The PCR and gel electrophoresis results for Soil organism #24 were unusable (program indicated an error of "no primers"). Therefore, we did not have a genetic sequence to enter into the BLAST database, and could not find more definite taxonomic information beyond knowing it is in the Domain Bacteria.
From the Gram Positive Cocci chart used in our Microbiology class, it appears to be most like Micrococcus luteus except for colony color and lack of growth on Mannitol Salt Agar. So, one really cannot tell very well from organisms studied in class what this one might be, in terms of possible Genus.
The difficulty in identifying this organism makes one appreciate the methods developed to identify bacteria!
Describe the location and conditions under which the organism was isolated.
Soil organism #24 came from soil that is park land in the Williamson Creek watershed in Austin, Texas. This site was chosen because it is park land. The idea was that park soil may present an interesting array of organisms, and contain less "fill" dirt.
Soil Sample Collection Information
Date/time soil sample collected: September 4. 2015, 7:05am
Temperature: 75 degrees F
24-hour rainfall: 0
Pressure: altimeter, 29.92 inches; Sea level, 1012.1 mb
Solar radiation: (mJm2) 22.09
Location of soil sample site, by coordinates: 30.212813, -97.7788033
According to the Web Soil Survey of U.S. Department of Agriculture, this soil area is 100% "LeB, Lewisville soils and Urban land, 0 to 2 percent slopes."
Collected soil sample using a clean stainless steel spoon. Dug 1 inch down into the soil and collected 2 spoons of soil. Placed in small plastic receptacle that was stored at room temperature until lab.
During lab, removed 1 gram of the soil sample, asceptically and added it to 9mL of PBS (Phosphate-buffered saline).Used this to create 4 serial dilutions, where each dilution is decreased by a factor of 10. Then, used these solutions to streak 5 LB (Lysogeny agar) agar plates.
Used growth from these plates to distinguish different bacterial growth from the soil sample. From one plate, chose 13 bacteria to add to a master patch plate. It was from the master patch plate that we selected soil organism #24. Seen here is a photo of the Master Patch Plate. Soil organism #24 is the large brown growth on the top left of the plate.
Description and Significance
Describe the appearance (colonial and cellular), possible antimicrobial activity etc. of the organism, and why the organism might be significant.
Colony morphology of Soil Sample #24:
- colony size = 1 cm. in diameter
- color: transluscent, light brown
- texture: glossy
- elevation: convex
- margin: umbonate
- shape: irregular (not perfect circle)
- colonies are not well defined, kind of grow into each other (result of streak plating?)
- stains Gram positive
- tiny size at 100x lens
- in clusters
The results of PCR and gel electrophoresis for Soil Sample #24 were not usable. The computer program stated there were "no primers" for this organism, so no sequence was generated for the soil sample.
To try to identify our organism, we studied cell morphology, stains, and biochemical test results. We also compared characteristics of Soil #24 to organisms described in our Microbiology class's Unknown bacteria charts, specifically the Gram Positive Cocci chart. We looked at organisms listed in our lab manual. We also tried using "Bergey's Manual of Determinative Bacteriology," but nothing could be concluded from that. Could some test results be inaccurate?
Cell Structure, Metabolism and Life Cycle
Interesting Features of Cell Structure:
Gram stain reaction: (+)
Results of whether Soil #24 produced antibiotics against Staphylococcus aureus or Escherichia coli:
Yes, zone of inhibition observed around Staphylococcus aureus growth on agar plate growth of Soil #24.
No, no zone of inhibition observed around Escherichia coli growth on agar plate growth of Soil #24.
These tests were done twice, with same results each time.
Physiology and Pathogenesis
Phenol Red Broth: Lactose (K), Glucose (-/-), Sucrose (K); No fermentation
Starch Hydrolysis: Weak (+); Amylase is present
Casein Hydrolysis: (-), no clearing
Gelatin Hydrolysis: (+), became liquid; Gelatinase is present
DNA Hydrolysis: Weak (+); DNase is present
Lipid Hydrolysis: (+), clearing; Lipase is present
Methyl Red & Vogus-Proskauer: MR (-), no color change, no acid-fermentation;
VP (-), no color change, acetonin is not produced
Citrate Test: (+), small color change to blue, Citrate is utilized by this organism
SIM Medium: Motility (+), Indole (-), Sulfur (+); Sulfur is reduced
Nitrate Reduction: (+) for reduction of Nitrate to Nitrite
Urea Hydrolysis: Weak (+); Urease is present
Triple Iron Sugar (TSI): (K/K): no fermentation, not from Enterbacteriaceae
Decarboxylation: Arginine (+), bright pink; Lysine (-); Ornithine (-)
Phenylalanine Deaminase: (-), no color change
Oxidase Test: (+), color change; Cytochrome c oxidase is present. #24 is an aerobe.
Eosin Methylene Blue Agar (EMB): pink growth (slow or weak fermentation); Organism ferments lactose with little acid production, possible coliform
Hektoen Enteric Agar (HE): brown growth and no black precipitate. Weak growth may mean not Gram-
MacConkey Agar (MAC): (+), but weak, pale pink & no halo. Weak fermentation and no bile precipitate.
Blood Agar: (+) Beta; complete hemolysis of sheep blood
Mannitol Salt Agar (MSA): (-), no growth in NaCL; organism most likely not Staphylococcus
Phenylethyl Alcohol Agar (PEA): (+) Good growth. #24 is Gram positive.
Catalase Test: (+), bubbles; enzyme Catalase is present
6.5% Salt Tolerance: (-), no turbidity; organism is most likely not Staphylococcus
Bile Esculin: (-), no color change; Lab manual presumes this result means it is not a member of Streptococcus or Enterococcus
Bacitracin/Optochin: (-) resistant to both
Antimicrobial/Kirby-Bauer: (-) for Sulfisoxazole; (+) for Azlocillin; (+) for Ticarcillin; (+) for Ceftazidime
Bauman, Robert W. "Microbiology With Diseases and Body Systems," 2015
Holt, J., Krieg, N., Sneath, P., Staley, J., Williams, S. "Bergey's Manual of Determinative Bacteriology," 2000.
Leboffe, M., Pierce, B. "Microbiology: Laboratory Theory & Application," 2015.
Natural Resources Conservation Service, "Web Soil Survey," United States Department of Agriculture, http://websoilsurvey.sc.egov.usda.gov/App/HomePage.htm. 2015.
Page authored by Annemarie Caporina and Jennifer Thomas, students of Prof. Kristine Hollingsworth at Austin Community College.