Transcript Biochemical tests

Fundamentals of Microbiology
Unit 4 Seminar:
Unknown Identification
Evelyn I. Milian
Instructor
2011
Microbiology: Unit 4 Seminar – Unknown Identification
Questions Assigned in Unit 4 Seminar Page
 Before seminar, take a look at the introduction in Bergey’s Manual.
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 http://www.archive.org/stream/bergeysmanualofd1957amer#page/n21/m
ode/2up
The Gram stain is typically the first test performed in a microbiology
laboratory in the process of identifying an unknown. WHY? Give some
examples of instances when a Gram stain would NOT be a useful first step.
What are biochemical tests and why are they performed?
Give 2 examples of biochemical tests and what the tests are used for.
Imagine that you are working in a microbiology lab and you are given a
sample of bacteria on an agar plate. You are told that it is either
Staphylococcus aureus or Streptococcus pyogenes. How would you
determine which one the sample contains? Are there any tests you could
do to differentiate them?
WHY must these steps be followed? Wouldn’t it be easier to just observe
the organism under a microscope and make an identification based on
observation?
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Microbiology: Unit 4 Seminar – Unknown Identification
Identification of Unknown Microorganisms
 Microorganisms are the most common and the most numerous group
of organisms on Earth. The world of Microbiology revolves around the
ability to categorize and identify these microorganisms. The
identification is especially important in the medical community to
determine the cause of disease and effectively treat patients.
 To identify unknown bacteria, Microbiologists use a diverse collection
of tests including stains, biochemical tests, and selective and
differential media.
 In general, the identification begins with a Gram stain and continues
with a scientific identification process, sometimes dependent upon
which identification is suspected. For example, if a child goes to the
pediatrician with a sore throat, the doctor might look specifically for
Streptococci in the child’s throat.
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Microbiology: Unit 4 Seminar – Unknown Identification
Identification of Unknown Microorganisms
 Given the massive number of bacterial species on Earth,
you can imagine that all of this information can pile up.
 For decades, Microbiologists have relied upon a collection
of books known as Bergey's Manual. This actually refers
to a collection of books that provide detailed information
on all recognized species of prokaryotes.
 Each chapter in Bergey's Manual is written by an expert,
contains tables, biochemical test results, and further
systematic information that is useful for identification
purposes.
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Microbiology: Unit 4 Seminar – Unknown Identification
Classification Schemes for Microorganisms
 A classification scheme provides a list of characteristics and a means for
comparison to aid in the identification of an organism. Once an organism is
identified, it can be placed into a previously devised classification scheme
(Tortora, 2010).
 Microorganisms are identified for practical purposes—for example, to
determine an appropriate treatment for an infection. They are not necessarily
identified by the same techniques by which they are classified. Most
identification procedures are easily performed in a laboratory and use as few
procedures or tests as possible.
 Protozoa, parasitic worms, and fungi can usually be identified
microscopically. Most prokaryotic organisms do not have distinguishing
morphological features or even much variation in size and shape.
Consequently, microbiologists have developed a variety of methods to test
metabolic reactions and other characteristics to identify prokaryotes.
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Microbiology: Unit 4 Seminar – Unknown Identification
Classification Systems in the Prokaryotes
 The original classification system was based on traits such as
morphology (shape), cell wall composition, motility, variations in
cellular arrangement, growth characteristics, and habitat.
 Today, classification schemes are based mainly on genetic and
molecular traits (such as comparing sequence of nitrogen bases in
ribosomal RNA) and their evolutionary relationships (phylogeny).
New groups are being identified and studied.
 Prokaryotes are divided into two domains: Archaea and Bacteria.
 Archaea share certain traits with bacteria and other traits with
eukaryotes.
 Bacteria and Archaea were grouped in Kingdom Monera in the past;
now they are separate domains because of important molecular
differences.
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Microbiology: Unit 4 Seminar – Unknown Identification
Classification Systems in the Prokaryotes
 Definitive published source for
bacterial classification:
 Bergey’s Manual – since 1923.
 The basis for early classification was
the phenotypic traits of bacteria
such as morphology, cell wall
composition, motility, growth features
such as oxygen use, and biochemical
reactions, including staining.
 Bergey’s Manual of Systematic
Bacteriology: Current version
combines phenotypic information
with molecular characteristics such
as rRNA sequencing.
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Microbiology: Unit 4 Seminar – Unknown Identification
Classification Systems in the Prokaryotes:
Diagnostic Scheme
 In clinical microbiology it is more useful to use a more informal
system to classify bacterial species based on their phenotypic
(observable) characteristics.
 This system is restricted to bacterial disease agents and is
based on readily accessible morphological and physiological
tests rather than on phylogenetic (evolutionary) relationships.
 It also divides the bacteria into gram-positive, gram-negative,
and those without cell walls.
 It subgroups bacteria according to cell shape, arrangement,
and certain physiological traits such as oxygen usage.
 Aerobic bacteria use oxygen, anaerobic bacteria do not use
oxygen, and facultative bacteria may or may not use oxygen.
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Microbiology: Unit 4 Seminar – Unknown Identification
Question 1: Gram Stain as a First Test
 The Gram stain is
typically the first test
performed in a
microbiology
laboratory in the
process of identifying
an unknown. WHY?
 Give some examples
of instances when a
Gram stain would NOT
be a useful first step.
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Microbiology: Unit 4 Seminar – Unknown Identification
Question 1: Gram Stain as a First Test
 The Gram stain helps differentiate bacteria into two broad groups based on
the structure of the bacterial cell wall. Most bacteria possess a cell wall
that contains either a thick peptidoglycan layer (Gram-positive; retain
crystal violet, a purple stain) or a thin peptidoglycan layer with an additional
lipopolysaccharide layer (Gram-negative; retain safranin, a red stain).
 The Gram stain is not be useful in identifying bacteria without a cell wall or
with unusual cell walls.
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Microbiology: Unit 4 Seminar – Unknown Identification
Questions 2 and 3: Biochemical Tests
What are biochemical tests and why
are they performed?
Give 2 examples of biochemical tests
and what the tests are used for.
(Chapter 5, Lab Manual)
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests for the Identification of Bacteria
 Simple, differential, and structural stains even if combined
with cultivation and observation of colony characteristics,
are not sufficient for the identification of bacterial isolates.
Results of staining and cultivation must be combined with
the results from biochemical tests.
 Biochemical tests evaluate the metabolic properties of
an isolate, which are unique for each species.
 A combination of biochemical tests can be used to
determine the biochemical pattern for an isolate. This
enables the identification of an isolate using an
identification scheme. (Alexander, 2001)
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests
 Biochemical activities
are widely used to
differentiate bacteria.
 Even closely related
bacteria can usually be
separated into distinct
species by subjecting
them to biochemical
tests, such as one to
determine their ability to
ferment an assortment of
selected carbohydrates.
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests
 In health care,
morphology and
differential staining
are important in
determining the
proper treatment for
microbial diseases.
 A clinician completes
the form to identify
the sample and
specific tests. In this
case, a genitourinary
sample will be
examined for sexually
transmitted infections.
A clinical microbiology lab report form
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests: Carbohydrate Utilization
 Tests used to determine whether an
organism has the ability to ferment
various carbohydrates (sugars)
and produce acid and gas.
 Inverted durham tube inside test
tube shows gas production.
 pH indicator: acid lowers pH
causing color change
 Yellow = fermentation (acid)
 Purple or red = negative for
fermentation
 Examples: phenol red broth;
purple broth
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 Bubbles in durham tube = gas
(from fermentation)
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests:
Citrate Utilization
 Tests for the enzyme citrase, produced
by some bacteria such as Enterobacter
aerogenes and Salmonella typhimurium,
but not by others, such as Escherichia
coli and Shigella flexneri.
 Medium: Simmon’s citrate agar,
containing citrate as the only carbon
source and pH indicator bromthymol
blue (blue if pH increases due to alkaline
products from citrate metabolism)
 Blue = positive
 Green = negative
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests: Indole Production
 Tests for presence of enzyme
tryptophanase, which breaks down
amino acid tryptophan to form
ammonia, pyruvic acid and indole.
 Medium: SIM, also used to detect
motility and hydrogen sulfide
production.
 Reagent: Kovac’s ( 5 drops added to
culture after incubation); reacts with
indole to form a red color (in alcohol
layer of Kovac’s).
Examples:
Negative = Enterobacter aerogenes
Positive = Escherichia coli
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 Red = positive
 No red = negative
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests: MR-VP Test
 Methyl Red–Voges Proskauer: combination medium used for two
tests for enteric bacteria; different reagents are added to do each test.
 Methyl Red (MR): To detect enteric bacteria capable of performing a
mixed acid fermentation, lowering the pH.
 Methyl red indicator added after incubation.
 Positive = red (stable acids produced)
 Negative = no color change (yellow/orange) (neutral end products)
Examples:
1) Enterobacter
aerogenes
2) Serratia
marcescens
Examples:
1) Escherichia coli
2) Proteus vulgaris
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests: MR-VP Test
 Voges Proskauer: for organisms able to ferment glucose and convert
acid products to acetoin and 2,3-butanediol.
 Voges-Proskauer reagents (alpha-naphthol and KOH) added after
incubation.
 Positive = red (2,3-butanediol fermentation; acetoin produced)
 Negative = no color change or copper color
Example:
Enterobacter
aerogenes
Example:
Escherichia coli
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests: Urea Utilization
 Tests for presence of urease, an
enzyme that breaks down urea (a
product of amino acid metabolism)
into ammonia (alkaline product)
and carbon dioxide.
 Distinguish Proteus from other
enteric bacteria.
Examples:
Positive: Proteus, Morganella,
Providencia (rapid urease-positive)
Negative: Escherichia coli
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 pH indicator: phenol red; changes
to pink when pH increases.
 Positive = pink
 Negative = no color change or
yellow (from acid products)
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Microbiology: Unit 4 Seminar – Unknown Identification
Questions 4 and 5: Biochemical Tests
 Imagine that you are working in a microbiology lab and you are given a
sample of bacteria on an agar plate. You are told that it is either
Staphylococcus aureus or Streptococcus pyogenes. How would you
determine which one the sample contains? Are there any tests you
could do to differentiate them?
 WHY must these steps be followed? Wouldn’t it be easier to just observe
the organism under a microscope and make an identification based on
observation?
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Evelyn I. Milian - Instructor
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Microbiology: Unit 4 Seminar – Unknown Identification
Biochemical Tests:
Catalase Test
 Detects the enzyme catalase,
possessed by most aerobic and
facultatively anaerobic bacteria.
 Catalase breaks down hydrogen
peroxide produced during aerobic
respiration. If H2O2 accumulates in
the cell, it becomes toxic.
 Some bacteria lack this enzyme:
Streptococcus , Enterococcus
 Reagent: 3% hydrogen peroxide
added to 18-24 hour culture on an
agar slant or glass slide
 Bubbles = positive (formed within
seconds from breakdown of
hydrogen peroxide into water and
oxygen). Example: Staphylococcus
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Microbiology: Unit 4 Seminar – Unknown Identification
Unit 5 Assignment – Example: CASE #1
 Following a round of water testing in rural Minnesota, contaminated
water was discovered in a stream. The source of the contamination is
most likely fecal contamination caused by run-off from a 600-acre dairy
farm close to the stream. Heavy rains in the area caused localized
flooding and the stream measured water levels well above normal.
 After isolating a species of bacteria from the water, the following
laboratory tests were completed. The lab notes are listed under
laboratory observations. You should record in the results column a
positive or negative for each test. To complete the Gram stain results,
simply interpret whether it is Gram positive or Gram negative and list the
gram reaction and shape. For example: Gram + rods in singles, Gram
negative cocci in chains.
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Microbiology: Unit 4 Seminar – Unknown Identification
Unit 5 Assignment – Example: CASE #1
TABLE 1.1
Laboratory Observations
Results
Gram Stain Pink rods
Glucose
Yellow media, gas bubble in durham tube
Indole
Red layer of reagents at the top of the test tube
Methyl Red Red
Urea
Pinkish-red color
Catalase
Bubbles
1. Interpret the tests in Table 1.1 and answer the questions below:
a. What is the result of the Gram stain?
b. Does this organism ferment glucose? How can you tell?
c. Would the catalase test be helpful to identify your unknown? Why or why not.
2. Using the unknown identification chart, compare the results recorded above. What
is the genus and species of the unknown organism?
3. If this water was ingested by humans, what type of infection could result? What
types of signs and symptoms would an infected individual display?
4. Is the only source of contamination the dairy farm? Give another possible
explanation for the water contamination.
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Microbiology: Unit 4 Seminar – Unknown Identification
Unknown Bacterium Identification Chart
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Microbiology: Unit 4 Seminar – Unknown Identification
References
 Alexander, S.K., & Strete, D. (2001). Microbiology: A Photographic Atlas for the
Laboratory. Pearson Education, Inc.-Benjamin-Cummings. CA, USA.
 Alters, S. & Alters, B. (2006). Biology, Understanding Life. John Wiley & Sons, Inc. NJ,
USA.
 Audesirk, T.; Audesirk, G. & Byers, B.E. (2005). Biology: Life on Earth. Seventh Edition.
Pearson Education, Inc.-Prentice Hall. NJ, USA.
 Black, J.G. (2005). Microbiology, Principles and Explorations. Sixth Edition. John Wiley &
Sons, Inc. NJ, USA. www.wiley.com/college/black.
 Campbell, N.A.; Reece, J.B., et al. (2008). Biology. Eighth Edition. Pearson Education, Inc.Pearson Benjamin Cummings. CA, USA.
 Cowan, M.K.; Talaro, K. P. (2009). Microbiology A Systems Approach. Second Edition.
The McGraw-Hill Companies, Inc. NY, USA. www.mhhe.com/cowan2e
 Dennis Kunkel Microscopy, Inc. (2010). http://www.denniskunkel.com
 Leboffe, M.J. & Pierce, B.E. (2010). Microbiology Laboratory Theory and Application.
Third Edition. Morton Publishing. Englewood, CO; USA.
 Mader, S.S. (2010). Biology. Tenth Edition. The McGraw-Hill Companies, Inc. NY, USA.
 Tortora, G. J.; Funke, B.R.; Case, C.L. (2010). Microbiology An Introduction. Tenth Edition.
Pearson Education, Inc.-Benjamin Cummings; CA, USA. www.microbiologyplace.com.
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