Microorganisms

A microscopic organism, typically a bacterium, virus, or fungus, is what is referred to as a microorganism or microbe. They were uncovered by Antonie van Leeuwenhoek in the 1670s and have given scientists a wealth of knowledge about the microscopic world, which has not yet been thoroughly explored. A precise and thorough comprehension of the organic world depends on the ability to identify and describe microorganisms. For instance, knowing a species' identity can assist researchers determine whether or not it currently threatens the human race, allowing them to develop an antibiotic to combat the potentially harmful microbe. Being able to identify an unknown bacterium is pertinent in many branches of science and medicine as it allows scientists, doctors, and researchers alike the ability to understand how a bacteria functions and its effects on humans.

The ability to accurately identify bacteria is based on the premise that all bacteria have a different genetic makeup and therefore carry out unique enzymatic reactions in an attempt to catalyse a particular molecule. By carrying out differential tests, such as biochemical testing, researchers can identify specific bacterium and observe how they react to certain chemical stimulus. Staining and bacterial tests are essential when differentiating bacterial species into two large groups, Gram-positive and Gram-negative, in that they are grouped based off of the physical and chemical properties of their cell walls. Within the study, a dichotomous key, indicated in Figure 2, is a tool used to determine the organisation of organisms within the physical and biochemical world, was constructed starting with Gram stain results and then divided into subgroups. This study was completed by applying all of the methods that have been learned thus far in the microbiology laboratory class for the characterization and identification of an unknown bacterium



Materials and Methods

An unknown sample substance labelled #2 was given out by the lab instructor as well as four pipets, a sterile swab, a ruler, and four test tubes. All previously learned methods and knowledge were applied accordingly to the unknown substance. The directions were read and followed as stated on the laboratory description sheet. After receiving the sample substance, the number given was recorded. The first step, as stated on the instructions, was to isolate the unknown from the soil by performing serial dilution. A dilution scheme was set up and precautions were taken in order to ensure correct and concise results; test tubes, media, and pipets were labelled prior to the beginning of the dilution (10(-2), 10(-4), 10(-5), 10(-6)) as indicated in Figure 1 below.



Figure 1. Indicated labelled test tubes along with soil sample and media

In order to ensure that each mixture was the same, each was vortexed equally. Using the first pipet, 0.1 ml of the unknown sample was transferred into a test tube containing 9.9 ml of sterile water thus becoming the 10(-2) dilution. Using the second pipet, 0.1 ml of the 10(-2) dilution was transferred into another test tube containing 9.9 ml of sterile water thus becoming the 10(-4) dilution. Using the third pipet, 1 ml of the 10(-4) dilution was transferred into another test tube containing 9 ml of sterile water thus becoming the 10(-5) dilution. Finally, using the fourth and final pipet, 1ml of the 10(-5) dilution was transferred into the last test tube containing 9 ml of sterile water, thus becoming the 10(-6) dilution. After serially diluting the soil sample, using the spread plate technique on a few select dilutions, the microorganisms were separated from the soil. This needed to be done carefully as to avoid improper results.

After ensuring that each mixture was vortexed to ensure homogeny, using a pipet, 0.1 ml of the 10(-4) dilution was transferred to the centre of a TSA plate and labelled 10(-4). Using a pipet, 0.1 ml of the 10(-5) dilution was transferred to the centre of a TSA plate and labelled 10(-5). Using the third pipet, 01 ml of the 10(-6) dilution was transferred to the centre of a TSA plate and labelled 10(-6). The cell spreader was flame sterilized, allowed to cool, then moved in an up and down motion, rotating 90 degrees and then the motion was repeated. This process was repeated four times. The TSA plates were then incubated at 37 degrees Celsius for 48 hours.

After dilution and incubation, a modified version of the Kirby-Bauer Disk Diffusion assay was performed to test the production of the antimicrobials. Using the cross streak method, one Gram negative and one Gram positive stain was streaked onto another TSA plate. Six antibiotic disks were then dispensed onto the surface of the TSA plate, inverted, and then incubated at 37 degrees Celsius for 48 hours. After incubation, signs of growth inhibition were compared to those of the controlled experiment.





Results







Figure 2. This is the dichotomous key used to organise natural organisms

Unknown #2 had the following morphology on the TSA plate: medium sized, orange/cream coloured, horizontal colony. After it was determined by the Gram stain that it was a Gram negative rod, the substance went through a sim tube, and lactose fermentation where it was determined that the unknown substances were in fact C. freundil and E. coli. The results shown in the dichotomous key in Figure 2 begin with each of the primary categories, Gram negative and Gram positive, and as the chart flows down the different substances are separated into sub categories of the main categories resulting in the identification of the unknown substance.

The dichotomous key allows the substances to be broken down and categorized in order for the researcher to easily identify an unknown substance based off of its chemical and physical characteristics. In Figure 2, highlighted in orange are the pathways of the substances identified in this experiment. Figure 3 lists all of the biochemical tests, their morphology, and results. The results are also shown in the dichotomous key in Figure2.



Figure3. Represents all biochemical tests, their morphology, and results.



Conclusion

After several experiments and differential testing, it was discovered from this laboratory experiment that unknown #2 was in fact E. coli and C. freundil. After performing the Gram strain in order to determine if the unknown #2 was a Gram negative rod, the microorganism was grown on a TSA plate for use in the biochemical tests. The TSA was repeated four times in order to ensure accuracy and it was concluded that the unknown was E. coli and C. freundil.

The advantages to utilizing biochemical testing to identify an unknown organism, at least in this experiment, were that grouping the organisms up and being able to determine their groups aided in finding out the unknown substance, however it was very limiting in that certain processes, the TSA procedure for example, had to be repeated due to an accuracy issue. Overall, though there were certain limitations, the experiment was a success and the substances were identified accurately and effectively.





















References

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https://www.ncbi.nlm.nih.gov/books/NBK8406/

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Retrieved April 15, 2017, from https://www.campdenbri.co.uk/services/microorganism-identification.php

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2017, from https://www.slideshare.net/Haiaykyu/identification-and-differentiation-of-microorganisms

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http://www.microbiol.org/resources/monographswhite-papers/the-gram-stain/