IDENTIFICATION OF
UNKNOWNS
Mon 11-3
IDENTIFICATION
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Over a four week period you will carry out a series of tests
and experiments to identify an initially unknown Gram
positive and Gram negative organism from a mixed broth
culture
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By accumulating results from all the various tests you will
identify each bacterium to a genus and species level.
PHENOTYPIC IDENTIFICATION METHODS

Morphological characteristics:
Isolated colony-appearance and morphology from streak plates
 Cell morphology, arrangement and size by staining and microscopy
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Gaseous requirements:
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observe growth patterns in broth and deeps
Differential staining:
Gram-staining
 Spore-stain (only applies to Gm + bacilli)
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Biochemical tests:
Differential and selective media: MacConkey, Mannitol Salt and
Blood agar;
 Biochemical tests of carbohydrate and protein metabolism
 Tests of cell respiration such as: oxidase, catalase and nitrate tests
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REFERENCES
Bergey’s Manual of Determinative
Bacteriology*
Provides identification schemes for
identifying bacteria and archaea
Morphology, differential
staining, biochemical tests
Bergey’s Manual of Systematic
Based on rRNA sequencing
Bacteriology
Provides phylogenetic information on
bacteria and archaea
We will use the Determinative manual.
Copies available in the microbiology lab; biology resource center and on reserve
at the CCSF library* On-line flow charts at:
http://www.uiweb.uidaho.edu/micro_biology/250/IDFlowcharts.pdf
Sample partial dichotomous key
Possible Gram- bacteria
Alcaligenes fecalis
 Citrobacter freundii
 Enterobacter aerogenes
 Escherichia coli
 Klebsiella pneumoniae
 Morganella morganii
 Proteus mirabilis
 Proteus vulgaris

Providencia stuartii
 Pseudomonas fluorescens
 Salmonella enterica
 Shigella flexneri

Possible Gram+ bacteria
Bacillus cereus
 Bacillus subtilis
 Corynebacterium xerosis
 Enterococcus fecalis
 Lactococcus lactis
 Micrococcus luteus
 Staphylococcus aureus
 Staphylococcus epidermidis

Week 1: Period 1
Begin with a mixed
culture; record number
 Streak plates to obtain
isolated colonies of gram
negative and positive
organisms on selective
and differential media
(MacConkey agar:
Mannitol Salt agar)
 Streak on Trypticase soy
(enrichment) agar

Week 1: Period 2
Record growth characteristics (colony
morphology, size, color, etc.)
 Transfer isolated colonies of two species onto 2
separate NA/TSA plates and keep them
separate from now on
 Confirm separation of mixed culture by gram
staining
 Transfer confirmed isolated bacteria to separate
TSA slants to incubate
 Subculture on fresh TSA slants in subsequent
weeks as needed

Week 2: Period 1
Perform biochemical
tests of carbohydrate
metabolism and
respiration on unknowns
 Inoculate and incubate
media
 Each bench include an
uninoculated control for
comparison

Week 2: Period 2
Get results and interpret
all biochemical tests (do
not simply record color
changes)
 Make note of any
reagents needed to
perform tests
 Fresh subcultures

Week 3: Period 1
Perform tests of protein
metabolism on each
unknown
 Inoculate and incubate
media
 Each bench include an
uninoculated control for
comparison

Week 3: Period 2
Get results and interpret
all biochemical tests (do
not simply record color
changes)
 Make note of any
reagents needed to
perform tests
 Fresh subcultures

Week 4
Perform any additional NECESSARY biochemical
tests (eg. spore stain). These will vary per student
 Compile your unknown report per lab manual
guidelines
 Presentation, organization, and completeness count!
No late reports accepted (Due 12/10/14)
 Note: if you lose your unknowns or do not keep them
growing you will only be reassigned organisms once

Guideline for unknown ID
Follow aseptic techniques at all times
 Label all cultures (date, name, section, organism)
and keep track of subcultures. Do not discard a
culture until you have new growth.
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Cultures < 2 days put at 37 C unless otherwise
advised
Cultures > 2 days put in RT incubator
Draw, label and color and/or photograph
observations as you go
Guideline for unknown ID
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Keep organized and detailed records of all materials,
methods, results and interpretations in a chronological
order
Work independently & be responsible for your work
Do NOT touch other students work and make sure it is
left in appropriate shelf for your lab section in the
incubator and the refrigerator
Lab report

Research paper format
Title: Should be descriptive and relevant to the
project. Do not use the word “unknown” in the
title.
 Abstract: Should state the topic of study and its
significance, describe general methods, highlight
major findings, and have concluding sentence on
what you learnt from your study.

Lab report
Introduction: Describe the main purpose and
goals of this project.
 Materials and Methods: Briefly outline the types of
media and methods used.
 Results: Include drawings of ALL your results and
include controls. Use a template for a petri plate or
test tube. Include a brief interpretation of the
results next to or below the figures. Do not just
state positive or negative outcomes.

Lab report
Discussion: Show your dichotomous keys to show
how you concluded the identity of each organism.
Once identified research each organism and discuss
any related diseases and symptoms (if any) and any
relevant prevention and treatment. If they have been
involved in a disease outbreak you can discuss that
information. Write about half a page for each
organism.
 Bibliography: Include a current bibliography and
at least 3 references in addition to your text and Lab
Manual

Lab report
Throughout the paper keep to the facts and avoid
repetition.
 Use the third person (passive voice) in reports and
avoid the use of “I” or “me”.
 Use the same tense (usually past) to be consistent.
 Check your spelling and grammar before submitting
your report

CHECKLIST OF RESULTS TO RECORD FOR
BOTH ORGANISMS
Isolated colony characteristics (enlarge image)
 Gram stain and cell morphology (enlarge image) and
document cell size
 Appearance on MAC/MSA/TSA media
 Biochemical tests of respiration, carbohydrate &
protein metabolism with controls
 Other: motility if present, gaseous requirements,
hemolytic outcome on blood agar, spore stain if needed

Mixed culture vs. pure culture
• Mixed culture: a microbial culture
consisting of two or more species
• Pure culture: a culture that contains only
one species of microorganisms
• Methods of isolation: achieve pure culture
from a mixed culture
Methods of isolation
• Isolation is a prerequisite to microbial ID
• Quadrant streak: during streaking, the cell
density decreases, eventually leading to
individual cells being deposited separately on
the agar surface
• Each colony comes from one individual cell,
so is a pure population (colony-forming units /
CFU)
Selective media
• Nutritional components:
• Selected to obtain optimal growth for certain bacteria
• Inhibitors:
• Inhibit growth of some bacteria
• Differentiating substrates:
• Substrates that only one group of organisms can
utilize
• Indicators:
• Make reactions visible
Mannitol Salt Agar
• Selective: for Staphylococcus, against all other
bacteria
• Differentiating substrate: Mannitol
• S. aureus ferments mannitol and forms bright yellow
colonies
• Other Staphylococcus bacteria don’t ferment
mannitol and form pink-red colonies
• Inhibitor: NaCl (7.5%)
• High salt kills most bacteria
• Indicator: phenol red
• Bright yellow at low pH (<6.8)
• Red or pink at high pH (>7.4)
Mannitol Salt Agar
S. aureus
S. epidermidis
Not Staphylococcus
https://c2.staticflickr.com/4/3470/3396459999_7d79cd3f62.jpg
MacConkey Agar
• Selective: for Gm- bacteria (enteric bacteria),
against Gm+ bacteria
• Differentiating substrate: lactose
• Coliforms: lactose fermentors
• Inhibitor: Bile salts and crystal violet
• Inhibit Gm+ bacteria
• Indicator: neutral red
• Red at low pH (<6.8)
• Colorless at high pH (>6.8)
MacConkey Agar
Gram+
Gramcoliform
Gram- coliform
(bile precipitate)
Gramnonfermenter
http://classconnection.s3.amazonaws.com/843/flashcards
/663843/jpg/picture31318207525204.jpg
Lab Report Grading Rubrics
• Download from Dr. Toebe’s website
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Total points: 75
Summary charts: 15 X 2
Actual results chart: 15
Dichotomous keys (flow chart): 15
Research of identified bacteria and
bibliography: 15
Lab Report Grading Rubrics
• Summary chart 1: due 11/17/14
• Should include all the carbohydrate
metabolism and respiration tests we will do
on 11/17
• Show protocols and expected results
• Summary chart 2: due 11/24/14
• Should include the protein metabolism
tests we will do on 11/24
• Show protocols and expected results