Introduction:
Part of the process of identifying bacteria is viewing them under the microscope. There are 3 common shapes of bacteria. Bacteria are prokaryotic cells and reproduce by binary fission.
Coccus:
spherical shape, may appear oval, elongated, or flattened
Size: 0.5 – 1.0 microns
Diplococci: pair of cocci, examples (Neisseria sp.)
Chains: Streptococcus species
Tetrads: square of 4
Sarcina: cube of 8
Clusters: Staphylococcus
Bacillus: rods
Streptobacillus - in chains
Coccobacillus – oval shaped
Size: 0.5-1.0 microns wide, 1-4 microns long
Spiral:
Vibrio: comma shape (Vibrio cholera)
Spirilium: thick, rigid, spiral
Spirochete: thin, flexible spiral
Size: width typically 0.25-0.5 microns,
Length: commonly 5-40 microns, some are over 100 microns
Yeasts: unicellular fungi, eukaryotic, spherical, reproduce by budding
Size: 3-5 microns in diameter
Measurement can be approximated using a microscope with an ocular micrometer (an eyepiece with a scale superimposed) or approximating the size in comparison to the pointer.
Basic Microsopy:
Refraction: bending of a light ray as it passes from 1 medium to the another
Index of refraction: ratio of the velocity of light in a vacuum to its velocity in a given medium
Resolving power: inversely related to resolution, increased by using a shorter wavelength of light, increasing the refractive index, and increasing the aperature of the objective. A typical microscope can resolve 2 points 0.27 microns apart, and 0.2 microns for oil (the limit of the eyes).
Immersion oil increases the refractive index, increases the angle of the aperature, and decreases the scatting of light.
Use of the compound light microscope:
PLEASE CARRY MICROSCOPES CAREFULLY WITH BOTH HANDS. Always clean off oil and turn off your microscope when putting it away. (financial aid does not cover the cost of replacing a microscope!)
1. There are 4 objectives – a scanning lens, a low power, a high dry, and an oil immersion objective. The lowest power has the largest aperature and the immersion is the smallest.
2. Turn on the light. Use the 4X objective to scan the slide and make sure it is under the objective. Rotate the low power 10X objective into place. In microbiology, the 4X is rarely used. As you become comfortable with microscopy you may begin with the 10X objective.
3. Move the condenser all the way up. This position is used for almost all microscopy, but can increase contract and decrease resolution in some wet mounts.
4. Use the iris diaphragm to adjust the light intensity.
5. Adjust the distance separating the eyepieces while looking through the microscope. You should see 1 complete circle.
6. Focus on the specimen. Raise the stage as far as it will go and slowly lower the stage with the coarse adjustment until the specimen is in focus. Always focus so that you can avoid crashing the lens into the slide!
7. Finish with the fine adjustment.
Swing the high dry lens in place after focusing with the low power 10X objective. The objectives should be parfocal so only minor fine adjustment is necessary.You may need to increase the light by opening the iris since the aperature is smaller in higher objectives.To focus with oil, first focus with the lower power objectives. Open the iris and place a drop of oil directly on the stained specimen. Swing the oil lens into place, rotating in the direction avoiding passing the high dry through the oil. Make sure the long lens does not touch any part of the mechanical stage. Only fine adjustment should be needed to focus on the specimen.Remove the oil from the lens with a flat piece of lens paper. Do not leave oil on the objective as it can seep in and harm the lens.
Procedures:
For bacteria slides- view under 100x with oil
For malaria- view under 10x with oil
Most parasites may be viewed under 40x
Prepare a wet mount of your hair using a clean slide, water, and a cover slip.
What to look for in the results?
