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Learning the identity of unknown bacteria is an essential part of Microbiology. First, identifying bacteria has medical significance. Several different species of bacteria cause many diseases. Therefore, doctors must identify bacteria in order to accurately diagnosis diseases. Also, bacteria have important environmental affects as well. It is important to quantify bacteria in sewage and drinking water to determine the purity of the water. Also, understanding the identity of bacteria that inhabit certain foods assists people in preparing edible food. Therefore, it is necessary to identify species of bacteria in order to maintain the safety of people.
Purifying a mixture of different bacteria ensures that accurate biochemical tests of the separated bacteria can be obtained. If the bacteria species are not separated, the results of every test can be compromised. Bacterial colony purification occurs by performing an isolation streak and noting the difference in appearance of colonies. A successful colony purification is noted by separate colonies forming on the plate. In contrast, an unsuccessful colony purification is indicated by a single colony forming on the plate.
(Temming,2018)
(Shmoop Editorial Team, 2018)
The catalase test determines the ability of a microbe to break down hydrogen peroxide into water and oxygen. This test differentiates between bacteria that produce the enzyme, catalase, and those that do not produce catalase. To perform a catalase test, two to three drops of hydrogen peroxide are added to a small amount of bacteria. After adding the hydrogen peroxide, a positive catalase test is indicated by the presence of bubbling by the bacteria culture. In contrast, a negative catalase test is indicated by no change in appearance to the bacteria. Therefore, if bubbling is present, the bacteria perform the reaction below.
The gram stain differentiates bacteria into groups based on their cell membrane, cell wall, and cell shape. Gram positive bacteria possess a thick peptidoglycan wall. In contrast, gram negative bacteria do not have a thick peptidoglycan wall, but they have an extra membrane around their cell wall. After incubation and the staining process, the results can be seen in the image below.
Similarly, the KOH test is used to confirm the presence of gram positive and gram negative bacteria. When a few drops of KOH are added to a prepared bacteria slide, gram negative and gram positive bacteria react differently. Gram positive bacteria will not stick to an inoculating instrument. However, gram negative bacteria will clump together and appear stringy while sticking to an inoculating device.
The hemolysis test differentiates bacteria that are in the genus Streptococcus based on their type of hemolysis. There are three major types of hemolysis that bacteria can perform: alpha-hemolysis, beta-hemolysis, and gamma-hemolysis. These processes are observable after incubation of bacteria on a blood agar plate. In alpha-hemolysis, hemolysin will create small pores in the agar and turn hemoglobin into biliverdin. This will create a green-metallic growth of bacteria. Beta-hemolysis occurs when the hemoglobin protein is complete destroyed and broken down into bilirubin. This process is indicated by yellow media when held in light. Finally, gamma-hemolysis is the absence of hemolysis, and this process is indicated by normal growth. The hemolysis test will determine the degree to which a bacteria produces hemolysin.
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To learn more about the Hemolysis test visit this link: http://www.vumicro.com/vumie/help/VUMICRO/Hemolysis_on_Blood_Agar.htm
KOH Test. Gram positive bacteria is present in block C, and gram negative bacteria are present in block D. (Vetbact, 2017)
Gram Stain.Gram negative appear pink, and gram positive appear purple under a microscope at 1000X magnification in oil immersion. (Hardy Diagnostics, 2019)
The antibiotic sensitivity test to bacitracin is used to differentiate species in the Streptococcus and Enterococcus genus. Bacitracin is effective in disrupting cell wall formation in group A Streptococcus. A positive bacitracin test indicates that the bacteria are sensitive to bacitracin. This sensitivity is indicated by a zone of inhibition around the antibiotic disc. In contrast, a bacteria species that is resistant to bacitracin grows without restraint around the antibiotic disc.
The lactose test determines if a microbe can ferment lactose and if a gas is produced during this process. A positive test indicates that a microbe can ferment lactose and is represented by a yellow broth. In contrast, a negative test is indicated by a red broth or no change in color. Finally, a positive test for production of gas during fermentation is noted by the presence of a gas bubble in the Durham tube. Overall, these results will demonstrate the fermentation of lactose by the bacteria and even the by-products of this metabolic process.
The H2S and motility test is used to determine the ability of bacteria to reduce compounds that contain sulfur into sulfides. Also, this test will demonstrate the motile capabilities of the microbe. The hydrogen sulfide production occurs in one of two pathways. In the first pathway, cysteine is reduced into hydrogen sulfide, pyruvic acid, and ammonia by cysteine desulfurase. In the second pathway, thiosulfate is reduced to hydrogen sulfide and sulfite by thiosulfate reductase. A positive and negative H2S and Motility Test is demonstrated below.
To learn more about the lactose test, visit this link: http://www.vumicro.com/vumie/help/VUMICRO/Lactose_Fermentation_Test.htm
H2S Test (Aryal, 2019)
Motility Test (lab 3rd test Flashcards, 2019)
(Rath & Padhy, 2015)
The indole test is used to select for bacteria in the family Enterobacteraciae and differentiate between bacteria that produce the enzyme, tryptophanase, and those that do not produce tryptophanase. This enzyme catalyzes the formation of indole, pyruvate, and ammonium by using water and tryptophan. After incubation, Kovac’s reagent reacts with indole to change the color of the solution. A positive and negative indole test is shown below.
(Tankeshwar, 2012)
Amrita Vlab. (2010). Streak Plate Method - Amrita University. Retrieved from
Aryal, S. (2019). Hydrogen Sulfide Test - Principle, Procedure, Uses and Interpretation. Retrieved from https://microbiologyinfo.com/hydrogen-sulfide-test/
Bio-Rad Laboratories. (2012). Gram Staining. Retrieved from
Hardy Diagnostics. (2019).Make Better Stains with the Gram Stain Advanced Kit from Hardy Diagnostics. Retrieved from http://hardydiagnostics.com/gramstainadvancedkit/
lab 3rd test Flashcards. (2019). Retrieved from https://www.easynotecards.com/notecard_set/54758
Rath, S., & Padhy, R. (2015). Antibacterial efficacy of five medicinal plants against multidrug-resistant enteropathogenic bacteria infecting under-5 hospitalized children. Journal Of Integrative Medicine, 13(1), 45-57. doi: 10.1016/s2095-4964(15)60154-6
Shmoop Editorial Team. (2019). Biology The Postulate and the Petri Dish - Shmoop Biology. Retrieved from https://www.shmoop.com/prokaryotes/postulate-petri-dish.html
Tankeshwar. (2012). Indole Test: Principle, Procedure and results -. Retrieved from https://microbeonline.com/indole-test-principle-procedure-results/
Temming, M. (2018). These disease-fighting bacteria produce echoes detectable by ultrasound. Retrieved from https://www.sciencenews.org/article/these-disease-fighting-bacteria-produce-echoes-detectable-ultrasound
VetBact. (2017). Retrieved from http://www.vetbact.org/index.php?displayextinfo=117'
All information obtained in this presentation was found in the Microbiology 321 Lab Manual 2nd edition written by Dr. Leahy and published by Pearson Learning.
First, an isolation streak using aseptic technique was performed on both a blood agar plate and a tryptic soy agar plate. To see how a isolation streak is performed, watch the video below. The streaked blood agar plate incubated at 37 degrees Celsius in the carbon dioxide incubator for 24 hours. The streaked tryptic soy agar plate was allowed to incubate at 37 degrees for 24 hours in a normal incubator. Once a purified colony was identified by appearance, a gram stain was performed (see slide 17). Using an inoculating needle and an isolation streak, the gram negative bacteria was plated on a tryptic soy agar plate, and the gram positive bacteria was plated on a blood agar plate.
To confirm the separation of bacteria, a bacterial smear and gram stain were performed. Two individual bacterial smears were performed for the tryptic soy agar colonies and the blood agar plate. Next, a gram stain was performed. To learn how the bacterial smear and gram strain were conducted, view the video below. Next, the microscope slide was blotted dry using bibulous paper, and the bacteria were viewed using an oil immersion technique at 1000x magnification. After the gram stain, the KOH test was performed. Using aseptic technique, the two types of bacteria were placed in separate spots on a microscope slide. Next, two to three drops of KOH was added to each bacteria spot. By touching the bacteria with the inoculation loop and viewing the appearance of the bacteria, the KOH test was completed.
(Amrita Vlab, 2010)
(Bio-Rad Laboratories, 2012)
In order to identify the gram positive bacteria, three tests must be performed. First, the hemolysis test was performed with the initial plating of the bacteria on a blood agar plate (see slide 16). The results of the hemolysis could be recorded based on color changes of the media.
Next, the catalase test was conducted. First, an isolated colony was taken from the gram positive stock culture that had been previously created and placed on a microscope slide using aseptic technique. Next, two to three drops of hydrogen peroxide were placed on the bacteria, and the bacteria were observed for bubbling.
Finally, a bacitracin antibiotic resistance test was implemented to confirm the identity of the gram positive bacteria. First, an isolation streak was performed using the gram positive bacteria on a blood agar plate. Next, forceps were placed in alcohol and heated over a flame. This procedure was repeated again to ensure the sterility of the forceps. After allowing the forceps to cool, a bacitracin disk was picked up using the forceps and placed on the beginning of the isolation streak. The forceps were again placed in alcohol and heated. This prepared plate was incubated at 37 degrees Celsius in the carbon dioxide incubator for 24 to 48 hours. Finally, the plate was observed for growth around the disk.
In order to learn more about these tests, visit these websites:
Hemolysis Test: http://www.vumicro.com/vumie/help/VUMICRO/Hemolysis_on_Blood_Agar.htm
Catalase Test: https://microbeonline.com/catalase-test-principle-uses-procedure-results/
Antibiotic Resistance Test: http://www.tmcc.edu/microbiology-resource-center/lab-protocols/antimicrobial-susceptibility-testing/
First, the lactose test was performed. In order to perform the lactose test, an inoculating loop was heated and cooled before taking from an isolated colony on the tryptic soy agar plate. Next, the isolated colony was gently swirled in a nutrient broth without disturbing the Durham tube. The inoculating loop was then heated and cooled again. Next, the tube was incubated at 37 degrees Celsius for 24 to 48 hours and the results were recorded.
Second, the hydrogen sulfide and motility test was conducted. First, an inoculating needle was heated over a flame and allowed to cool. Next, a gram negative isolated colony was inoculated on the needle before removing the cap of the SIM agar deep. The neck of the tube was flamed and the needle was carefully stabbed in a straight motion through two-thirds of the agar. After carefully removing the needle, the neck of the tube was re-flamed, and the inoculating needle was heated and cooled again. Finally, the cap was placed back on the tube, and it is was allowed to incubate at 37 degrees Celsius for 24 to 48 hours before observing results.
The third test conducted was the indole test. In the indole test, an inoculating loop is heated and cooled. Next, bacteria from the gram negative stock culture were gently swirled in a 1% tryptone broth using aseptic technique. Then, the bacteria were incubated at 37 degrees Celsius for 24 to 48 hours. Next, ten drops of Kovac’s reagent were added, and the solution was allowed to react for a couple of minutes before observing results of color.
To learn more, visit these websites:
Lactose Test: http://www.vumicro.com/vumie/help/VUMICRO/Lactose_Fermentation_Test.htm
H2S and Motility Test: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/SIMMedium.htm
Indole Test:https://microbeonline.com/indole-test-principle-procedure-results/
The unknown gram negative bacteria's white appearance, gram stain, cell morphology, and KOH test helped prove that the bacteria was successfully purified from the other gram positive bacteria. Since these tests proved the bacteria to be gram negative, the biochemical tests could be performed to successfully identify the unknown. The first test performed was the lactose test because about half of the bacteria that are gram negative ferment lactose and the other half do not. Since this gram negative unknown bacteria produced a positive lactose test, this left six possible unknown bacteria. Therefore, to further differentiate the bacteria, the hydrogen sulfide test was selected because it would further half the possible choices of bacteria. Since this gram negative unknown exhibited a positive hydrogen sulfide and motility test, only three possible choices of bacteria were possible. The positive indole test would prove that Proteus vulgaris was the gram negative unknown. Proteus vulgaris was the unknown gram negative bacteria because it is the only bacteria that produces a positive hydrogen sulfide test, positive indole test, and positive lactose fermentation.
The gray appearance, gram stain, cell morphology, and KOH test confirmed that the bacteria in the blood agar plate was a purified gram positive cocci. The unknown gram positive bacteria demonstrated Beta-hemolysis from the hemolysis test which instantly made the total number of choices of bacteria become three. In order to determine if the bacteria was Staphylococcus aureus, the catalase test was performed. A negative catalase test indicated that the gram positive bacteria was either Streptococcus agalactiae or Streptococcus pyogenes. To further differentiate between the two species, a bacitracin resistance test was performed, and it indicated that the bacteria was sensitive to this antibiotic. With the results of this test, the unknown bacteria species must be Streptococcus pyogenes.