In this exercise, we are going to discuss how the various experiments we performed in class led to the identification of an unknown bacterium. The labs that we performed were : Morphology of the unknown and control organisms, Isolation of pure cultures from mix cultures by streaking, Gram stain, Effects of temperature, Motility and other biochemical tests, Effects of salt, Oxygen requirements, effect of UP light, and effects of anti microbe drugs and disinfectants on the unknown.
The first lab we performed that was helpful in identifying the unknown was the streak plate? pure culture technique. In this particular experiment, the aim was to observe single colonies of bacteria and recognize the morphology and arrangement of bacteria cells. With this In mind, we discovered that our unknowns colony was white and round and that the colony morphology was cuscus?circular clusters of cells. In the second lab to identifying unknown, we performed the simple stain to stain our unknown. This second lab is similar to the former lab in the sense that it conformed that the unknown was truly Cisco in structure.
It also made us to discover what its gram reaction was. The objective of this lab was to learn a simple way of staining and understand arrangements of bacteria using correct biologically terms. By the end of these different labs, we have discovered that the unknown Is a gram positive bacterium that Is Cisco In structure The third lab we performed was the differential staining technique. This technique was done to understand gram reaction of bacteria? how we could differentiate gram negative from gram positive bacteria and the steps involved in doing so.
We, again, discovered that the unknown was gram positive because it stained purple with rascal violet. In the fourth lab we performed, the sole aim was to determine what temperature our unknown grew best in. It was discovered , while Incubating It In different temperatures that it was mesospheric?growing best at 20 to 45 degrees. To be very precise, it grew best at 22 degrees. Understanding that our unknown was mesospheric was helpful because it did help to narrow our search; there are many bacteria that exists as gram positive and Cisco in structure, with difference in preferred growth temperatures.
Another lab performed was the biochemical tests. It was used to determine what our known energy source was, and the motility test was used to determine if the unknown bacteria had flagella. With biochemical tests, we tested for the presence of cytosine displeases indicated by hydrogen sulfide, transposes indicated by indolent, citrate permeate indicated by a Prussian color, catalane which produced bubbles in an agar, and the oxides test that changes agar color from colorless to purple. This biochemical tests helped plan-pilot what enzymes our unknown had.
However, these salinity, up light effect, and anti-microbial drugs that destroys the whole- being of this particular bacteria. In another similar lab, we wanted to know the effect of oxygen on our unknown. This lab helped us to determine if the unknown was arroba, anaerobe, macrophage, facultative or rattlebrain. We, however, realized that the unknown could respirator in oxygen and non-oxygen conditions, making it a facultative bacterium. The salinity test was performed to know if the unknown was a helpline and to determine the level of salt it tolerated.
The unknown does not like very saline conditions as water comes out from its cells and it shriveled, during extreme salty conditions. The unknown grew in 0. % NCAA, not very much in 5% NCAA and nothing in the 10% NCAA. Thus, the unknown does not tolerate alkaline conditions. We tested the effect of up light on the unknown? Why? Because we wanted to know how long it took it to die under such heat and even how it reacted to it. It took 10 miss for our unknown to be completely consumed under the UP light.
We performed the effect of antiseptic, disinfectant and anti microbe drugs on unknown. The purpose was to determine the agents that killed the unknown and we came to the conclusion that hydrogen peroxide and triple sulfa was very harsh on the unknown. All the experiments performed towards finding the unknown were very helpful. The streak plate -a pure culture technique- was a way to grow individual bacterial colonies. By streaking a first portion and sterilizing the loop in the flame, we spread a small portion from the previous streaked area unto another area several times.
This method ensured that we obtained a pure culture by the end of the experiment. The lab is significant because we came to see what the morphology of the unknown was and even what a colony looked like with the physical eyes. The control, Seriate was calculus and had a pink colony and doing these controls ensured that our tests were viable. Remember we had performed the simple staining technique that required crystal violet. In this other experiment, we put the unknown on a slide, spread water on the slide and allowed it to dry. After this procedure, we did the heat fixation step.
The essence of heat fixing the slide was to eliminate the possibility of coagulation. In other words, if we did not heat fix, we would lose the unknown cells during staining. We performed this lab because we wanted to differentiate bacterial arrangements ND morphologies from each other. Our controls, stash and Seriate produced cuscus and bacillus morphologies respectively. They both helped to differentiate these morphologies from each other. Performing the differential stain was like repeating what we had done for he simple stain.
However, this test was used to show the gram reaction?gram positive cell walls stained purple and gram negative cell walls stained pink. Our controls Stash and Seriate stained purple and pink respectively, indicating that the former is gram positive while the latter is gram negative. In this test, we used the primary stain crystal violet), the iodine as the mordant that binds with crystal violet and locks it into the pedagogical of bacteria, the decontrolling agent?due to high lipid content positive cell wall, the counter stain was responsible for staining decolonize gram negative cell walls pink.
With all the procedures of washing and applying stains, the controls were something to fall back on when we messed up the unknown staining. When we performed the effect temperature had on the unknown, we were trying to find out what temperature was bactericidal and biostatistics on unknown. We put he unknown in N.B. tubes of different temperatures and incubated for 16 to 24 hours. By the end of this procedure, no growth was observed for 4 degrees and 55 degrees. There was so much growth in the 22 degrees tube, moderate in 37 degrees and very little in 45 degrees.
This test is important because we discovered the unknown is not a bacteria that likes to infect humans because its optimum growth temperature is 22 degrees- body temperature is 37 degrees. The control, seriate, was used to show pigment production. At 22 degrees, seriate released a red- pigmented color. The biochemical tests was one of the interesting labs that we performed. We had to know what energy source the unknown used so that we can know its classification. We did a variety of tests that involved color changes and the unknown responded negative to all these tests.
Moreover, it did not posses a flagella for motility. The control, stash and seriate proved positive for the catalane test, positive and negative for oxides test, and negative and positive for motility. These controls help to ensure that were always in the right track, doing what was right. Another test performed was the unknowns growth in the presence and absence of oxygen. We used a fluid technological media for this experiment because it was emission and had a gradient of oxygen; there is complete oxygen saturation at top and almost none at bottom, thus, any kind of bacteria could metabolize in this media.
We put the unknown in this special tube for about 16 hours and we noticed growth through out the tube. This confirmed that the unknown was facultative?would live in presence or absence of oxygen. The controls, seriate and stash, were robes and confirmed that our experiment was right. We have discussed many things about the unknown, but not its salt concentration. The unknown did not tolerate salt at all. We put the unknown in 0. % salt, 5% salt and 10% salt and it grew in the 0. 5, 5 percent moderately and nothing in the 10 percent.
The controls, stash and seriate, did not like salt either but not as much our unknown. There were there to equally serve as results we can compare the unknown results to. Another lab performed was the effect of UP light on bacteria. We streaked 7 Petri plates with the unknown and exposed each of them under UP light for seconds, seconds, 1 minute, 2 minutes, 5 minutes and 10 minutes respectively. After this time, growth was visible on all except the 10 miss plate. Thus, the time required to ill the unknown under UP light was 10 minutes.
UP light destroys bacteria by destroying their DNA, it can also result in epinephrine dimmers making transcription and translation difficult. There were no controls for this experiment. We performed the anti-microbe, antiseptic and disinfectant effect on the unknown. Plate of unknown, put disinfectants. Between 16 to 24 hours, the unknown had died in the presence of hydrogen peroxide and tripe sulfa, a disinfectant. What might have accounted for this differences? The unknown was extremely sensitive to triple sulfa and hydrogen peroxide. T was also sensitive to pantomimic , but not very much.
There were no controls for this experiment. We had discovered what agents and disinfectant destroyed our unknown bacteria which makes our search for the unknown straightforward. After carefully studying and researching what the unknown was, I have come to a certain conclusion. I have looked at the reactions that the unknown did undergo and a chart of unknown reactions and their identity, I believe I am right. At least it made me to know the genus first, before I narrowed my search to its species using the Burgers manual of determinative biology.