Transcript Lecture 2 – Week 7 Control of Microbial Growth

Lab 10
BACTERIOLOGY OF THE
GASTROINTESTINAL TRACT
Lab Manual (p.67-82)
THE GASTROINTESTIONAL TRACT
THE GASTROINTESTINAL TRACT
HOST DEFENSE MECHANISMS
Anatomical characteristics/mechanisms to prevent/fight
infection:
•High acidity of stomach prevents most microbes from
surviving, making it almost sterile.
•Normal microbiota – a variety of microorganisms that
compete with each other for space and nutrients, aid in
digestion, immunity and preventing infection from
pathogens.
– Enterobacteriaceae (enteric Gram-negative rods),
Staphylococcus spp., Streptococcus spp.,
Enterococcus spp. and various anaerobic bacteria
ENTEROBACTERIACEAE
(ENTERIC Gram-negative RODS)
• Enterobacteriaceae are a family of Gramnegative rods that include both harmless
and pathogenic bacteria:
– Characteristics:
• Gram-negative rods
• Facultative anaerobic
• Ferment glucose
• Oxidase negative
• If motile, peritrichious flagella
ENTEROBACTERIACEAE
(ENTERIC Gram-negative RODS)
• Enterobacteriaceae are commonly found as 1.)
Normal flora or 2.) Pathogenic in the
gastrointestinal tract (termed “enterics”)
• Non-pathogenic in the gastrointestinal tract:
– E.coli (exception: Shiga toxin-producing E. coli)
– Klebsiella spp.
– Proteus spp.
– Citrobacter spp.
• Pathogenic in the gastrointestinal tract:
– Salmonella sp.
– Shigella sp.
INFECTIONS OF THE
GASTROINTESTINAL TRACT
Colitis
Diarrhea or
Gastroenteritis
Cholera
THE GASTROINTESTINAL TRACT
Specimens & Diagnosis
• Fresh stool or stool in preservative (CaryBlair transport)
– Routine Pathogens detected:
• Salmonella, Shigella, Campylobacter,
and Shiga toxin-producing bacteria
– Toxigenic Clostridium difficile (CDIFF)
– Special bacteria requests: Yersinia
enterocolitica, Aeromonas spp., Vibrio
spp.
THE GASTROINTESTINAL TRACT
Specimens & Diagnosis
• Parasites (requires special preservative
[EcoFix]):
– Ova and Parasite screening:
• Giardia spp. and Cryptosporidium spp.
– Ova and Parasites extended testing:
• Other than above, including all
protozoa, eggs and worms in stool.
• Viruses
– Not routinely tested
BIOCHEMICAL
TESTS
BIOCHEMICAL TESTS
• SIM (Sulfide, Indole and
Motility
• MRVP (Methyl Red and Voges
Proskauer)
• Citrate
• TSI (Triple Sugar Iron) Agar
SIM TEST
• This is a multipurpose medium that can evaluate 3
different physiological characteristics in one tube
• The SIM (Sulfur Indole Motility) test is used to
determine if the bacteria:
– (S): to determine the production of sulfide
– (I): to determine the presence of an enzyme
tryptophanase, which is responsible for
producing the end product indole
– (M): to determine if the bacterium is motile
METHYL RED / VOGES PROSKAUER
(MRVP) TEST
• 2 Biochemical tests performed in the same
media (MRVP broth) that contains glucose
MR – Methyl red
VP – Voges-Proskauer
• Both of these tests are performed on MR-VP
Medium (contains glucose)
METHYL RED TEST
• The MR TEST determines if a bacterium is
capable of mixed acid fermentation
• Mixed acid fermentation results when
glucose is fermented to produce acids,
which lowers the pH of the media
• The isolate is inoculated to a MRVP broth
and incubated overnight
• Methyl Red is added to the tube to detect
whether or not large amounts of acid has
been produced
VOGES-PROSKAUER TEST
• The VP TEST determines if a bacterium
ferment sugars to produce the neutral
alcohol end products acetoin and
butanediol
• The isolate is inoculated to a MRVP
broth and incubated overnight
• Barritt’s reagents are added to the tube
to detect whether the neutral products
have been produced
CITRATE TEST
• Citrate agar contains citrate (carbon
source), ammonium phosphate (nitrogen
source), and bromthymol blue (pH indicator)
– Used to determine if a bacterium can transport citrate
into their cell to utilize citrate as a source of energy
– Nitrogen (from a source such as ammonium - NH4+) is
also a requirement for growing bacteria
• Utilization of citrate and nitrogen release
alkaline by-products which raise the pH that
is detected by a color change in the citrate
agar slant
CITRATE TEST
TSI (Triple Sugar Iron agar)
• Triple-Sugar Iron agar is a differential
medium used to identify Gramnegative enteric bacteria based on
their ability to ferment selected
carbohydrates and produce hydrogen
sulfide (a metabolic by-product)
• Many biochemical tests are performed
simultaneously in a single tube
TSI (Triple Sugar Iron agar)
Each TSI slant contains:
• 1 part glucose and another carbohydrate/sugar
• 10 parts glucose
• 10 parts lactose
• 10 parts sucrose)
• Phenol Red (pH indicator)
• Two sources of sulfur (used to detect
the production of sulfur)
• Cysteine and thiosulfate
• An iron salt
• Peptone
TSI (Triple Sugar Iron agar)
The bacterium is inoculated and incubated overnight at 35C
• Glucose utilized within 8 – 12hrs
• Butt remains acidic after 18 –24 hrs
• Slant reverts to alkaline (red) due to oxidation of the
fermentation products under aerobic conditions on the
slant results in:
• CO2 and H20 and the oxidation of peptones in the
medium to alkaline amines
• Slant reverts back to yellow
• This means that the fermentation of lactose and/or
sucrose has occurred, yielding acidic products
• Slant stays red
• No fermentation of lactose and/or sucrose
TSI (Triple Sugar Iron agar)
• Production of gas
• Positive for gas (CO2 or H2) - Cracks or
bubbles in the agar
• Negative for gas – No cracks or bubbles
in the agar
• Black precipitant in the butt
• Desulfurization of sulfur-containing amino
acids
• Indicates the production of ferrous sulfide
and H2S gas
BIOCHEMICAL
TESTS
SET UP
SETUP: SIM TEST
SETUP: MR-VP TEST
SETUP: CITRATE TEST
SETUP: TSI SLANT
Loose cap on all tubes!
BIOCHEMICAL
TESTS
RESULTS
RESULTS: SIM TEST
RESULTS: MR-VP TEST
METHYL RED TEST
– POSITIVE MR TEST: Large amounts of acid =
medium remains RED (pH below 4.4)
– NEGATIVE MR TEST: Neutral products produced
= medium turns YELLOW (pH above 6.2)
VOGES-PROSKAUER TEST
– POSITIVE TEST: Production of RED color
– NEGATIVE TEST: No RED color
RESULTS:CITRATE TEST
IMViC
(Indole, Methyl Red, Voges-Proskaeur, Citrate)
• The IMViC tests to identify enteric
Gram-negative rods to the Genus or
even Species level
RESULTS: TSI SLANT
1. Slant (Lactose and/or sucrose)
Acid = yellow
Alkaline = red
2. Butt (Glucose)
Acid = yellow
Alkaline = red
3. Gas production
Positive = bubbles or cracks in the media
Negative = intact agar with no air bubbles
4. Hydrogen sulfide production
Positive = black to dark charcoal gray insoluble precipitate
Negative=no black color detected