FINAL LAB REPORT MICROBIOLOGY 2420-40742 IDENTIFYING UNKNOWN ORGANISM
#6 INTRODUCTION The purpose of this lab was to identify an unknown bacteria culture using different tests. The identification of these unknown culture was accomplished using the methods learn in the microbiology lab and based on specific biochemical characteristics to help recognize the specific unknown culture. It is important for students to understand that identifying an unknown organism provides essential information about the organism and the way that it behaves in certain environment. Unknown #6 was subsequently identified as Micrococcus luteus. M. luteus can be found in many places such as the human skin, water, dust, animals, and soil. Micrococcus is generally thought of as harmless bacterium, but there have been rare cases of Micrococcus infections in people with compromised immune systems, as occurs with HIV patients.(1) Defining characteristics of Micrococcus are the ability to aerobically produce acid from glucose glycerol, masculine hydrolysis, arginine di-hydrolase, major pigment production, motility, and conversion of nitrate to nitrite.(2) Micrococcus is generally thought to be a saprotrophic or commensal organism, though it can be an opportunistic pathogen. This is particularly true in hosts with compromised immune systems.(1) They are generally strict aerobes and can generally reduce nitrate. M. luteus oxidizes carbohydrates to CO2 and water, and it does not produce acid from glucose as well as it does not make arginine di-hydrolase or bgalactosidase.(2) M. luteus has been shown to survive in oligotrophic environments for extended periods of time. Recent work by Greenblatt et al. demonstrate that Micrococcus luteus has survived for at least 34,000 to 170,000 years on the basis of 16S rRNA analysis, and possibly much longer.(2) It has been recently sequenced and has one of the smallest genomes of freeliving actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp.(4) Most Micrococcus infections are discovered through process of elimination (all other bacterial, fungal, etc. tests showing up negative) along with the presence of abundant Micrococcus tetrads in the lesions or cysts (which would normally have been overlooked because Micrococcus is a natural part of the skin’s microbial flora)(3). Recently, this organism was recognized as an opportunistic pathogen and has been implicated in recurrent bacteremia, septic shock, septic arthritis, endocarditis, meningitis, intracranial suppuration, and cavitating pneumonia in immunosuppressed patients.(4) METHODS Each student was given an unknown sample with which to work with. The first step was to conduct a Gram Stain to determine if the organism was Gram+ or Gram- and identify the morphology and arrangement of the sample using microscope. Results were recorded based on observations under the microscope by each student. The second step was to prepare a Streak Plate isolation of the organism on BHI agar. This was later stored in the incubator until the following lab day.
The materials and method used for the Gram Stain and the Streak Plate are listed below. Gram Stain Materials: Unknown #6 Bunsen burner, water, Wire inoculating loop, microscope slide, crystal violet, iodine, Gram decolorizer, microscope, immersion oil, Test tube rack, safari, bibulous paper, Methods: 1. Follow the protocol of heat fixing a smear for Unknown #6. 2. Smear the a small sample of each of the organism into a water drop of water on a slide 3. Let the smear dry completely 4. Heat fix each of the smear by passing it across the flame 3 times 5. Saturate the slide with crystal violet and leave for approximately 1.5 mins 6. Rinse with water 7. Saturate the slide with iodine and leave for 1.5min 8. Rinse with water 9. Slowly decolorize the slide with a decolorizer for about 10-20secs till the edge of the slide is clear 10. Immediately rinse with water 11. Saturate the slide with safari and leave it for 1.5min 12. Rinse slide with water 13. Blot dry and view with microscope 14. Dispose of slide properly and thoroughly clean microscope using lens paper and ethanol before storing. Streak plate test Materials: Unknown #6, inoculating loop, flint striker, Bunsen burner, 1 plate. Methods: 1. Label the back of each plate I, II, III, IV. 2. Flame sterilize the inoculating loop, allow to cool, sample the organism from the tube 3. Spread the organism into the area I 4. Heat sterilize your loop 5. Drag the loop from area I to II in a zigzag pattern across the area 6. Heat sterilize the loop 7. Drag the loop from area II to area III in a zigzag pattern 8. Heat sterilize the loop 9. Drag the loop from the area III to IV in a zigzag pattern 10. Invert the plate and place in the incubator under 370 C to view later. Each student collected their sample and agar plate from the incubator and observations were made. The results were recorded and noted on the flow chart. Based on the results for the Gram Stain and the Streak plate, the following tests were carried out. Catalase Test Materials: Unknown #6, 1 microscope slides, inoculating loop, Bunsen burner, flint sticker. Methods: 1. Aseptically transfer a copious amount of the organism onto the clean microscope slide. 2. Place a drop of catalase reagent (hydrogen peroxide) into the slide. 3. Examine for bubbles and record results. Motility test: Indirect method (Motility Medium with TTC). Materials: Unknown #6, 1 TTC motility tube, inoculating needle, Bunsen burner, flint striker, test tube rack, bacterial culture. Methods: 1. Aseptically transfer the organism into the motility medium by carefully performing a straight stab approximately two thirds of the way into the agar. 2. Incubate tubes at 37o C. 3. View to record result Nitrate Reduction test Materials: Unknown #6, 1 nitrate broth tube, Nitrate A&B solutions, zinc dust, Bunsen burner, flint striker, inoculating loop. Methods: 1. Aseptically inoculate each tube of nitrate broth with a single organism. 2. Incubate at 37o C. 3. Following incubation, add 10 drops of reagent A and 10 drops of reagent B to each tube. 4. Add a small amount of zinc into each tube that did not turn red after the addition of reagent A and B. 5. Wait about 5 to 10 minutes to read the results. PR glucose test Materials:
Unknown #6, 1 tube of PR Glucose, test tube rack, labels, sterile disposable transfer pipettes, Bunsen burner, flint striker, inoculating loop, gloves Methods: 1. Aseptically inoculate the organism into the sugar broths 2. Incubate broth cultures at 37o C. 3. Record results – Yellow Broth: Positive for carbohydrate fermentation(Acid/A) – If Yellow: check for Air bubbles in Durham tube = Negative for carbohydrate fermentation (G) – Same as negative control: Negative for carbohydrate fermentation (Neutral) – Pinker than control: Negative for carbohydrate fermentation (Alkaline/K) RESULTS: Sample #6 Gram Stain Color Morphology Arrangement Interpretation Purple Coccus Tetrads Gram(+) Coccus Streak plate test Organism Configuration Margin Elevation Color Hemolysis M. luteus Round with raised margin Smooth Raised Yellow Partial (Alpha) Catalase Test Organism Catalase test Conclusion M. luteus Bubbles (+) catalase Motility test Organism Visual Result Conclusion M. luteus A solid line of red remained in the stab line. The solid line of red indicates that the organism M. luteus is negative for motility Nitrate Reduction test Organism Color after adding A and B Color after adding zinc Interpretation Conclusion M. luteus Colorless Red negative for nitrate (NO2) negative for nitrate reduction PR glucose test Organism Color Conclusion M. luteus Same color (neutral) It is negative for carbohydrate but negative for the production of gas CONCLUSIONS: Unknown #6 was a Gram positive organism with purple color. It was concluded that this unknown is Micrococcus luteus. The following results were performed; streak plate technique which was positive for growth with I most growth seen in section two. The growth had round shapes and grey in color. Gram stain had round dots of tetrads arrangement and purple color. Organism was positive for catalase with bubbles. The motility test indicted negative, nitrate test was negative for nitrate reduction, as well as the PR glucose test show negative for carbohydrate but negative for the production of gas. After all these tests were performed it was concluded that unknown #6 is Micrococcus luteus. WORK CITED 1. Madigan M; Martinko J (editors). (2005). Brock Biology of Microorganisms (11th Ed.). Prentice Hall 2. Greenblatt, CL, J.Baum, B. Klein, et al., 2004. Micrococcus luteus – Survival in Amber. Microbial Ecology. 48; 120-127. 3. Smith, K. J., R. Neafie, J. Yeager, and H. G. Skelton. 1999. “Micrococcus folliculitis in HIV-1 disease.” British Journal of Dermatology, vol. 141, no. 3. British Association of Dermatologists. (558-561) 4. Shuhua Yang, Shunji Sugawara, Toshihiko Monodane, Masahiro Nishijima, Yoshiyuki Adachi, Sachiko Akashi, Kensuke Miyake, Sumihiro Hase, and Haruhiko Takada. Micrococcus luteus Teichuronic Acids Activate Human and Murine Monocytic Cells in a CD14- and Toll-Like Receptor 4-Dependent Manner. Infection and Immunity, April 2001, p. 2025-2030, Vol. 69, No. 4
