Lab 12: Microbial unknown

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Lab 12 Microbial unknown
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Objective of the exercise

• Isolate and identify bacteria from an unknown
  culture with minimal help
• Practice the standard laboratory techniques
• Prepare a formal lab report

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Suggested time frame

• First day (Monday)
• Streak for isolation (three streak technique) on
  Mannitol Salt and MacConkey agar plates.
• Second day (Tuesday)
• Observe results
• Record colony growth, colony color, plate color.
• Take one isolated colony from each plate and
  streak on separate TSA. Name, section,
  unknown, date and which plate you transferring
  from Mannitol or MacConkey

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• Third day (Wednesday)
• Perform Gram stain from each TSA plate.
• Follow the chart for Gram and the chart for
  Gram negative
• Inoculate appropriate biochemical test and/or
  antibiotic test if required.
• Students are responsible for putting their own
  test in incubator and refrigerator.
• Fourth day (Thursday)
• Observe results
• Determine the Bacteria unknown based on the
  biochemical results and Gram stain
• Remember
• The bacteria you use in your tests must be from a
  pure culture (1 isolated colony)
• No body can have a repeated tube number

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Mixed culture Gram stain and streak for
isolation MAcConkey
Day 1
Day 2
TSA
Gram stain and select a Gram negative colony
Oxidase (-)
Oxidase ()
Lac positive on MAC
Lac negative on MAC
Lac negative on MAC
Day 3
Green sheen on EMB
Dark purple on EMB
Urea ()
Glucose oxidation () on O/F
Citrate (-)
Citrate ()
H2S ()on TSI
Lipase () on tributyrin
Day 4
Escherichia coli
E. aerogenes
Proteus vulgaris
Ps. aeruginosa
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Mixed culture Gram stain and streak for
isolation MSA
Day 1
Day 2
TSA
Gram stain and select a Gram positive colony
Catalase ()
Positive for mannitol on MSA
Negative for mannitol on MSA
Day 3
Nitrate ()
Nitrate (-)
Trehalose(-)
Novobiocin sensitive
Novobiocin resistant
Novobiocin sensitive
Day 4
Staphylococcus aureus
Staphylococcus saprophyticus
Staphylococcus epidermidis
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Lab report

• Maximum 5 pages in length (excluding cover page
  and references) double spaced, 12pts fonts
• Attach all figures, chart, drawing at the end of
  the report (must be referenced in text)
• Cover page
• Title
• Author
• Number of your tube

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Introduction

• Introduction
• Background information about microbial
  metabolism. e.g.. Enzymes, catabolism,
  fermentation and oxidation.
• Biochemical tests in bacteria identification,
  focused in the biochemical test used to identify
  your unknown bacterium.
• Presentation of the problem Bacterial unknown

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Materials and methods

• The steps you followed to identify your
  bacterium.
• What media did you use in this assay
• How was the inoculation process
• What tool did you use to inoculate
• What reagents did you use after incubation
  process to confirm each test
• Incubation temperature

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Results and discussion

• Draw your own flow chart of your biochemical
  results
• Interpret your results
• Give the identity of your unknowns
• Bacteria name must be written in italic
• E.g. Escherichia coli
• Make sure that you include the unknown number in
  this section
• Some background about the clinical importance of
  your bacteria unknowns

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References

• Required each time that you use information that
  is not common knowledge
• You can use trustworthy website (university,
  college etc)
• Journal Article Steward, K. K. and W. H. Ornes.
  1975. The ecology of sawgrass in the Florida
  Everglades. Ecology 56162-171.
• In the text (Steward and Ornes, 1975)
• Website can be referred to in the text or as foot
  print
• Lab report due
• April 14th 2008