Observing Bacteria and Blood

Purpose: The purpose of this experiment is to gain knowledge of the functions and operations of the compound light microscope and an immersion oil lens by observing prepared slides of various bacteria and blood slides. We are also learning to indentify and observe the various shapes and characteristics of bacteria, as well as, yogurt cultures (fresh and prepared) and blood samples under a microscopic view. We will also be able to distinguish between blood cultures and bacteria specimens.

Procedure:

Exercise 1: Viewing Prepared Slides

The first step is to assemble the compound light microscope. Secondly, we cleaned the slides and cover slips for dust and other particles. Once assembled, we begun by placing the prepared letter “e” slide on the stage and making adjustments to the condenser, diaphragm, and course/fine adjustment knobs until the image came into clear focus under 10x resolution and again under 40x resolution. Next, affix the immersion oil lens to the nosepiece. Place a drop of immersion oil onto the slide and slowly rotate the immersion oil lens into place. Use only the fine adjustment knob with the immersion oil lens to bring the object into focus. Repeat these steps with six prepared slides of different microbes and note observations.

Exercise 2: Observing Bacteria Cultures in Yogurt

Firstly, we created an incubator using a Styrofoam cooler with a removable lid and a small hole in the top which was used for placing a 7 watt light bulb in order to maintain a Microbe growing temperature of 35-37° Celsius. We also monitored the temperature with the use of a thermometer. A teaspoon of yogurt was then placed into a clean plastic container with lid. The container was then placed in the Styrofoam incubator, which was not disturbed for 24hrs. After removing the yogurt from the incubator, a toothpick was used to obtain a sample of the yogurt, which was then smeared onto a clean slide. A cover slip was used to cover the slide, which was then observed for bacteria under the microscope with the resolutions 10x, 40x, and 100x with the use of immersion oil. Observations were noted and we replaced that yogurt slide with the prepared, stained yogurt slide. We compared and noted the observations between the two yogurt samples.

Exercise 3: Preparing and Observing a Blood Slide

Firstly, we washed our hands to remove any germs or contaminants and further sterilized our little fingers with alcohol. A pin sterilized by flame was used to prick the little finger quickly and lightly. The finger was squeezed and a drop of blood was placed on a clean slide, smeared, and covered with a cover slip. The blood sample was viewed under low power then adjusted to 40x and 100x resolutions respectively. Observations were noted.

Observations/ Data Table(s):

Exercise 1: Viewing Prepared Slides.

Slide
10x
40x
100x
Bacterial Cocci
Very scarce, tiny specks
A few ~ 40-60 purple specks, looks like paint platter. Mostly very small dots Many scattered small dots/spherical shapes and a few short dashes. Some of the dots have a white dash inside of them. A few have two dashes. Bacterial Bacillus

Small purple ink blot with a small area of a spray of tiny dots surrounding (this could be an excess of dye on the slide and not the bacteria at all) Many scattered tiny dots, with a lot of white background

A few ~ 40-60 scattered small, short dashes (rods?) with a lot of white background Bactrial Spirillum
Black, tightly woven net with very small holes throughout, resembles a light coat of spray paint Thousands of close cells that look like a thick afro or a plate of black pepper with a small sprinkle of salt A few scattered black dots and many dashes, some curved, some spiral shaped, and some in clusters. There is something yellow –not sure what it is hard to make out a discernable image Amoeba w.m

Pink and purple cell bodies with irregular edged circles with a darker sphere inside each, random curved strands Cell body shaped like irregular starfish, one of the cells has a darker sphere within made up of several smaller dark dots Solid bodies with smaller dark sphere inside.

Penicillum, w/conidia
Green with many web-like connections. Looks like a pile of thin strands of thread jumbled together with thicker green tops that resemble grass Many strands with tops that look like broccolini bunched together Many groups of vertical branches resembling broccolini with several small dots along each branch Anabaena, w.m

Many pink spheres and rod shapes (maybe Staphylococci and Streptobacillus), a few clusters with small dots (maybe ribosomes, rings of spherical chains, star shaped body Vibrio shapes, large vein shaped structure with internal lines Bunches of spherical shapes (Staphylococci) looks mosaic, Streptobacillus, the veined structure is more pronounced, Vibrio shapes (slightly curved, but not distinctly spiral) Ascaris eggs, w.m

Clear branches and networks with green spheres (eggs?) scattered about in clusters, very few singular spheres Larger branch, looks like a flower with a dark outline and green hue on the inner lining, some of the spheres are transparent Some of the green spheres have jagged edges, the branch now looks grey

Exercise 2: Observing Bacteria Cultures in Yogurt
Slide
10x
40x
100x
Fresh Yogurt
Thousands of close knit black and white specks that resemble coarse gravel or the wind making waves in the ocean or a lot of bubbles Resembles a dry scab, with networks of white veins flowing throughout Cells are moving in a stream, circular dark blots and lt. grey areas Prepared Yogurt

Blue large resembles an ink blot with random dark areas and small specks Sheet of short filamentous fibers, some tightly clustered, large dark blot Smaller clusters of fibers, some look like little worms, clusters of spheres and specks

Exercise 3: Preparing and Observing a Blood Slide
Slide
10x
40x
100x
Blood Smear
Many yellow shapes that resemble cheese curls (Spirochete?), very little white background Many sphere shapes with little lines that look like one large patch The cells are moving, it is pale yellow and grey with little white background area

Results/Analysis: We were able to successfully utilize the compound light microscope and immersion oil lens to view various samples of microorganisms and document our observations. I observed different bacterial shapes and morphologies through preparation and examination of fresh yogurt and blood slides.

Questions:
Exercise 1: Viewing Prepared Slides

A. Identify the following parts of the microscope and describe the function of each.

A. Ocular Lens – the lens on the top of the microscope that are closest to the eyes and are used to view or further magnify objects with 10x or 15x power. B. Body Tube – the long tube that connects the eyepiece (oculars) to the revolving nosepiece that holds the objective lenses. C. Revolving Nosepiece – holds two or more objectives lenses and can be rotated easily to change power D. Objective lenses – the microscope is equipped with two or more objective lenses with magnifications of 4x, 10x, 40x, and 100x. E. Stage – The flat plate where the slides are placed for observation. F. Diaphragm – Generally a five hold disc placed under the stage. It is used to vary the intensity and size of the cone of light to see the slide. G. Illuminator – A light source, used to reflect light from an external light source up through the bottom of the stage. H. Coarse Focus Knob – Rough focus knob on the microscope used to move the objective lenses towards or away from the specimen. I. Fine Focus Knob – Knob used to fine tune the focus on the specimen, used after the coarse focus knob. J. Arm – part of microscope that connects the tube to the base, and is used in the transport of the microscope. K. Stage Clip – clips on the stage used to hold the slide into place L. Base – the part of the microscope that holds everything in place.

B. Define the following microscopy terms:

Focus – A means of moving the specimen closer or further away from the objective lens to render a sharp image.

Resolution – ability of a lens system to show fine details of the object being observed.

Contrast – When imaging specimens in the optical microscope, differences in intensity and/or color create image contrast, which allows individual features and details of the specimen to become visible. Contrast is defined as the difference in light intensity between the image and the adjacent background relative to the overall background intensity.

C. What is the purpose of immersion oil? Why does it work?

The purpose of immersion oil is to increase the resolution of a microscope. Immersion oil works well because it has almost the same refractive index as the glass slide; therefore, the refraction of light entering the lens becomes minimal and gives a better view of the specimen under the microscope.

Exercise 2: Observing Bacteria Cultures in Yogurt

A. Describe your observations of the fresh yogurt slide.

The microscope at a resolution of (10x) showed many black and white specks resembling coarse gravel or waves or bubbles. At a resolution of (40x) the sample resembled a dry scab, with a system of white veins going through. However, with the microscope at a resolution of (100x) the cells were seen moving in a stream like pattern with the presence of circular dark blots and grey areas.

B. Were there observable differences between your fresh yogurt slide and the prepared yogurt slide? If so, explain.

The prepared yogurt resembled short filamentous fibers with tightly clustered spheres and specks that appeared to be dormant, while the fresh yogurt had a tight network of active cells moving in a stream like manner. The fresh yogurt appeared to have less growth than the prepared slide of yogurt.

C. Describe the four main bacterial shapes.

Coccus – spherical shaped bacteria
Bacillus – bacteria that are rod shaped.
Spirillum – bacteria that have a small, curving, or spiraled shape. Vibrios – comma-shaped spirillum bacteria

D. What are the common arrangements of bacteria?

Paired – bacteria are arranged in pairs e.g., diplococci, diplobacilli Chained – bacteria are arranged in chains e.g., streptobacilli, streptcocci Clusters – bacteria arranged in clusters e.g., sarcinae, tetrads, staphylococci

E. Were you able to identify specific bacterial morphologies on either yogurt slide? If so, which types?

The bacterial morphologies were not as clear to us as we would have liked; however, the clusters of spheres we saw in the prepared yogurt slide signifies the presence of coccus bacteria.

Exercise 3: Preparing and Observing a Blood Slide

A. Describe the cells you were able to see in the blood smear. The microscopic view of the blood smear revealed hundreds of tiny spherical shaped cells, which we identified as red blood cells. The white blood cells were not as visible as the red blood cells; however, we were able to identify plasma cells.

B. Are the cells you observed in your blood smear different than the bacterial cells you have observed? Why or why not?

Yes, Bacteria have a number of shapes, which include spheres, rods, spirals, and vibrios (comma shaped) while the red blood cells were only spherical in shape.

Conclusions:

The outcome of this experiment was positive. I learned the proper way to use a compound light microscope in addition to an immersion oil lens to view prepared slides of specimens under 10x, 40x, and 100x. I was also able to prepare my own smears of fresh yogurt and blood and observe the various shapes and characteristics of their cells under the microscope. I learned that bacteria have different shapes, arrangements, and morphologies that may affect how they function.

Lab Pictures:

Microscope, lab kit contents, prepared slides, and blood Slide

Incubator, prepared yogurt, prepared yogurt slide, fresh yogurt slide