The bacterium examined was Escherichia coli and Bacillus subtilis.
Method – See Handout
Results Gram Staining
Gram positive Gram negative
Bacillus Subtilis Escherichia Coli
(x100) (x100)
Small rod shaped bacteria Small rod shaped bacteria
Purple Red
Results Endospore staining
Bacillus Subtilis (x100) Escherichia Coli (x100)
Results Hanging drop method
Conclusion and Discussion
Grams Method
Upon completion, the experiment concluded that using Grams method the staining procedure made it a lot easier to distinguish between both Gram positive Bacillus subtilis and Gram negative Escherichia coli whilst observing the bacterium under the light microscope.
One of the downfalls to light microscopy is the lacking of contrast. As this is the case dyes can be used to stain the cells which increases their contrast so that they are more easily seen. Many dyes commonly used in microbiology are positively charged (methylene blue, crystal violet and safranin) which react with the negatively charged cellular parts i.e. nucleic acids. These dyes combine with the structures of the cells, which is really the whole purpose of staining, to observe the cells and their contents.
This was because the Gram positive cell wall lies above the cell membrane, and there is no outer membrane. These cells stain purple as the crystal violet primary stain and iodine mordant form an insoluble precipitate which is trapped as the acetone-alcohol destain dehydrates the cell wall. The results showed Bacillus subtilis to be Gram positive bacteria. Medical research would prove this bacterium easily killed by Penicillin.
The Gram negative cell wall on the other hand is very thin, and composed of about 10% peptidoglycan, lipoproteins, lipopolysaccharides, and proteins associated with the peptidoglycan layer. The stain could not penetrate this outer membrane to get to the peptidoglycan layer to colour it. It is too thin to retain most of the crystal violet-mordant complex, but it could retain the safranin counterstain, so gram negative cells stain red. As shown in the results Escherichia coli was stained red this enabled a conclusion that E-coli is a Gram negative bacteria.
The space between the peptidoglycan layer and the outer membrane is called the periplasm and it contains many different proteins. The outer membrane also contains porins, which are proteins that form pores in the membrane and allow small hydrophilic molecules to pass into or out of the cell. Hydrophobic or larger molecules cannot pass through the porins and this is how the Gram stain is prevented from reaching the peptidoglycan layer to colour it.
Endospore Method
The results conclude that Escherichia coli do not produce endospores yet Bacillus subtilis do. The endospores were seen as oval blue/green structures, and the red rods were classified young vegetative cells.
Unique to bacteria endospores are highly resistant dehydrated cells with thick walls and additional layers. The thick wall consists of two membranes containing peptidoglycan, a thick spore coat that contains proteins which form around the membrane thus enabling the endospore to be highly resistant.
The endospore can be smaller, the same size or larger than the vegetative cell. The endospores were located in the middle of the cells (central), at the end (terminal), or between the end and the middle of the cells (subterminal). The endospores themselves were oval.
Endospores do not take up the crystal violet-iodine mordant complex or the safranin counter stain during gram staining, so they are made visible by utilizing another differential staining technique exactly like the performed technique used in this experiment.
Endospores can survive temperatures above 100 degrees as opposed to a normal bacterium which is usually killed by heating to 80 degrees for ten minutes. They are also resistant to desiccation, freezing, toxic chemicals and radiation. As this is the case it can be concluded that the purpose of endospores are for bacterial survival.
The formation of endospores by bacteria is important both to medicine and to the food industry because of the endospore resistance to heat and most chemicals. The majority of endospore - forming rods and cocci are gram - positive and can be aerobic, facultative anaerobic, obligate anaerobic or microaerophilic. Bacillus subtilis is part of this group of bacteria. It is known as the “endospore – forming Gram positive rods.
Hanging Drop
Research indicated Escherichia coli to have flagella in the peritrichous arrangement. This means the bacterial cells has several flagella which are located at various sites on the cell surface. Bacterial flagella are long, thin appendages which are free at one end and are joined to the cell at the other. These are so thin that they cannot be seen under a light microscope. The peritrichous arrangement is known to have several flagella inserted at many places around the cell. It was this arrangement which created the movement of the bacterium Escherichia coli in a tumbling motion.
Under the light microscope in this experiment the movement did not seem to have a certain direction, yet it was always moving, thus indicating the tumbling motion. This indicated that directional control was quite poor. This bacterium is known to move in response to chemicals (the favourable condition), this is called Chemotaxis as opposed the Phototaxis, in which case the bacterium would be moving in response to light.
On the other hand the Bacillus Subtilis also known to have a peritrichous flagella arrangement was alternating between Swimming and tumbling which shown that they had much more directional control towards their attractant.
Typically microbes that live in aqueous environments (like the two bacteria observed here) will continually move around looking for nutrients, and as described in this case sometimes this movement is random, but in other cases it is directed toward or away from something. In other words, bacteria are capable of showing simple behaviour that depends upon various stimuli. In Bacillus subtilis, chemotaxis’ attractant is asparagines. Escherichia is attracted to alanine. They both however repel against Methanol.
In peritrichous flagellate bacteria the flagella rotate independently of one another 95% of the time the flagella rotate counter clockwise, 5% of the time the flagella switch directions and rotate clockwise. When the flagella are all rotating counter clockwise, the flagella are bundled together and the bacteria travel in a straight line (i.e. it swims). When one flagella switches direction the bundle disassociates, and the bacteria tumbles, alternative swimming and tumbling results in a three-dimensional random walk. Chemo-attractants and repellents can interaction with receptor proteins in the cell envelope, which influence the rate of tumbling when the cell is moving in a given direction.
An electron microscope would have shown a more detailed motility, as this was not available the results did not show as many of these facts stated in this conclusion.
Not all microorganisms get around by using bacterial flagella. Eukaryotic microbes will use flagella, but they are larger and more complex than bacterial flagella. Also, other bacteria use gliding motility that depends upon contact with a solid surface. Under the microscope it seems as if they are sliding along the surface.
Enterobacteriaceae (Enterics)
Include a group of bacteria that inhabit the intestinal tract of humans and other animals. They include motile and nonmotile species; those that are motile have peritrichous flagella. The facultative anaerobic E. coli is one of the most common inhabitants of the intestinal tract. It is not usually pathogenic; it can be a common cause of urinary tract infections and can also cause “traveller’s diarrhoea”.
References
Brock Biology of Microorganisms. 10th Edition. 2003. Michael T. Madigan et al. Pearson Education, Inc.
Microbiology an Introduction. 6th Edition.1997. Tortora, Funke, Case. Benjamin/ Cummings Publishing Company.
Basic Microbiology. Information Booklet provided by St. Helens College. Interpreted by Paul Kowabnik.
