Identifying an Unknown Bacterial Sample with Biochemical Testing
Unknown #18
Bacillus subtilis
Sarah Koif
Submitted to
Swatantra Neupane
Nursing Microbiology 2460-003
Spring 2018
Department of Biology
University of Texas at Arlington
Abstract
The purpose of the unknown project was to present nursing microbiology students with an opportunity to demonstrate their ability to identify an unidentified bacterial sample by observing macroscopic and microscopic morphologies, utilizing biochemical tests, and performing a gram stain. Immediately after being given the unknown bacterial sample, which was labeled as #18, a gram stain was performed. The unknown tested as gram positive, which determined which biochemical tests were utilized. The tests and/or agars included two Tryptic Soy Agar plates, which
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After conducting these biochemical tests, it was determined that the identification of unknown #18 was Bacillus subtilis.
Introduction
The beginning of this project was marked by receiving an unidentified sample of bacteria. Since the end goal was to identify the specific genus and species of bacteria that was given, it only made sense to start eliminating bacteria by using broad tests and later utilizing more specific tests as the process continued, until finally the bacteria could be identified with confidence. The first test conducted was the Gram stain, which eliminated about half of the possible bacteria that the unknown could have been, as the results are either gram-positive or gram-negative. The difference between these two results, gram-positive and gram-negative, is their cell envelope makeup, as gram-positive cells have a thick cell wall and lack an outer membrane, while gram-negative cells have a very thin
The first day an unknown sample was assigned to each group of students. The first test applied was a gram stain to test for gram positive or gram-negative bacteria. The morphology of the two types of bacteria was viewed under the microscope and recorded. Then the sample was put on agar plates using the quadrant streak method for isolation. There were three agar plates; one was incubated at room temperature, the second at 30 degrees Celsius, and the third at 37 degrees Celsius. By placing each plate at a different temperature optimal growth temperature can be predicted for both species of bacteria.
Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
The Gram positive bacteria has been nicknamed Posi. The Gram positive species’ morphology includes having an opaque opacity with a smooth margin. The moisture content of the Gram positive species is shiny and the pigmentation is gold. The Gram positive species grows at an optimal temperature of 37°C. The shape of the Gram positive species is a cocci, with an arrangement of grapelike clusters. The Gram positive species’ size ranges from .5-1.5 µm. Oxygen requirement of the Gram positive species is facultative, and has complete lysis of red blood cells. All results are summarized in Table
The purpose of this study is to identify an unknown bacterium from a mixed culture, by conducting different biochemical tests. Bacteria are an integral part of our ecosystem. They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species. These biochemical tests are process of elimination that relies on the bacteria’s ability to breakdown certain kinds of food sources, their respiratory abilities and other biochemical conditions found in nature.
The results of the gram stain test were cocci and purple. This indicated that the unknown bacteria were gram positive. The gram stain test eliminated Escherichia coli, Klebsiella pneumonia, Salmonella enterica, and Yersinia enterocolitica as choices because these bacteria are gram negative. Next a Blood Agar plate was used because in order to do a MSA or a Catalase test there needs to be a colony of the bacteria. The result of the Blood Agar plate was nonhemolytic.
The purpose of this laboratory is to learn about cultural, morphological, and biochemical characteristics that are used in identifying bacterial isolates. Besides identifying the unknown culture, students also gain an understanding of the process of identification and the techniques and theory behind the process. Experiments such as gram stain, negative stain, endospore and other important tests in identifying unknown bacteria are performed. Various chemical tests were done and the results were carefully determined to identify the unknown bacteria. First session of lab started of by the selection of an unknown bacterium then inoculations of 2 tryptic soy gar (TSA) slants, 1 nutrient broth (TSB), 1 nutrient gelatin deep, 1 motility
The eighteenth exercise of the laboratory manual titled Unknown Identification and Bergey’s Manual is an experiment to identify an unknown bacterium. In this exercise, a student must randomly choose a numbered bacterium available to the class. The keys in Appendix H, located on the last pages of the book, are the major helpful tools in this exercise because it provides completed steps of tests that needs to be performed in order to distinguish certain bacteria. This means that in this exercise, various types of tests and techniques must be performed to identify the chosen unknown bacterium. The unknown bacterium that I selected was number thirty-nine in which I discovered as the Bacillus megaterium after conducting several tests.
Phenotypic methods of classifying microorganisms describe the diversity of bacterial species by naming and grouping organisms based on similarities. The differences between Bacteria, Archaea and Eukaryotes are basic. Bacteria can function and reproduce as single cells but often combine into multicellular colonies. Bacteria are also surrounded by a cell wall. Archaea differ from bacteria in their genetics and biochemistry. Their cell membranes are made with different material than bacteria. Just like bacteria, archaea are also single cell and are surrounded by a cell wall. Eukaryotes, unlike bacteria and archaea, contain a nucleus. And like bacteria and archaea, eukaryotes have a cell wall. The Gram stain is a system used to characterize bacteria based on the structural characteristics of their cell walls. A Gram-positive cell will stain purple if cell walls are thick and a Gram-negative cell wall appears pink. Most bacteria can be classified as belonging to one of four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, and Gram-negative bacilli) (Phenotypic analysis. (n.d.).
I was given unknown organism #14, in order to find out what organism I had, I had to perform several different biochemical tests to identify it. Starting with the Gram stain test, which is performed to differentiate Gram-positive and Gram-negative cells. After staining, when observed through the microscope Gram-positive cells are a purple color with thick peptidoglycan cell walls. Gram-negative cells are a pinkish/red color with thinner cell walls. (handout G. s.) My organism was observed to be pinkish rod shaped meaning it is Gram-negative bacteria.
The dichotomous keys show process of elimination taking place and several steps that were performed to the two unknown bacteria. Bacteria number one was concluded to be either S. aureus or E. faecals. As shown, a gram stain was performed to bacteria 1, because it helped in separating the bacteria into Gram negative or positive. Bacteria 1 was Gram positive bacteria, so the next step was to eliminate the bacteria that were bacilli, because under the microscope the bacteria was shown to be cocci. Then a lactose test was performed, because it helped me in determining the bacteria metabolism (if lactose was utilized as a sugar by the bacteria) (“Phenol Red Broth”, n.d). Lastly, with the help of process of elimination a MSA test was performed to
Bacteria play a large role in our health, the environment, and most aspects of life. They can be used in beneficial ways, such as decomposing wastes, enhancing fertilizer for crops, and breaking down of substances that our bodies cannot. However, many bacteria can also be very harmful by causing disease. Understanding how to identify bacteria has numerous applications and is incredibly important for anyone planning to enter the medical field or begin a career in research. Having the background knowledge of identifying an unknown bacteria may one day aid healthcare professionals diagnose their patient with a particular bacterial infection or help researchers determine various clinical, agricultural, and numerous other uses for bacteria.
Planaria are one of many free-living flat worms that can be found in marine, aquatic, and terrestrial environments. Certain characteristics of planaria worms include an acoelomate body, a gut with no anus, lack of a blood vascular system, and a simple nervous system. The main reason as to why planaria are subjected to many studies is because of their unique ability to regenerate. Regeneration is the ability to re-grow lost body parts that may have been cut off. This is possible because the organism has the ability to form a blastema, which is an accumulation of undifferentiated cells, at the site of the wound. Regeneration is capable of occurring at various degrees throughout the animal kingdom. This unique process would never be able to be seen in human beings. Humans and other mammals
Talaro , K., & Chess, B. (2012). Foundations in microbiology. (8th ed., pp. 563-564). New York, NY:
I identified the genus and species of an unknown bacterial culture, #16, and I applied the following knowledge of morphologic, cultural and metabolic characteristics of the unknown microorganism according to the laboratory manual as well as my class notes and power point print outs. I was given an incubated agar slant labeled #16 and a rack of different tests to either examine or perform myself; the tests are as follows: Gram Stain; Nutrient Gelatin Test; Carbohydrate Fermentation; Dextrose, Lactose and Sucrose; IMVIC tests; Citrate, Indole, Mythel-Red and Vogues Proskauer test; as well as a Urease and TSI Test.
Handbook of Laboratory and Diagnostic Tests with Nursing Implications (3rd edition). Philadelphia: F.A. Davis Company.