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Bacterial identification techniques

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Title: Bacterial identification techniques


1
Bacterial identification techniques
2
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)

3
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

4
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

5
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

6
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

7
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

8
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

9
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

10
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

11
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

12
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

13
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

14
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

15
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
16
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
17
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

18
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

19
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

20
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

21
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

22
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

23
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
24
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

25
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

26
Oxidation/fermentation reaction
O-/F- B. pertussis
O/F- N. gonorrhoeae
O-/F C. perfringens
O/F E. coli
27
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

28
Second stage tests forGram positive cocci
29
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

30
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)

31
Carbohydrate fermentation
No acid/no gas Negative
Acid/no gas Positive
Acid and gas Positive
32
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

33
Hemolysis
Alpha
Beta
Gamma
Comparison
34
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
35
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

36
Mannitol salt agar
37
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

38
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

39
Nitrate test
40
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

41
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)

42
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
43
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

44
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
45
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

46
Novobiocin resistance
  • S. epidermidis zone of inhibition
  • SUSCEPTIBLE
  • S. saprophyticus no zone of inhibition
  • RESISTANT

47
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

48
Drug differentiation tests
  • S. pneumoniae S. viridans

49
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

50
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

51
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
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