BIO488L: Medical Microbiology Lab Exercise 2

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BIO488L Medical Microbiology Lab Exercise 2

• Spring 2006
• T.A. Sami Kemper

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Today

• Safety Quiz
• Scores from last week
• Exercise 2
• Staining
• Biochemicals
• Selective and differential media

• Open hours for this week
• Tuesday
• Wednesday
• Thursday

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The Enterics

• Enterobacteriaceae
• Gram negative rods
• Ubiquitous
• Mostly opportunistic pathogens
• Some overt pathogens
• Some E. coli strains (STEC, enteropathogentic,
  enterohemorrhagic etc)
• Some Salmonella strains (Typhi, paratyphi,
  Choleraesuis)
• Yersinia pestis, and Yersinia pseudotuberculosis

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Escherichia coli

• Well studied common commensal organism.
• Evolved with mammals
• Great genetic diversity.
• Shigella spp. and Salmonella enterica
• Truly pathogenic strains evolved recently.
• STEC/VTEC enterohemorrhagic, enteropathogenic
  and enteroinvasive

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Laboratory Indications

• Lysine
• Citrate -
• Indol
• Acetate
• Lactose
• Motile
• Urease -
• Hydrogen sulfide -
• Growth on MacConkey's, EMB and blood
• Acid slant and acid butt in TSI, gas

E. coli on EMB
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Shigella sonnei

• Multiple origins from within the E. coli
• Causes bacillary dysentery occaisional HUS

Shigella sonnei on EMB
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Virulence Factors

• Cytotoxin
• Endotoxin
• Enterotoxin
• Siderophore
• Shigatoxin
• Invasin

• Induced phagocytosis
• Lysis of phagosome
• Induced endocytosis by epithelial cells
• Intracellular growth

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Laboratory Indications

• Lysine -
• Non-motile
• -/ TSI reaction (no gas)
• Acetate -
• Lactose -
• Serotype D
• Growth on MacConkey's and EMB
• Hydrogen Sulfide -
• Urease -

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Salmonella typhimurium

• Biovar of Salmonella enterica
• Cuases gastroenteritis (Salmonellosis) and
  enteric fever
• Causes a typhoid-like fever in mice

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Serovars
Role in Virulence
Locus
All
Invasion of epithelials, macrophage apoptosis
SPI-1
All but S. bongori
Intracellular prolif., systemic disease
SPI-2
All, some have large deletions
Intramacrophage survival
SPI-3
All
Intramacrophage survival, ???
SPI-4
Dublin, Typhimurium, Enteritidis, Choleraesuis,
Gallinarum, Pullorum
Enteropathogenesis
SPI-5
All non-typhoidal
Intramacrophage survival??
Plasmid
All
Multiple, adhesion
Islets
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Laboratory Indications

• Lysine
• Hydrogen sulfide
• Indole
• Citrate
• ONPG -
• Malonate -
• Hydrogen sulfide
• -/ TSI reaction (with gas)
• Growth on MacConkey's, EMB, HEK

Salmonella typhimurium on HEK
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Klebsiella pneumoniae

• Most clinically important species of the genus
• Pathogenicity attributed to the production of
  heat stable enterotoxin and endotoxin (lipid A)
• May carry resistance plasmids (R-plasmids)
• Produces a capsule
• Pneumonia, septicemia, wound infection, burn
  infection, UTI

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Laboratory Indications

• Growth on MacConkey's with pink colonies
• Growth on EMB
• Lysine
• Citrate
• Indol -
• / TSI (with gas)
• Non-motile
• Ornithine -
• Hydrogen sulfide -

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Proteus vulgaris

• Causes wound infections and UTI's
• Second most common cause of non-hospital acquired
  UTI's
• The enzyme used to break down urea raises the pH
  of urine and potentiates the formation of
  urinatry stones in which the bacteria can hide
  during treatment
• P. vulgaris is less common than P. mirabilis and
  can be differentiated by its positive indole
  formation

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Virulence Factors

• Adhesin
• Endotoxin
• Urease
• Motility

Proteus vulgaris
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Laboratory Indications

• Lysine -
• Hydrogen sulfide
• Swarming motility
• Urease
• Indole
• Growth on MacConkey and EMB
• Gas in TSI

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Biochemical Tests

• Carbohydrate Fermentation
• Urease
• Motility
• Triple Sugar Iron Agar
• Catalase
• Oxidase

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Definitions

• Selective Media Suppresses the growth of
  certain organisms while permitting the growth of
  others. This occurs because an ingredient is
  inhibitory or lethal only to certain organisms.
• Differential Media (can also have selective
  properties) Media on which specific
  physiological characteristics of microbes can be
  demonstrated. This makes it possible to
  recognize differences between related microbial
  groups. For example, Staphylococcus aureus
  fermentation of mannitol (on Mannitol Salt Agar
  plates) produces acid, which diffuses into the
  agar that surrounds its colonies. A red pH
  indicator has been added to the medium, and the
  acid changes the color of the indicator to
  yellow. Staphylococcus epidermidis does not
  degrade mannitol. Therefore, there would be no
  color change of the agar around the colonies.

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Carbohydrate Fermentation

• Ability to ferment various carbohydrates
• Glucose, sucrose, lactose, mannose
• Check for production of acid (yellow color
  change), and bubbles (trapped in Durham tube) at
  24 and 48 hours

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Urease

• Presence of urease enzyme which breaks down the
  waste product urea
• Color chance to bright pink indicates positive,
  any other color change is negative
• Check at 24 and 48 hours

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Motility

• Detect motility in bacteria
• Tetrazolium indicator shows where bacteria have
  grown
• Stab the media- growth outside of stab line
  indicates motility

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Triple Sugar Iron Agar (TSIA)

• Contain lactose and sucrose in 1 concentrations
  and glucose in a 0.1 concentration.
• Used to differentiate among the different genera
  of Enterobacteriaceae, and to distinguish the
  Enterobacteriaceae from other Gram-negative
  intestinal bacilli.
• Differentiation is made based on differences in
  carbohydrate fermentation patterns and H2S
  production.
• The acid-base indicator (Phenol Red) is also
  incorporated turns yellow (acidic) when
  fermentation occurs.
• Sodium thiosulfate may be metabolized to produce
  H2S and ferrous sulfate. Following incubation,
  only cultures of organisms capable of producing
  H2S will show an extensive blackening of the butt
  due to the precipitation of the insoluble ferrous
  sulfide.
• Read at 24 and 48 hours.

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Catalase

• Presence of catalase enzyme which breaks down
  peroxide radicals
• Place a few drops of hydrogen peroxide solution
  on a loopful of your isolate on a glass slide and
  watch for the appearance of bubbles- they may be
  very small
• Bubbles positive/ no bubblesnegative

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Oxidase

• Presence of cytochrome c in the electron
  transport chain
• Phenylenediamine solution on filter paper- dont
  touch!
• Positive blue/ negativegrey

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Eosin Methylene Blue Agar (EMB)

• EMB contains protein, buffer, two dyes (Eosin Y
  and Methylene Blue), agar, and two carbohydrates
  (lactose and sucrose).
• Eosin Y and Methylene Blue are inhibitory to most
  species of Gram-positive bacteria, but have
  little toxicity against G- bacilli.
• Differentiation of G- bacilli based on lactose
  fermentation.
• Escherichia coli produces colonies with a
  metallic sheen.
• Lactose and sucrose fermenters form dark-colored
  colonies. This dark precipitate is Methylene
  Blue Eosinate, which is precipitated as the
  result of the low pH generated
• Non-fermenters usually raise the pH of the
  surrounding medium by oxidative deamination of
  protein, which solubilizes the Methylene
  Blue-Eosin complex and results in colorless
  colonies.

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Hektoen Enteric Agar (HEK)

• HEK contains protein, bile salts, sodium
  thiosulfate, ferric ammonium citrate, two
  carbohydrates (lactose and sucrose), salicin, and
  pH indicators (Acid-Fuchsin and Thymol Blue).
• Selective and differential medium for direct
  isolation of enteric pathogens. The selectivity
  is based upon the presence of bile salts that
  inhibit the growth of Gram bacteria and retard
  the growth of many strains of normal intestinal
  Gram- flora.
• Gram- enteric pathogens and non-pathogens are
  differentiated according to their ability to
  ferment lactose and sucrose and to produce H2S.

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• Most non-pathogens ferment at least one of the
  carbohydrates and produce bright orange to
  salmon-pink colonies due to the combination of
  the yellow color from the Bromthymol Blue and the
  red color from the Acid-Fuchsin when acid is
  produced.
• Organisms that do not ferment lactose and sucrose
  typically appear green or blue-green.
• H2S-producing species (e.g. Salmonella)
  generate H2S gas from sodium thiosulfate. The
  gas reacts with ferric ammonium citrate to yield
  a black precipitate that accumulates within the
  colonies and forms a black center.

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Salmonella typhimurium
Escherichia coli
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MacConkey Agar (MAC)

• Contains protein, bile salts, NaCl, lactose,
  agar, and two dyes (Crystal Violet and Neutral
  Red).
• A selective and differential medium for the
  cultivation of aerobic or facultatively anaerobic
  Gram- bacilli.
• Selective action of MAC agar is attributed to
  Crystal Violet and bile salts, which are
  inhibitory to most species of Gram bacteria.
  Gram- bacteria usually grow well on the medium,
  and are differentiated by their ability to
  ferment lactose.

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• Lactose fermenting strains grow as red or pink
  colonies and may be surrounded by a zone of
  acid-precipitated bile. The red color is due to
  production of acids from lactose, absorption of
  Neutral Red, and a subsequent color change of the
  dye when the pH of the medium falls below 6.8.
• Non-lactose-fermenting strains, such as Shigella
  and Salmonella, are colorless and transparent.
  They typically do not alter the appearance of the
  medium.

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Salmonella Shigella Agar (SS)

• SS agar contains protein, bile salts, an H2S
  indicator and sulfur source (sodium thiosulfate),
  lactose, agar, and two dyes (Brilliant Green and
  Neutral Red).
• Used for isolation of Salmonella spp. and many
  strains of Shigella spp..
• Selective action of SS agar is attributed to
  Brilliant Green dye, which is inhibitory to most
  species of intestinal bacteria other than
  Salmonella, and bile salts and sodium citrate,
  which are inhibitory to most species of Gram
  bacteria.

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• The high concentration of bile salts is also
  inhibitory to many lactose-fermenting normal
  intestinal flora.
• Sodium thiosulfate is reduced by certain species
  of enteric bacteria to sulfite and H2S gas.
  Production of H2S gas is detected as an insoluble
  black precipitate (ferrous sulfide), which is
  formed upon reaction of H2S with the ferric irons
  of ferric citrate.
• Upon fermentation of lactose by the few
  lactose-fermenting organisms that can grow on SS
  agar, acid is produced and the pH indicator
  (Neutral Red) changes from yellow to red. Thus,
  these organisms grow as red-pigmented colonies.
  Non-lactose-fermenting organisms grow as
  colorless and translucent colonies with black
  centers (Salmonella) or without black centers
  (Shigella).

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Salmonella sonnei
Klebsiella pneumoniae
Escherichia coli