Abstract/Summary: Changing our focus from the properties and functions that take place within the cell to the actual cell itself, we observed three specific bacterial types within this lab: Gram-positive bacteria, Gram-negative bacteria, and cyanobacteria. We closely observed the features of cyanobacteria and were able to differentiate them from other bacteria. We specifically observed gloeocapsa, merismopedia, anabaena, and oscillatoria. In addition to this we observed the different characteristics of prokaryotic organisms referring to size, form, color, elevation and texture, which helped us to understand the diversity of these cell types in the kingdom Monera. Through culturing samples from both the toilet and our own mouths, we were able to observe Gram-negative bacteria. We also sketched our two examples of Gram-negative bacteria. Though we failed to observe Gram-positive bacteria, this may have been due to our sampling and staining procedure. Because of the concentration of the sample towards the center, bacteria within this region are hard to see under oil immersion microscopy. If Gram-positive bacteria had been present, it was covered by layers of both Gram-positive and Gram-negative bacteria making it extremely difficult to differentiate while viewing under a light microscope.
Introduction: Most of the experiments thus far in this laboratory have covered many of the functions carried out within the cell such as enzyme activity, photosynthesis, reproduction, and genetics. This experiment gave us a chance to examine the overall single prokaryotic cell in its entirety instead of just the functions it can carry out. We will encounter 4 groups under the kingdom Monera: archaeobacteria, Gram-negative bacteria, Gram-positive bacteria, and Mycoplasms. We will also examine the diversity of prokaryotic cell types. For example some are pathogenic, causing diseases, while other are saprophytic, breaking down organic material, and further still some bacteria are autotrophs, making their own food through photosynthesis or chemiosmosis (Lab Manual, 2001).
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We will also examine the characteristics of these prokaryotic organisms such as the visible size, form, color, elevation, and texture of a colony or colonies. To get detailed observations of these various organisms we will view their occurrence through the culturing of samples by adding microbes onto a sterile medium within Petri dishes and allowing them to multiply. Finally we will also examine the features of cyanobacteria that distinguish them from other bacteria, and their role in nature.
Materials/Methods: First examine the plates prepared in the previous week and observe as many of your other classmates plates as possible. While observing keep in mind the number of organisms or colonies formed as well as the types of organisms. Also note the number of different colony types based in colony size, shape, color, elevation, and texture. Move on to the second part of the procedure involving Gram staining. First wash a microscope slide with soap and water and dip the slide in alcohol and allow to air dry. Put two drops of deionized water on the slide. Flame a bacterial loop to kill any foreign organisms then use it to scoop a small portion of the bacterial colony off the agar plate and spread it evenly on the slide’s center. Resterilize the loop. Allow the slide to air dry. Using a clothespin, gently heat the slide passing it three times through low flames to allow the cells to adhere to the surface. After it is cooled flood the surface with Gram’s stain. After waiting a minute rinse off the stain with water. Now flood the slide with aqueous iodine to enhance color development, then rinse the slide with water again after waiting another minute. Squirt 95 percent ethanol over the surface of the stained slide carefully until the runoff is clear.
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Rinse with water. Now flood the surface with safranin, a counterstain. After waiting a minute carefully rinse your slide with water. Blot the edges of your slide dry with a paper towel and examine under oil immersion. Record your observations and make a sketch. If time permits examine your own oral bacteria. Scrape the tartar between your teeth and smeer it on a slide and heat the slide to promote adhering. Add a drop of crystal violet stain and wash off with water after a minute then blot the slide edges dry. Allow your slide to air dry and then examine under oil immersion and sketch your observations. For the third portion of our experiment we will observe cyanobacteria. Prepare a wet mount of Gloecapsa. Then place a drop of India ink to one side of the coverslip and promote diffusion by placing absorbent tissue on the opposite side of the coverslip. Describe the color by shade distribution and comment whether it is localized evenly spread throughout the cell. Prepare a wet mount of Merismopedia. Look for the dividing cell and describe the division process. Now prepare a wet mount of Anabaena and apply India ink as in step 1. Try to identify the Heterocyst and Akinete. Do you observe nuclei? Finally prepare a wet mount of Oscillatoria. Note the feel by rubbing it between your fingers prior to mounting it. Observe the filaments under a microscope, observe and describe any movements. Find short chains of cells joined to the filament by dead cells—they fragment the filament and grow, reproducing asexually.
Agar Plate 1 (Control) – No formation of bacteria visible.
Agar Plate 2 (Toilet) – There are three observable colony types. One is a cluster of around 24 colonies in punctiform. It is convex in elevation and the margin is undulated. These colonies are around 1.1 mm in diameter and have a shiny yellow appearance. In addition to these there are 3 very large irregular convex colonies in entire form. They are slightly transparent white. The diameter ranges between 5 – 9mm and appears mucoid. There are also 5 colony groups in punctiform. They are white, and pulvinate with undulated margins. Each individual colony is less than 1mm in diameter and are found in clusters of 5-12 individual colonies.
... gram-staining and endospore staining procedures (see Appendix). Results Fortunately or unfortunately, there were no coliform bacteria observed from the samples. The samples ... plates, the presence of red colonies was observed. These colonies were possible coliform bacteria and were subjected to the last ... because the stains might splatter out. Then the slide was flooded with malachite green. This was ...
Agar plate 3 (Oral bacteria) – There is one observable colony type that appears in a cluster of around 500-600 individuals. They are in punctiform and are convex. Ranging from a pinpoint to around 2mm these yellow mucoid bacteria is marginally entire.
Observations of Cyanobacteria
Gloeocapsa – 40x Blue green brown in
Merismopedia – 40x Mostly bluish green
Yellowinsh brown in
Anabaena – 40x Hairy in appearance and
grayish green in color.
Oscillatoria – 40x Blue green in color.
Toilet Gram Negative
Oral Gram Negative
Conclusion: “More prokaryotes inhabit a handful of dirt or the human mouth or skin than the total number of people who have ever lived. Prokaryotes are not only the most numerous organisms by far but also the most pervasive” (Campbell, 1999).
Within this lab we came across a comparatively small number of these prokaryotic organisms, namely Gram positive and Gram negative bacteria along with several examples of cyanobacteria. We observed 4 samples of cyanobacteria at 40x magnification, which were gloeocapsa, merismopedia, ananbaena, and oscillatoria. We also observed various other microbes that we cultured from both oral and toilet samples in Petri dishes. We were able to thoroughly examine these cultures and describe the various colonies and forms these microbes multiplied into. Most appeared slightly transparent and either yellow or white in color. Most were also in punctiform, or colonies clustered throughout their culturing medium. In addition we observed two samples from our cultures and found Gram-negative bacteria in both instances. It seems as though Gram-positive bacteria did not form in each of the two samples. One possible reason for this may be traced to our staining of the bacteria. When the first sample was viewed under oil immersion, most of the central mass of the sample was concentrated in the center of the slide making it difficult to observe under the light microscope, forcing us to center our attention on the outer radius, which may have explained why we were unable to identify Gram-positive bacteria.
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Laboratory Manual: Biological Sciences 110, University of California Department of Biological
Sciences, Fall 2001.
Campbell, N. A., Reece, J. B., & Mitchell, L. G. Biology: Fifth Edition. Addison Wesley
Longman, Inc. Menlo Park, 1999.