UNKNOWN LAB REPORT 1 Experiment 25 Unknown Lab Report Microbiology UNKNOWN LAB REPORT 2 Identification of The Unknowns The ability to differentiate media and identify unknown bacteria is a necessary skill used in labs and clinical settings. For a medical professional to understand how to treat an ill patient, they must first determine the species of the organism. Experiment 25 allows students to have hands on experience applying their comprehensive knowledge to demonstrate proper techniques learned throughout the semester. Students will receive two unknown bacteria, one Gram-positive and one Gram-negative, and use different growth media and biochemical tests to identify their unknowns. For this experiment, unknown ID #10 and #35 were obtained. #10 and #35 were dyed following the proper gram stain techniques and placed under the microscope for better viewing. Due to thick cell walls and low lipid content, gram positive bacteria will hold on to the crystal violet and will appear purple. Gram negative bacteria have thin cell walls and a lipid outer membrane that will be dissolved through the decolorizer and counter stained. The counter stained used is Safranin, staining Gram-negative bacteria pink. When viewed under the microscope, #10 appeared purple and #35 appeared almost clear with a slight hue of pink, indicating #10 is Gram Positive and #35 Gram Negative. Cell morphology was also viewed, showing #10’s shape being cocci and #35’s being bacillus. Due to #10 being a Gram-positive cocci shaped bacterium, the unknown organism’s identity was narrowed down to Staphylococcus aureus, Streptococcus salivarius, Staphylococcus epidermidis, and Enterococcus faecalis. Gram-positive #10 was properly inoculated in Bile Esculin agar. Bile Esculin agar is used to differentiate between Enterococci and group D Streptococci through the presence of bile UNKNOWN LAB REPORT 3 esculin hydrolysis. Group D streps will hydrolyze esculin in the presence of bile. Due to byproducts of hydrolysis and iron salts in the medium reacting, a black color change will indicate a positive reaction. The bacteria had heavy growth in this medium with no color change. The result signifies the organism does not use esculin. Because #10 has significant growth with no use of esculin, the unknown’s identity was narrowed down between Staphylococcus aureus and Staphylococcus epidermidis. MacConkey agar is used to isolate or differentiate between Enterobacteriaceae members based on lactose fermentation. When a bacterium is acidic due to lactose fermentation, pH indicator, Neutral red, will color shift to a red hue. Unknown bacteria #35 was inoculated into MacConkey agar. The bacterium grew in this medium but did not ferment as there was no color change. The lack of color change indicates that this bacterium cannot ferment lactose. Because #35 had growth with no fermentation, the unknown was narrowed down to Pseudomonas aeruginosa, Proteus mirabilis, and Serratia marcescens. # 10 was inoculated into blood agar, which is used to grow bacteria that is fastidious. Hemolysins in certain bacteria can cause lysis of the red blood cell. By studying the degree of hemolysis, specific Gram-positive cocci can be identified. Hemolysis is sorted into three groups: Beta hemolysis, complete lyse and complete clearing; Alpha hemolysis, partial lyse and discoloration; Gamma hemolysis, no change. When the bacterium grew in the blood agar, it showed complete clearing around the colonies. Complete clearing around colonies is indicative that #10 can lyse red blood cells through beta hemolysis. This narrowed down #10’s identity to Staphylococcus aureus. # 10 was inoculated into Mannitol salt agar. As a selective and differential medium, it can differentiate between members of the genus staphylococcus and Micrococcus species. If an UNKNOWN LAB REPORT 4 organism grows in this medium, it indicates that it is saline tolerant. If the bacteria ferments mannitol, the pH indicator, Phenol red, will cause a color change of red to yellow in the medium. #10 showed growth in this medium and turned the entire plate from red to yellow. The results signify that the organism is salt tolerant, acidic, and can ferment mannitol. This result confirms the identity of #10 to be Staphylococcus aureus. #35 was inoculated into Triple Sugar Iron agar slant using the fishtail streak method. This medium can differentiate between bacteria of Enterobacteriaceae, based on their ability to ferment sugars and reduce sulfur to hydrogen sulfide. If a bacterium ferments carbs, Phenol red will turn yellow due to production of acid. Organisms that do not ferment any sugars will break down peptones and produce ammonia in the media, turning the agar violet due to its alkaline nature. Blackening of the butt of the tube indicates iron sulfide, which is produced through a reaction of hydrogen sulfide gas and iron. When the bacterium grew in this medium, it had turned the butt of the tube black. Gas production was also seen at the butt of the tube through an air pocket in the agar. The professor of this course had set out result samples to be used for comparison, and upon comparison, this result means that this specific bacterium can ferment glucose and form iron sulfide. Due to #35’s ability to ferment glucose and form iron sulfide, its identity was narrowed down to P. mirabilis. #35 was inoculated into a plate of Citrate agar. This medium is used to determine an organism’s ability to use citrate as a carbon source and is used to differentiate bacteria in the Enterobacteriaceae family. Based on the bacteria’s acidity, the pH indicator, Bromthymol blue, will color shift from neutral green to alkaline blue when pH levels are higher than 7.6. The positive blue color shift indicates that citrate is being used. The bacteria was inoculated in this UNKNOWN LAB REPORT 5 medium and showed no growth or color shift, indicating that this bacterium does not use citrate as a carbon source. This confirms the identity of #35 to be P. mirabilis. #35 was inoculated into 3 separate broths, individually containing lactose (red cap), sucrose (white cap), and glucose (blue cap). These broths are used to determine if an organism can ferment specific carbs. Each test tube contained a Durham tube, ferrous sulfate, and pH indicator, Phenol red. Due to fermentation of carbs, the pH indicator will shift from red to yellow when met with acidic conditions. Gas production will be seen through trapped bubbles in the Durham tube. In the medium containing glucose, a gas bubble in the Durham tube and color shift from red to yellow was seen, indicating the sugar was utilized. In the sucrose and lactose medium, no color shift or gas bubble was seen, indicating the sugars were not utilized. Due to #35 solely fermenting glucose, this result confirms the identity to be P. mirabilis. Activity 5 is used to further differentiate Gram-positive bacteria that are cocci shaped. #10 was smeared on two different glass slides, one was tested with catalase and the other coagulase. The purpose of the catalase test is to test which bacteria produce the enzyme catalase. Bacteria that produce catalase can break hydrogen peroxide into water and gas, forming bubbles in a positive reaction. When bacteria #10 was tested with a drop of hydrogen peroxide, bubbles formed, indicating that this organism produces the enzyme catalase. The purpose of the coagulase test is to detect if the bacteria can clot plasma. Bacteria that is coagulase positive will react with plasma, forming clumps of white precipitate. When plasma was mixed into bacteria #10, white precipitate formed, indicating that this organism produces coagulase. #10 had the ability to produce both catalase and coagulase, confirming that the identity is Staphylococcus aureus.
