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Microbiology Unknowns Stephanie Everitt, Carly McMacken, Quiana Neilson, & Heather Peterson BIOL 201C – Spring 2008 OVERVIEW Introduction Inoculation Introduction to unknowns Identification G+ species G- species Conclusion Best Guess Future Research

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microbiology unknowns

Microbiology Unknowns

Stephanie Everitt, Carly McMacken,

Quiana Neilson, & Heather Peterson

BIOL 201C – Spring 2008

overview
OVERVIEW
  • Introduction
    • Inoculation
    • Introduction to unknowns
  • Identification
    • G+ species
    • G- species
  • Conclusion
    • Best Guess
    • Future Research

Image Source: Missouri University of Science and Technology; http://web.mst.edu/~microbio/Bio221.html

specimen collection
Specimen Collection
  • Sample source:
    • tree bark on campus

Image Source: Microshield Environmental Services; http://www.microshield-es.com/airqualitymoldtesting.html

inoculation
Inoculation
  • Both colonies:
    • Small
    • Circular

Beige colonies

(white)

Orange colonies

Figure 1. Culture of tree sample on TSA; incubated at 37° for 24 hrs.

  • Smooth edges
  • Convex elevation
  • Mucoid consistency
unknown orange
Unknown Orange
  • G+
  • Rod shape
  • Asporical
  • Random arrangement
  • ~2μm

Figure 2. Gram Stain of unknown orange bark sample at 1000x.

unknown white
Unknown White
  • G-
  • Slender rod shape
  • Asporical
  • Random arrangement
  • ~2 μm

Figure 4. Gram Stain of unknown white bark sample at 1000x.

g unknown white
G- : Unknown White
  • Tests Positive for:
    • Growth in high salinity of bile salts
    • Hydolysis of:
      • Casein
      • Gelatin (anaerobically)

Figure 5. Gram Stain of unknown orange bark sample at 1000x using a zoom lens camera. The black on the right is the microscope pointer.

g unknown white10
G- : Unknown White
  • Tests negative for:
    • Fermentation of: manitol, lactose, glucose, maltose, and sucrose
    • Hemolysis
    • Citric acid as a C source
    • Indole production
    • N reduction
    • Mixed acid & 2,3-butanediol fermentation

Figure 5. Gram Stain of unknown orange bark sample at 1000x using a zoom lens camera. The black on the right is the microscope pointer.

g unknown white11
G- : Unknown White
  • Most similar to Pseudomonas aeruginosa
  • Eliminating test
    • Milk Nutrient Agar

Figure 6. Pseudomonas aeruginosa on Pseudomonas Isolation Agar has a similar appearance to its growth on Milk Nutrient Agar.

Image Source: The Environmental Reporter; http://www.emlab.com/s/sampling/env-report-03-2007.html.

g unknown white12
G- : Unknown White
  • Is it actually G+?
    • Same test results on average:
      • 2.9 – G-
      • 3.6 – G+
    • Pallisades
      • Corynebacterium
        • G+ rods
        • pallisades

Figure 5. Gram Stain of unknown orange bark sample at 1000x using a zoom lens camera. The black on the right is the microscope pointer.

g unknown white13
G- : Unknown White
  • Is it actually G+?
  • Most conclusive cell wall test?
    • Antibiotic results

Image Source: The Microbial World; http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/penicill.htm

g unknown white14
G-: Unknown White

Table 1. Antibiotic clearing results (mm) for Unknown White, G+ known species, and G- known species. * indicates resistant strains present within the clearing.

g unknown white15
G- : Unknown White
  • Other Pseudomonas?
    • Non-fermenters
      • Only 15% of G-, 70% Pseudomonas
      • Yes
    • Unknown white has high salinity tolerance
    •  No!

Figure 5. Gram Stain of unknown orange bark sample at 1000x using a zoom lens camera. The black on the right is the microscope pointer.

g unknown white16
G- : Unknown White
  • Pseudomonadaceae
    • Frateuria
      • if tests + for acidity tolerance

Figure 5. Gram Stain of unknown orange bark sample at 1000x using a zoom lens camera. The black on the right is the microscope pointer.

unknown orange enrichment cultures
Unknown Orange Enrichment Cultures
  • Can grow in high salinity
  • Tests negative for:
    • Fermentation of: manitol, lactose, glucose, maltose, and sucrose
    • Hydolysis of: starch, casein, gelatin, and urea
    • Hemolysis

Figure 3. Gram Stain of unknown orange bark sample at 1000x using a zoom lens camera.