BIO488L Medical Microbiology Lab Exercise 2 by drg59916


									BIO488L: Medical Microbiology
       Lab Exercise 2

            Spring 2006
         T.A.: Sami Kemper
●   Safety Quiz              ●   Open hours for this
●   Scores from last             week
    week                         –   Tuesday
●   Exercise 2                   –   Wednesday
                                 –   Thursday
    –   Staining
    –   Biochemicals
    –   Selective and
        differential media
                     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,
         ●   Yersinia pestis, and Yersinia pseudotuberculosis
               Escherichia coli
●   Well studied common commensal
    –   Evolved with mammals
●   Great genetic diversity.
●   Shigella spp. and Salmonella enterica
●   Truly pathogenic strains evolved recently.
    –   STEC/VTEC: enterohemorrhagic,
        enteropathogenic and enteroinvasive
Type                     Disease                  Virulence Factors
Enterotoxigenic (ETEC)   Watery to cholera-like   LT, ST, CF
Enteroinvasive (EIEC)    Watery diarrhea to       Ipas, VirG/IcsA
Enteropathogenic         Watery diarrhea          Intimin, Tir, BFP
Enterohemorrhagic        HC, HUS                  EPEC factors +
 (EHEC)                                            shigatoxin(s), EspP and
Enteroaggregative        Watery to mucoid         AAF adhesins, EAST-1,
 (EAEC)                  diarrhea                  Pet, Pic, hemolysin
Diffusely adhering       Watery diarrhea          Adhesins and Esp-like
 (DAEC)                                            proteins
Uropathogenic (UPEC)     UTI’s                    Type I pili, P pili, Afa,
                                                   hemolysin, CNF-1
Septic (SEC)             Neonatal sepsis,         Capsule, type I pili, S-
                          meningitis               fimbrial adhesin, IbeA,
                                                   and IbeB invasins
         Laboratory Indications
●   Lysine +                      E. coli on EMB
●   Citrate -
●   Indol +
●   Acetate +
●   Lactose +
●   Motile
●   Urease -
●   Hydrogen sulfide -
●   Growth on MacConkey's, EMB and blood
●   Acid slant and acid butt in TSI, gas +
             Shigella sonnei
●   Multiple origins
    from within the E.
●   Causes bacillary
    occaisional HUS

                         Shigella sonnei on EMB
              Virulence Factors
●   Cytotoxin          ●   Induced
●   Endotoxin              phagocytosis
●   Enterotoxin        ●   Lysis of phagosome
●   Siderophore        ●   Induced
                           endocytosis by
●   Shigatoxin             epithelial cells
●   Invasin            ●   Intracellular growth
        Laboratory Indications
●   Lysine -
●   Non-motile
●   -/+ TSI reaction (no gas)
●   Acetate -
●   Lactose -
●   Serotype D
●   Growth on MacConkey's and EMB
●   Hydrogen Sulfide -
●   Urease -
      Salmonella typhimurium
●   Biovar of Salmonella enterica
●   Cuases gastroenteritis (Salmonellosis) and
    enteric fever
●   Causes a typhoid-like fever in mice
Locus     Role in Virulence          Serovars

SPI-1     Invasion of epithelials,   All
           macrophage apoptosis
SPI-2     Intracellular prolif.,     All but S. bongori
           systemic disease
SPI-3     Intramacrophage survival All, some have large
SPI-4     Intramacrophage            All
           survival, ???
SPI-5     Enteropathogenesis         Dublin, Typhimurium,
                                      Gallinarum, Pullorum
Plasmid   Intramacrophage            All non-typhoidal
Islets    Multiple, adhesion         All
        Laboratory Indications
●   Lysine +
●   Hydrogen sulfide +
●   Indole +
●   Citrate +
●   ONPG -
●   Malonate -
●   Hydrogen sulfide +            Salmonella typhimurium on HEK
●   -/+ TSI reaction (with gas)
●   Growth on MacConkey's, EMB, HEK
        Klebsiella pneumoniae
●   Most clinically important species of the
●   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
        Laboratory Indications
●   Growth on MacConkey's with pink colonies
●   Growth on EMB
●   Lysine +
●   Citrate +
●   Indol -
●   +/+ TSI (with gas)
●   Non-motile
●   Ornithine -
●   Hydrogen sulfide -
            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
               Virulence Factors
●   Adhesin
●   Endotoxin
●   Urease
●   Motility

                         Proteus vulgaris
        Laboratory Indications
●   Lysine -
●   Hydrogen sulfide +
●   Swarming motility
●   Urease +
●   Indole +
●   Growth on MacConkey and EMB
●   Gas in TSI
               Biochemical Tests
●   Carbohydrate Fermentation
●   Urease
●   Motility
●   Triple Sugar Iron Agar
●   Catalase
●   Oxidase
●   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.
    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
●   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
●   Detect motility in bacteria
●   Tetrazolium indicator shows where bacteria
    have grown
●   Stab the media- growth outside of stab line
    indicates motility
    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.
                                                  T S IA S la n t T u b e s
                                             In te r p r e ta tio n o f R e s u lts
                               (L e b o ffe &     P ie r c e P h o to g r a p h ic A tla s – p . 8 0 )

R e s u lts (S la n t / B u tt)                                     S ym bol                           In te r p r e ta tio n
R e d / Y e llo w                                                   K    / A                          G lu c o s e fe r m e n ta tio n o n ly ,
                                                                                                      P e p to n e c a ta b o liz e d

Y e llo w / Y e llo w                                               A    / A                          G lu c o s e + la c to s e a n d /o r
s u c ro s e
                                                                                                      fe r m e n ta tio n

R ed / R ed                                                         K    / K                          N o fe r m e n ta tio n ,
                                                                                                      P e p to n e c a ta b o liz e d

R e d / N o c o lo r c h a n g e                                 K       / N C                        N o fe r m e n ta tio n ,
                                                                                                      P e p to n e c a ta b o liz e d

Y e llo w / Y e llo w , w ith b u b b le s                          A    / A , G                      G lu c o s e + la c to s e a n d /o r
s u c ro s e
                                                                                                      fe r m e n ta tio n , G a s p r o d u c e d

R e d / Y e llo w , w ith b u b b le s                              K    / A , G                      G lu c o s e fe r m e n ta tio n o n ly ,
                                                                                                      G a s p ro d u c e d

R e d / Y e llo w , w ith b u b b le s                          K    / A , G , H              2   S   G lu c o s e fe r m e n ta tio n o n ly ,
 a n d b la c k p r e c ip ita te                                                                     G a s p ro d u c e d , H 2S p ro d u c e d

R e d / Y e llo w , w ith b la c k p r e c ip ita te            K       / A , H   2   S               G lu c o s e fe r m e n ta tio n o n ly ,
                                                                                                      H 2S p ro d u c e d

Y e llo w / Y e llo w , w ith b la c k p r e c ip ita te         A      / A , H       2   S           G lu c o s e + la c to s e a n d /o r
s u c ro s e
                                                                                                      fe r m e n ta tio n , H       2   S   p ro d u c e d

N o change / N o change                                          N C       / N C                      O r g a n is m   is g r o w in g v e r y
s lo w ly
                                                                                                      o r n o t a t a ll

A = A c id p r o d u c tio n ,      G    = G a s p r o d u c tio n ,        K     = A lk a lin e r e a c tio n ,       H   2   S   = S u lfu r
r e d u c tio n
●   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 bubbles=negative
●   Presence of cytochrome c in the electron
    transport chain
●   Phenylenediamine solution on filter paper-
    don’t touch!
●   Positive= blue/ negative=grey
    Eosin Methylene Blue Agar
●   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.
    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.
●   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
●   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.
Salmonella typhimurium   Escherichia coli
       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 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
●   Non-lactose-fermenting strains, such as Shigella
    and Salmonella, are colorless and transparent.
    They typically do not alter the appearance of the
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+
●   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).
Salmonella sonnei                      Klebsiella pneumoniae

                    Escherichia coli

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