Bacterial identification techniques

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Bacterial identification techniques
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Bacterial nomenclature

• Bacteria are given a genus and a species name,
  which have taxonomic standing
• The genus provides a broad umbrella under which
  to place a group of related organisms
• The species further refines the individuals in
  the genus
• There is also sub-species nomenclature, which is
  often based on antigenic characteristics
• Salmonella enterica subgroups (gt2,463)
• E. coli is typed on the basis of its LPS (O157)

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Bacterial nomenclature

• For the most part, bacterial names consist of a
  genus and a species
• Both of these are written in italics (or
  underlined if italics are not possible)
• The genus name is ALWAYS capitalized
• The species name is NEVER capitalized

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Bacterial nomenclature

• When writing a bacterial name, the first time you
  use the name it is spelled out in full
• Every time after that, the genus name is
  abbreviated to (usually) one letter with a period
  and the species name is spelled out in full (all
  are italicized)
• Escherichia coli or E. coli
• Clostridium perfringens or C. perfringens

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Bacterial nomenclature

• As well as taxonomically correct names, there are
  trivial names, e.g., staphylococci or
  corynebacteria
• These names have no taxonomic standing and can be
  thought of as bacterial nicknames
• These names are never italicized or capitalized
  (unless at the beginning of a sentence)
• Some staphylococci are pathogenic for humans
• Staphylococci are often human pathogens

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Bacterial classification

• There have been numerous ways in which bacteria
  have been classified
• Older methods relied on descriptive properties
• Gram stain reaction
• Motility
• Spore formation
• Aerobic and/or anaerobic growth

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Bacterial classification

• Other methods include biochemical properties
• E.g., the ability to express a certain enzyme
  which can be measured in vitro
• An example of this is the enzyme catalase
• Catalase breaks down H2O2 (hydrogen peroxide)
  into water and oxygen
• The O2 (oxygen gas) is detected as bubbles

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Bacterial classification

• Other classification techniques employ antigenic
  properties
• These antigenic differences are detected through
  the use of specific antibodies
• Antigenic differences usually split members of a
  single species into sub-species or serovars
• E. coli (O and H antigen typing)
• Salmonella enterica (gt2463 types)
• Salmonella enterica subsp. enterica serovar Typhi

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Bacterial classification

• Now, we rely on molecular methods for bacterial
  classification
• The most popular technique is 16S rRNA typing
• Allows differentiation at the single nucleotide
  level in the bacterial DNA (DNA differences)
• Bacterial classification is changing all the time
• In this class, we will not always use the most
  up-to-date classification, but (in a few cases)
  we will retain the more familiar names
• Shigella dysenteriae vs. E. coli Dysenteriae

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Bacterial classification

• However, just because 16S rRNA typing is a more
  powerful tool doesn't mean that standard staining
  techniques and biochemical tests have no
  relevance
• The Gram stain is still one of the most commonly
  used identification techniques in medical
  bacteriology

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Bacterial identification

• About 40 years ago, two British scientists, Cowan
  and Steele, developed a scheme for the
  identification of medically important bacteria
• We will use these principles in the bacteriology
  portion of this class
• Cowan and Steele noticed that a few simple tests
  could define the genus of the unknown bacterium
• They called these the first stage tests
• Remember, all these bacterial identification
  techniques assumes that you have a PURE culture

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First stage tests

• Consist of
• Gram stain (reaction and cell shape)
• Acid fast cell wall
• Spores
• Motility
• Aerobic and/or anaerobic growth
• Catalase
• Oxidase
• Acid from glucose
• Oxidation/fermentation reaction

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Gram stain

• Still the most widely used staining procedure in
  bacteriology
• It is a differential stain since it
  differentiates between Gram-positive and
  Gram-negative bacteria
• Bacteria which stain purple are Gram-positive
• Bacteria which stain pink are Gram-negative

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Acid fast cell wall

• This test measures the ability of a Gram stained
  bacterial cell wall to resist decolorization with
  acid alcohol
• This test is only positive for Mycobacterium spp.
• We will not use this test in the labs

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Spore stain

• Spores are resistant to heat, drying,
  radiation and chemical disinfectants
• Therefore, they are difficult to stain and
  staining requires heat to allow the stain to
  penetrate
• Once stained, spores are equally hard to
  decolorize
• Only Gram positive rods produce spores, so the
  spore stain is a good diagnostic test for
  Bacillus and Clostridium spp.

B. subtilis
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Motility

• We will measure motility with a semi-solid
  motility test medium
• Non-motile organisms growth occurs along the
  line of inoculation and is very sharp and defined
• Motile organisms swim away from the stab line
  and growth occurs throughout the tube rather than
  being concentrated along the line of inoculation
• Dyes, which change color as the bacteria grow,
  can make the test easier to read

Nonmotile
Motile
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Aerobic and/or anaerobic growth

• Microorganisms vary in their requirements for and
  tolerance of oxygen
• Aerobes grow only in the presence of oxygen
• Grow in ambient air (18-20 oxygen, lt1 CO2)
• Microaerophiles grow only in decreased oxygen
  concentrations (these are still aerobes though)
• Grow in a candle jar
• Oxygen is consumed by the candle flame and
  replaced with CO2
• CO2 increases to between 5-10

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Aerobic and/or anaerobic growth

• Anaerobes are inhibited or killed by oxygen
• Grow in a Brewers jar the gas pack generates
  H2 which reacts with oxygen via a catalyst to
  form H2O
• How much oxygen an organism prefers determines
  how we culture bacteria
• More oxygen in air (regular incubator)
• Less oxygen (microaerophile) candle jar
• No oxygen Brewers jar
• You need to know the difference between a candle
  jar and a Brewers jar

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Aerobic and/or anaerobic growth

• Perform this test using media that will support
  the growth of all of your suspect organisms
• Use a rich medium such as Tryptic Soy Agar (TSA)
  or Sheep Blood Agar (SBA) for fastidious
  organisms
• As most organisms are facultative anaerobes,
  positive growth under both conditions doesnt
  tell us much
• However, either obligate aerobic or anaerobic
  growth can be diagnostic for a number of species
• Pseudomonas spp. are obligate aerobes, while
  Clostridium spp. are obligate anaerobes

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Catalase

• Aerobic respiration results in the formation of
  hydrogen ions (H) which are converted to toxic
  hydrogen peroxide (H2O2) by part of the electron
  transport system
• Catalase breakdowns H2O2 into non-toxic H2O
  and O2
• Therefore, most bacteria are catalase-positive,
  with the exception of bacteria that do not carry
  out aerobic respiration
• Streptococcus and Clostridium spp. are
  catalase-negative

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Catalase

• Do not perform this test using a nichrome wire
  loop as the metal can cause false positive
  reactions
• Use a toothpick
• If possible, do not perform the catalase test on
  colonies grown on blood-containing media as this
  can also cause false positives

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Oxidase

• This test determines whether an organism makes
  cytochrome oxidase
• This enzyme is involved in the electron transport
  chain
• Oxidase reacts with O2 and oxidase reagent form a
  blue/purple colored compound, indophenol
• A positive reaction should occur within 3 minutes
• If the color change does not occur within 3
  minutes, the test is negative

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Oxidase

• Use fresh cultures
• Do not perform this test using a nichrome wire
  loop as the metal can cause false positive
  reactions
• Wet the Taxo N disk (contains dried reagent) with
  a couple of drops of water
• Use a toothpick to transfer the colony to the wet
  disk
• A positive reaction is indicated by a dark
  blue/purple color within 3 minutes

Negative Positive
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Acid from glucose

• The medium used for this test contains
• Glucose the sugar is broken down by
  fermentation and/or respiration, providing a
  source of energy
• Peptone partially digested proteins (peptides)
  which supplies amino acids that are a source of
  nitrogen, carbon, sulfur and energy can also be
  used to support growth if the sugar is not
  utilized
• pH indicator changes color under acidic
  conditions, such as occurs during sugar breakdown
• All carbohydrate (sugar) fermentation tests use
  similar principles

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Oxidation/fermentation reaction

• Fermentation
• An energy yielding, catabolic process whereby
  organic molecules (not oxygen) serve as both
  electron donors and electron accepters
• This is NOT the same thing as anaerobic growth
• Fermentation reactions produce energy and result
  in the formation of acids
• Oxidation
• An energy yielding process involving oxygen and
  the formation of acid end products
• Production of acid reduces the pH of the medium
  and the indicator changes from green to yellow

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Oxidation/fermentation reaction
O-/F- B. pertussis
O/F- N. gonorrhoeae
O-/F C. perfringens
O/F E. coli
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Second stage tests

• The choice of second stage tests depends on the
  genus of the bacterium
• These tests are used to identify most species of
  clinically relevant bacteria (pathogens and
  normal flora) with as few tests as possible
• Common second stage tests include
• Carbohydrate fermentation
• Hemolysis
• Growth in the presence of inhibitors high salt,
  bile
• Species-specific tests e.g., coagulase for S.
  aureus

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Second stage tests forGram positive cocci
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Carbohydrate fermentation

• Carbohydrates (sugars and starches) can be broken
  down by fermentation, resulting in acid
  production (pH indicator change)
• Specific enzymes are required to break down each
  carbohydrate, so if the bacteria has the enzyme,
  it can use the carbohydrate
• Carbohydrate fermentation patterns are commonly
  used to identify unknown bacteria

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Carbohydrate fermentation

• Carbohydrate fermentation media contains
• Peptone to support growth if the carbohydrate
  can not be utilized
• Carbohydrate sugar to be tested
• pH indicator bromthymol blue, changes from
  purple to yellow to indicate acid production as a
  by-product of carbohydrate fermentation
• Glass tube inverted tube collects any gas
  formed
• We detect carbohydrate fermentation by looking
  for
• Acid end products (pH indicator change)
• Acid and gas end products (pH and gas bubbles)

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Carbohydrate fermentation
No acid/no gas Negative
Acid/no gas Positive
Acid and gas Positive
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Hemolysis

• Hemolysis is measured on agar containing blood
  and is a result of lysis of the red blood cells
• The most common medium used is sheep blood agar
• There are two main types of hemolysis
• Alpha hemolysis the zone around the colony is
  green as a result of incomplete lysis
• Beta hemolysis the zone around the colony is
  clear as a result of complete lysis of the red
  blood cells
• Gamma hemolysis is actually NO hemolysis

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Hemolysis
Alpha
Beta
Gamma
Comparison
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Coagulase

• Coagulase is an enzyme expressed by certain
  pathogenic staphylococci which can coagulate
  blood plasma
• Bacteria are mixed with rabbit plasma and
  coagulase on the cell surface coagulates or
  clumps the plasma
• Usually definitive for S. aureus

Negative
Positive
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Mannitol salt agar

• Used to identify staphylococci
• Medium contains
• 7.5 NaCl inhibits the growth of non-salt
  tolerant bacteria
• Mannitol sole carbon source if the organism
  can utilize mannitol, the pH of the medium will
  change (red to yellow)
• For the test to be positive, the organism must
  grow (be salt tolerant) AND ferment mannitol (pH
  change)
• S. aureus is positive in this test

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Mannitol salt agar
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Nitrate test

• The ability to reduce nitrate to nitrite is a
  commonly used test in identification of bacterial
  unknowns
• The nitrate test medium contains
• Peptone and beef extract for growth
• Potassium nitrate

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Nitrate test

• Add equal parts of reagent I and II to the slant
• A red color indicates the presence of nitrite
• Positive reaction
• If color does not appear within 30 seconds, add a
  pinch of zinc dust (with a toothpick)
• If no color appears, complete nitrate reduction
  occurred and no nitrate or nitrite are present
  positive reaction
• If a pale red color appears, it means that the
  nitrate was present in medium negative reaction
• An uninoculated nitrate slant should also be
  tested to make sure the reagents are working

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Nitrate test
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Hippurate hydrolysis

• 0.2ml water is heavily inoculated with the strain
  to be tested (visibly turbid)
• A hippurate disk added and incubated at 37oC for
  2 hours
• Ninhydrin reagent is added and the broth is
  incubated at 37oC for 30 min
• Glycine is detected by formation of a dark
  blue/purple color
• A faint purple tinge is considered negative
• Hippurate hydrolysis is considered presumptive
  for identification of Group B streptococci, such
  as S. agalactiae

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CAMP test

• This test is used to identify S. agalactiae
• S. agalactiae strains release a diffusible,
  extracellular compound
• In conjunction with a specific beta-hemolysin of
  S. aureus, this compound causes complete lysis of
  sheep red blood cells in an agar medium
• The test is performed by streaking an S. aureus
  down the plate
• Strains to be tested are streaked at a 90o angle
  to the S. aureus (close, but not touching)

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CAMP test

• The test is positive if an arrow-shaped zone of
  complete red blood cell lysis is observed
• CAMP is an acronym for Christie, Atkins, Munch,
  Petersen, the discoverers of this phenomenon

S. aureus
S. agalactiae Positive
S. bovis Negative
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Bile esculin agar

• The medium contains
• 40 bile a detergent found in the gut toxic to
  many bacteria, especially at this high
  concentration
• Esculin a sugar by-products of esculin
  hydrolysis react with iron in the medium, turning
  it black
• A positive result is growth AND blackening of the
  medium
• Used to distinguish enterococci from streptococci
• Enterococci are usually found in the gut, so can
  grow in the presence of bile, whereas most
  streptococci can not

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Bile esculin agar

• For the test to be positive
• Growth must occur on the medium
• Organism can grow on bile salts
• AND
• The medium turns black around the growth
• Hydrolysis of esculin (a sugar) to esculatin

Negative (just growth)
Positive
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Drug differentiation tests

• A drug-impregnated disk is applied to the primary
  streak on an agar plate
• If the organism is resistant, it can grow to the
  edge of the disk
• If the organism is susceptible, a zone of
  inhibition (no growth) is seen around the disk
• Novobiocin
• An antibiotic that interferes with DNA synthesis
• Most clinically important staphylococci are
  susceptible to novobiocin, whereas S.
  saprophyticus is resistant

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Novobiocin resistance

• S. epidermidis zone of inhibition
• SUSCEPTIBLE
• S. saprophyticus no zone of inhibition
• RESISTANT

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Drug differentiation tests

• Bacitracin
• A peptide antibiotic which acts on the bacterial
  cell wall
• S. pyogenes is susceptible, whereas other
  Streptococcus spp. are resistant
• Optochin
• Ethylhydrocupreine hydrochloride - a quinine
  derivative with detergent-like action
• S. pneumoniae is susceptible, whereas other alpha
  hemolytic streptococci are resistant

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Drug differentiation tests

• S. pneumoniae S. viridans

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Third stage tests

• Third stage tests can be used to further
  differentiate closely related species or
  sub-species
• These tests often rely on the antigenic
  properties of the strain
• LPS (O-antigen) or flagella (H-antigen)
• Detection of antigens requires specific
  antibodies
• E. coli typing (H7O157)
• We will not do any third stage tests is the lab
  exercises

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Points to remember

• First stage tests will identify the genus of an
  unknown bacterium
• Or at least, will narrow it down to two closely
  related genera
• Second stage tests will identify the species of
  an unknown bacterium
• See the caveat above
• Third stage tests will further differentiate the
  species into sub-species or sub-types
• All the tests require pure cultures

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Points to remember

• First stage tests consist of
• Gram stain, acid fast cell wall
• Spores, motility
• Aerobic and/or anaerobic growth
• Catalase, oxidase
• Oxidation/fermentation reaction, acid from
  glucose
• Remember, it may not be necessary to perform all
  the first stage tests, depending on the Gram
  stain result