1.1 Setting up and using the microscope
Introduction
A microscope is an instrument used to see objects too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope. There are many types of microscopes, the most common and first to be invented is the optical microscope which uses light to image the sample. Despite advances in other area of microscopy, the light microscope is still the instrument most frequently used for viewing microorganism. Therefore, we using microscope to observe Penicillium conidia, Rhizopus sporangia and Bacillus gram positive.
Part of Microscope
Part of microscope | Function |
Eyepiece Lens | the lens at the top that you look through. They are usually 10X or 15X power. |
Tube | Connects the eyepiece to the objective lenses |
Arm | Supports the tube and connects it to the base |
Base | The bottom of the microscope, used for support |
Illuminator | A steady light source (110 volts) used in place of a mirror. If the microscope has a mirror, it is used to reflect light from an external light source up through the bottom of the stage. |
Stage | The flat platform where the slides is placed. Stage clips hold the slides in place. Mechanical stage able to move the slide around by turning two knobs. One moves it left and right, the other moves it up and down. |
Revolving Nosepiece or Turret | This is the part that holds two or more objective lenses and can be rotated to easily change power. |
Objective Lenses | There are 4 objective lenses on a microscope. They almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. To have good resolution at 1000X, you will need a relatively sophisticated microscope with an Abbe condenser. The shortest lens is the lowest power, the longest one is the lens with the greatest power. Lenses are color coded and if built to DIN standards are interchangeable between microscopes. The high power objective lenses are retractable (i.e. 40XR). This means that if they hit a slide, the end of the lens will push in (spring loaded) thereby protecting the lens and the slide. All quality microscopes have achromatic, parcentered, parfocal lenses. |
Rack Stop | This is an adjustment that determines how close the objective lens can get to the slide. It is set at the factory and keeps students from cranking the high power objective lens down into the slide and breaking things. Adjust this if very thin slides been using and the specimen unable to focus on high power. |
Condenser Lens | The purpose of the condenser lens is to focus the light onto the specimen. Condenser lenses are most useful at the highest powers (400X and above). Microscopes with in stage condenser lenses render a sharper image than those with no lens (at 400X). |
Diaphragm or Iris | Many microscopes have a rotating disk under the stage. This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide. There is no set rule regarding which setting to use for a particular power. Rather, the setting is a function of the transparency of the specimen, the degree of contrast you desire and the particular objective lens in use. |
Magnification and Resolution
Total magnification=objective lens power x eyepiece lens power
Objective lens | Eyepiece | Magnification |
4 X | 10X | 40X |
10X | 10X | 100X |
40X | 10X | 400X |
100X | 10X | 1000X |
The resolution of a microscope is the ability to clearly determine two separate points, or objects, as singular, distinguished entities. If closer together than appropriate for your resolution, the objects or points will blur together, making it impossible to differentiate. Adjust the resolution with the resolving power of the lens of the microscope. Resolution is not magnification, which is a microscope's ability to increase size--not clarity. Magnification also utilizes lenses, but if the resolving power is poor, increasing magnification will only magnify a blurry specimen
What happens as the power of magnification increases?
What happens as the power of magnification increases?
Power = 10 x 4 = 40
Power = 10 x 10 =100
Power = 10 x 40 = 400
Objective
Please refer to the lab manual
Materials and reagent
Please refer to the lab manual
Procedure
Please refer to the lab manual
Result
 |
 |
Rhizopus sporangia (400x magnification) |
 |
Bacillus gram positive. (400x magnification) |
Discussion
- We observed the specimen using lowest magnification that is 40X to the highest magnification that is 1000X (oil immerse).This is because when light passes from a material of one refractive index to material of another, as from glass to air or from air to glass, it bends. Light of different wavelengths bends at different angles, so that as objects are magnified the images become less and less distinct. With "dry" objective lenses this loss of resolution prevents using magnifications of above 400x or so.
- When we observed the specimen, we used the fine adjustment knob and moved the stage to focus the specimen.
- The light intensity was adjusted using the brightness control.
- The diaphragm was adjusted to get a clearer view of specimen.
- We had observed 3 microorganism that is Penicillium conidia, Rhizopus sporangia and Bacillus gram negative.
(a) Penicillium conidia- filamentous fungi have branched conidiophores and is round in shape and show gram negative.
(b) Rhizopus sporangia (black bread mold) –is a type of fungi with pinhead-like structure and contains spores inside the cell. Shown gram positive under microscope.
(c) Bacillus gram positive.- rod shape bacteria
- (a) Penicillium conidia :
· The name Penicillium comes from the word "brush"; this refers to the appearance of spores in Penicillium.
· Conidia are round and unicellular
· Penicillium species tend to have small hyphae.
· The conidia of all species studied were shown to have an outer layer made up of "rodlets" which formed distinctive patterns.
(b) Rhizopus sporangia
· Rhizopus is a genus of common saprobic fungi on plants and specialized parasites on animals. They are found on a wide variety of organic substrates, including "mature fruits and vegetables", faeces, jellies, syrups, leather, bread, peanuts and tobacco
· the tip swells up to form a spherical head
· Rhizopus has large sporangial heads (green) and therefore needs a very large columella to supply the large mass of developing sporangiospores.
(c) Bacillus gram positive
· Gram-positive, capable of growth in the presence of oxygen, and forms a unique type of resting cell called an endospore
· Endospore formation, universally found in the group, is thought to be a strategy for
survival in the soil environment, wherein these bacteria predominate
- A species can be classified as Gram positive, Gram negative, or Gram variable depending on the ability if cells to retain the blue dye. Gram negative bacteria do not retain the dark blue color, but can be counterstained a light red so that they can be seen in bright field microscopy.
Conclusion
A light microscope is an instrument to observe small objects using visible light and lenses. It can be used to view living or dead samples’s shapes and colors.It can be maximize these samples up to one thousand times (1000x) their actual size.Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain (safranin or fuchsine) and appearing red or pink. Gram- positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Gram-positive cell walls typically lack the outer membrane found in Gram-negative bacteria.
Reference
sciencespot.net/Media/microparts.pdf（2011）
JSTOR provided by Universiti Sains Malaysia Library
http://en.wikipedia.org/wiki/Rhizopus
http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/bio%20102/bio%20102%20lectures/fungi/fungi.htm
http://www.uoguelph.ca/~gbarron/MISCELLANEOUS/rhizopus.htm
Todar's Online Textbook of Bacteriology(2011) Kenneth Todar, PhD University of Wisconsin
1.2 Examination of cells
Introduction
I. Oil Immersion
In light microscopy, oil immersion is a technique used to increase the resolution of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens. Immersion oils are transparent oils that have specific optical and viscosity characteristics necessary for use in microscopy. An oil immersion objective is an objective lens specially designed to be used in this way. Many condensers also give optimal resolution when the condenser lens is immersed in oil. Therefore, we using microscope to observe E.coli in a stained cell and Bacillus gram positive.
II. Wet mount
Wet mount is a glass slide holding a specimen suspended in a drop of liquid (as water) for microscopic examination. Wet mounts are necessary for examining living organisms under a microscope. Without the liquid, the organism would die. Water also allows the observer to watch the motility of the microorganism.
Objective
Please refer to the lab manual
Materials and reagent
Please refer to the lab manual
Procedure
Please refer to the lab manual
Result
Discussion
Lactobacillus
Lactobacillus is a genus of Gram-positive facultative anaerobic or microaerophilic bacteria. They are a major part of the lactic acid bacteria group, named as such because most of its members convert lactose and other sugars to lactic acid. They are common and usually benign. In humans they are present in the vagina and the gastrointestinal tract, where they are symbiotic and make up a small portion of the gut flora. Many species are prominent in decaying plant material. The production of lactic acid makes its environment acidic, which inhibits the growth of some harmful bacteria. Several members of the genus have had their genome sequenced.
Bacillus gram positive
Bacillus gram positive are gram-positive bacteria are those that are stained dark blue or violet by Gram staining. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Gram positive bacteria have a very thick outer cell layer made of peptidoglycan which stains dark purple. The bacilli part simply means the shape of the bacteria. A bacillus is a rod-shaped bacteria
Conclusion
During the experiment, we found out that Lactobacillus is gram positive cell. Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes Stains cells are colored bacterial cells and increase their contrast so they can be seen more easily with a microscope. While, mounting usually involves attaching the samples to a glass microscope slide for observation and analysis. Wet mount is a preparation of a culture to observe motility (movement) or structure of microorganisms.
Reference
http://en.wikipedia.org/wiki/Oil_immersion(2011)
http://en.wikipedia.org/wiki/Staining
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