lab 10 bacteriology of the gastrointestinal tract lab manual (p.67-82)
TRANSCRIPT
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Lab 10BACTERIOLOGY OF THE
GASTROINTESTINAL TRACTLab Manual (p.67-82)
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THE GASTROINTESTIONAL TRACT
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THE GASTROINTESTINAL TRACTHOST 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
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ENTEROBACTERIACEAE(ENTERIC Gram-negative RODS)
• Enterobacteriaceae are a family of Gram-negative rods that include both harmless and pathogenic bacteria:–Characteristics:•Gram-negative rods• Facultative anaerobic• Ferment glucose•Oxidase negative• If motile, peritrichious flagella
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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.
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INFECTIONS OF THE GASTROINTESTINAL TRACT
Cholera
Diarrhea orGastroenteritis
Colitis
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THE GASTROINTESTINAL TRACTSpecimens & Diagnosis
• Fresh stool or stool in preservative (Cary-Blair 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.
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THE GASTROINTESTINAL TRACTSpecimens & 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
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BIOCHEMICAL TESTS
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BIOCHEMICAL TESTS
• SIM (Sulfide, Indole and Motility•MRVP (Methyl Red and Voges Proskauer)• Citrate• TSI (Triple Sugar Iron) Agar
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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
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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)
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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
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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
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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
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CITRATE TEST
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TSI (Triple Sugar Iron agar)
• Triple-Sugar Iron agar is a differential medium used to identify Gram-negative 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
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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
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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
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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
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BIOCHEMICAL TESTS
SET UP
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SETUP: SIM TEST
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SETUP: MR-VP TEST
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SETUP: CITRATE TEST
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SETUP: TSI SLANT
Loose cap on all tubes!
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BIOCHEMICAL TESTS
RESULTS
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RESULTS: SIM TEST
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RESULTS: MR-VP TEST
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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)
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VOGES-PROSKAUER TEST
–POSITIVE TEST: Production of RED color
–NEGATIVE TEST: No RED color
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RESULTS:CITRATE TEST
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IMViC(Indole, Methyl Red, Voges-Proskaeur,
Citrate)• The IMViC tests to identify enteric Gram-
negative rods to the Genus or even Species level
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RESULTS: TSI SLANT
1. Slant (Lactose and/or sucrose)Acid = yellow Alkaline = red
2. Butt (Glucose)Acid = yellowAlkaline = red
3. Gas productionPositive = 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
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