Abstract: Microscopes are fragile instruments that must be handle with extreme caution as they can produce high quality results when observing the smallest specimens on earth. A microscope must be properly cleaned before use and storage. The different objectives allow for a range of observations. At the highest objectives, the resolution can easily be lost which is why the oil immersion lens is used to minimize refraction. While observing specimens, both dead and active, their shapes and arrangements can be observed. For example, the oil immersion lens can be used to observe the active Streptobacillus bacteria in yogurt.
Purpose: The purpose of this lab was to display knowledge of the use of a compound microscope with and without an oil immersion lens while observing and identifying various bacterial shapes and arrangements, including a self prepared yogurt culture.
Exercise 1: Viewing Prepared Slides
1. After setting up and cleaning all lenses, place the prepared e slide properly on the stage. 2. View the slide under the10x objective by moving it around with the X and Y stage travel knobs then focus it by first using the coarse adjustment followed by the fine adjustment until the view is clear. 3. Adjust the diaphragm to allow enough light for good resolution. 4. After a micrograph is taken, rotate the 10x objective away from the specimen and the 40x over it. Use the fine adjustment knob to bring the specimen back into focus. 5. Repeat the above steps for 6 more specimens. Those viewed and micrographed in this lab are:
Part 2 of Exercise 1:
1. View 6 more prepared slides by using the oil immersion lens. Follow steps 1-5 above to locate, center, and focus each slide at 10x and 40x. 2. Then swing the 40x objective to its half way position with the 100x objective nearing the slide. 3. Add a drop of provided oil to the slide cover’s surface and slowly swing the 100x objective over. 4. Using the fine adjustment knob bring the specimen into focus and take a micrograph. 5. Repeat this process for 5 more specimens. Those that were micrograph in this lab were:
Exercise 2: Observing Bacteria Cultures in Yogurt
1. Using a clean sealable glass jar, place a teaspoon of yogurt in the container. 2. Cover the jar and place in a dark, relatively warm areas fro 12-24 hours. 3. Place a sample of the yogurt specimen in a clean slide using a toothpick and cover with a cover slip. 4. Repeat the above steps for viewing the slide at the 10x, 40x, and 100x oil immersion with the microscope. Keep the diaphragm low, as the bacteria will be transparent. 5. Repeat the viewing process with the prepared yogurt slide from the lab kit. Compare the two specimens. 6. Clean all items used in this lab: specimen vials, slides, and microscope. Carefully cover and store microscope.
Specimens observed in Exercise 1 Part 1 with the 10x objective:
Amoeba Proteus at 100x
Anabaena w.m. at 100x
Ascaris Eggs, w.m. at 100x
Paramecium Conjugation at 100x
Yeast, w.m. at 100x
Pencillium with conidia, w.m. at 100x
Specimens observed in Exercise 1 Part 2 & Exercise 2 with the 40x objective:
Bacteria Bacillus form at 400x
Bacteria Coccus form at 400x
Bacteria Spirillum form at 400x Yoghurt Bacteria at 400x
Fresh Yogurt Specimen at 400x
Specimens observed in Exercise 2 with the 100x Oil Immersion
Fresh Yogurt Specimen at 1000x
A. Identify the following parts of the microscope and describe the function of each.
A. Eyepiece lens
D. Objective Lens
H. Coarse Adjustment knob
I. Fine Adjustment knob
B. Define the following microscopy terms:
Focus: The point at which the light from a lens comes together.
Resolution: The closest two objects can be before they are no longer detected as separate objects.
Contrast: The difference in light intensity between the image and the adjacent background relative to the overall background intensity.
C. Describe your observations from the fresh yogurt slide you prepared in Exercise 2. D. Were there observable differences between your fresh yogurt slide and the prepared yogurt slide? If so, explain.
C&D: Observation of the prepared slide was made easier by the purple staining and the dead bacteria. The observation of the fresh specimen was harder to see in a focused manner because it continued to move. The prepared slide is an obvious Bacillus bacterium. While the fresh sample is harder to focus so it is not as easily observed as Bacillus bacterium. There are more bacteria present in the fresh specimen than in the prepared slide.
E Describe the four main bacterial shapes.
Cocci: a spherically shaped bacterium
Bacillus: a rod shaped bacterium
Spirillum: spirally shaped bacterium
Vibrio: comma or S shaped bacteria
F. What are the common arrangements of bacteria?
Cocci: occurring as a single sphere
Diplococci: occurring as spheres in pairs
Streptococci: chains of linked spheres
Staphylococci: spheres grouped in grape like clusters
Bacillus: a single rod arrangement
Diplobacillus: pairs of rods
Streptobacillus: Chain-linked rods
G. Were you able to identify specific bacterial morphologies on either yogurt slide? If so, which types?
In both samples Streptobacilluss arrangements and shapes were observed. They were more prevalent and easy to distinguish in the prepared slide since that bacteria was not actively moving. At times the fresh specimen at 400x looked like Staphylococci; however, upon observing the bacteria closer at 1000x it was obvious that there were no sphere shaped bacterium present.
H. What is the purpose of immersion oil? Why does it work?
Normally, the quality of an observed specimen decreases with the number of lenses, glass, etc that the light travels through. With the oil immersion lens, one of the strongest microscope lenses at 100x, the oil restricts the light refraction allowing for a clear focused image at such a high resolution. The oil mixes with the specimen and the oil itself has the same refractive index to that of glass. This gives the specimen a finer resolution and brightness than would have otherwise been observed.
Conclusion: In conclusion this lab taught one how to properly use, clean, and store compound microscope. The lab assessed the ability to observe a specimen both provided and freshly prepared with and without an oil immersion lens. This allowed for hands on learning, observation, and identification of various bacterial shapes and arrangements. One was able to extended his/her learning while ageing yogurt and making a fresh specimen on a slide to be observed at all objectives with and without oil.
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