be seen by our naked eyes because of their size is too small. Microscopic means the things that invisible to our eyes unless aided by a microscope.

There are parts in microscope that makes microscope functioning:

Eyepiece Lens: the lens that used to look the specimen. They have 2 types of power lens that are 10x and 15x.
Arm: supports the eyepiece lens holder on the base.
Illuminator: light used in order to replace sunlight.
Stage: platform that has hole at the middle to allow light from illuminator to pass through the slide contain specimen. This stages can be move.
Objective Lenses: function to magnify the specimen into particular lenses power to obtain excellence view. It contain 3 to 4 objectives lenses that are 4x, 10x, 40x and 100x.
Condenser Lens: function to focus light from illuminator direct to specimen.
Coarse and Fine Focus: function to adjust the stage to get the best image view.

Nowadays there are so many types of microscope created in order to help scientist, researcher or people to discover new things more easier.

That are:

Compound Microscope

Compound microscope is an optical device used to magnifying image and use system of combining of lenses.

Digital Microscope

Digital microscope use camera at the eyepiece to capture the image produce from the slide.

Fluoresence Microscope

Fluorescence microscope use reflection and fluorescence to light up the specimen to get excellence result.

There are many types of microscope rather than examples given. So that, this machine can help people to make research and discover new things.

Think about what you are looking for

It is a lot harder to find something when you have no expectations as to its appearance. How big is it? Will it be moving? Is it pigmented or stained, and if so what is its color? Where do you expect to find it on a slide? For example, students typically have a lot of trouble finding stained bacteria because with the unaided eye and at low magnifications the stuff looks like dirt. It helps to know that as smears dry down they usually leave rings so that the edge of a smear usually has the densest concentration of cells.

Focus, locate, and center the specimen

Start with the lowest magnification objective lens, to home in on the specimen and/or the part of the specimen you wish to examine. It is rather easy to find and focus on sections of tissues, especially if they are fixed and stained, as with most prepared slides. However it can be very difficult to locate living, minute specimens such as bacteria or unpigmented protists. A suspension of yeast cells makes a good practice specimen for finding difficult objects.

Use dark field mode (if available) to find unstained specimens. If not, start with high contrast (aperture diaphragm closed down).
Start with the specimen out of focus so that the stage and objective must be brought closer together. The first surface to come into focus as you bring stage and objective together is the top of the cover slip. With smears, a cover slip is frequently not used, so the first thing you see is the smear itself.
If you are having trouble, focus on the edge of the cover slip or an air bubble, or something that you can readily recognize. The top edge of the cover slip comes into focus first, then the bottom, which should be in the same plane as your specimen.
Once you have found the specimen, adjust contrast and intensity of illumination, and move the slide around until you have a good area for viewing.

Adjust eyepiece separation, focus

With a single ocular, there is nothing to do with the eyepiece except to keep it clean. With a binocular microscope (preferred) you need to adjust the eyepiece separation just like you do a pair of binoculars. Binocular vision is much more sensitive to light and detail than monocular vision, so if you have a binocular microscope, take advantage of it.

One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus it. Since very few people have eyes that are perfectly matched, most of us need to focus one eyepiece to match the other image. Look with the appropriate eye into the fixed eyepiece and focus with the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not the microscope.

Select an objective lens for viewing

The lowest power lens is usually 3.5 or 4x, and is used primarily for initially finding specimens. We sometimes call it the scanning lens for that reason. The most frequently used objective lens is the 10x lens, which gives a final magnification of 100x with a 10x ocular lens. For very small protists and for details in prepared slides such as cell organelles or mitotic figures, you will need a higher magnification. Typical high magnification lenses are 40x and 97x or 100x. The latter two magnifications are used exclusively with oil in order to improve resolution.

Move up in magnification by steps. Each time you go to a higher power objective, re-focus and re-center the specimen. Higher magnification lenses must be physically closer to the specimen itself, which poses the risk of jamming the objective into the specimen. Be very cautious when focusing. By the way, good quality sets of lenses are parfocal, that is, when you switch magnifications the specimen remains in focus or close to focused.

Bigger is not always better. All specimens have three dimensions, and unless a specimen is extremely thin you will be unable to focus with a high magnification objective. The higher the magnification, the harder it is to "chase" a moving specimen.

Adjust illumination for the selected objective lens

The apparent field of an eyepiece is constant regardless of magnification used. So it follows that when you raise magnification the area of illuminated specimen you see is smaller. Since you are looking at a smaller area, less light reaches the eye, and the image darkens. With a low power objective you may have to cut down on illumination intensity. With a high power you need all the light you can get, especially with less expensive microscopes.

When to use bright field microscopy

Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms. It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections. Here is a not-so-complete list of specimens that might be observed using bright-field microscopy, and appropriate magnifications (preferred final magnifications are emphasized).

Prepared slides, stained - bacteria (1000x), thick tissue sections (100x, 400x), thin sections with condensed chromosomes or specially stained organelles (1000x), large protists or metazoans (100x).
Smears, stained - blood (400x, 1000x), negative stained bacteria (400x, 1000x).
Living preparations (wet mounts, unstained) - pond water (40x, 100x, 400x), living protists or metazoans (40x, 100x, 400x occasionally), algae and other microscopic plant material (40x, 100x, 400x). Smaller specimens will be difficult to observe without distortion, especially if they have no pigmentation.

Care of the microscope

EVERYTHING on a good quality microscope is unbelievably expensive, so be careful.
Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder, for example.
Hold the plug (not the cable) when unplugging the illuminator.
Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done.
Always make sure the stage and lenses are clean before putting away the microscope.
NEVER use a paper towel, a kimwipe, your shirt, or any material other than good quality lens tissue or a cotton swab (must be 100% natural cotton) to clean an optical surface. Be gentle! You may use an appropriate lens cleaner or distilled water to help remove dried material. Organic solvents may separate or damage the lens elements or coatings.
Cover the instrument with a dust jacket when not in use.
Focus smoothly; don't try to speed through the focusing process or force anything. For example if you encounter increased resistance when focusing then you've probably reached a limit and you are going in the wrong direction.

Result

Species: Bacillus sp.

Magnification: 400X

Species: Clostridium Perfringens sp.

Magnification: 400X

Species: Aspergillus sp.

Magnification: 100X

Species: Penicillum sp.

Magnification: 100X

Disscusion

Gram staining (or Gram's method) is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical, primarily the presence of high levels of peptidoglycan, and physical properties of their cell walls. The Gram stain is almost always the first step in the identification of a bacterial organism. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram-variable and Gram-indeterminate groups as well.

The word Gram is always spelled with a capital, referring to Hans Christian Gram, the inventor of Gram staining.

Gram staining is a bacteriological laboratory technique used to differentiate bacterial species into two large groups (Gram-positive and Gram-negative) based on the physical properties of their cell walls.Gram staining is not used to classify archaea, formally archaeabacteria, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.

The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, however. It is of extremely limited use in environmental microbiology, and has been largely superseded by molecular techniques even in the medical microbiology lab. Some organisms are Gram-variable (that means, they may stain either negative or positive); some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are far more specific and information-rich than differential staining.

Gram-negative bacteria

Main article: Gram-negative bacteria

The proteobacteria are a major group of Gram-negative bacteria. Other notable groups of Gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria.

These also include many medically relevant Gram-negative cocci, bacilli, and many bacteria associated with nosocomial infections.

Gram-positive bacteria

Main article: Gram-positive bacteria

In the original bacterial phyla, the Gram-positive forms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Bacillus, Listeria,Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms.

Bacillus sp.:

Bacillus sp. Is genus of gram postive od shape bacteria. It has oth free living and pathogenic species. It form class Bacilli, family Bacillaceae and genus Bacillus. Two species are considered medically significant, B. anthracis cause anthrax and B. thuringiensis is an important pathogen. We can control the diesease by:

Sanitary: Decontamination of infected animal products, deep burial of animal carcasses and the use of protective clothing can reduce the incidence of anthrax. Proper food handling, preparation and storage are essential to preventing food poisoning.
Immunological: An avirulent spore vaccine for animals and those at high risk is available against anthrax.
Chemotherapeutic: Penicillin, erythromycin or tetracycline are drugs of choice for anthrax.

Clostridium Perfringen sp.

Clostridium perfringens sp. Is a gram positive, rod shaped, anaerobic. It come from kingdom bactria, class Clostridia, famiy Clostridiaceae, genus Clostridium and form species C. Perfringens. C. perfringens is a human pathogen sometimes, and other times it can be ingested and not cause any harm. Clostridium perfringens is commonly encountered in infections as a component of the normal flora. In this case, its role in disease is minor.

Penicillum sp.

Penicillium is a genus of ascomycetous fungi of major importance in the natural environment as well as food and drug production. It produces penicillin, a molecule that is used as an antibiotic, which kills or stops the growth of certain kinds of bacteria inside the body. It from class Eurotiomycetes, Order Eurotiales, family Trichocomaceae and genus Penicillium sp.

Aspergillus sp.

Aspergillus is a genus consisting of several hundred mold species found in various climates worldwide. Aspergillus was first catalogued in 1729 by the Italian priest and biologist Pier Antonio Micheli. Viewing the fungi under a microscope, Micheli was reminded of the shape of an aspergillum (holy water sprinkler), from Latin spargere (to sprinkle), and named the genus accordingly. Today "aspergillum" is also the name of an asexual spore-forming structure common to all Aspergilli; around one-third of species are also known to have a sexual stage. It from class Eurotiomycetes, Order Eurotiales, Family Trichocomaceae and genus Aspergillus sp.

Conclusion

This report has identified the correct way to view sample of microorganisms by using simple bright-field microscope. Different species of micrrorganisms was observed under different magnification to examine the structure and size of micrrorganisms.

Reference