Microbio Lab Exam 3

IMViC test

4 separate media that are used to identify enteric bacteria

SIM/Indole test

used to determine if bacteria produce tryptophanase which produces indole

also determines if sulfide is being produced and if bacteria is motile

indole test

test apart of SIM that differentiates Enterobactericae

motility test

test apart of SIM that tests wide variety of organisms

SIM medium

medium that contains nutrients (peptone which contains amino acid - tryptophan), iron, and sodium thiosulfate

evaluates 3 physiological characteristics

produced indole/positive

pink/red section on top of yellow medium

did not produce indole/negative

all yellow medium

Salmonella typhimurium

E. coli

Klebisella pneumoniae

bacteria used in SIM/indole test

SIM/indole test procedure

1. needle is used to stab bacteria to the bottom of the tube

2. tubes are incubated in 37 degrees Celsius incubator

SIM + motility

media is semi-solid and allows bacteria to move

inoculated with needle by stabbing bottom of tube and carefully retract needle along same streak line

positive SIM + motility test

motile, swims away from stab line

negative SIM + motility test

nonmotile, grow in dense line

SIM + hydrogen sulfide production

medium contains an iron salt and thiosulfate

when thiosulfate is reduced, hydrogen sulfide (H2S) gas produced reacts with iron salt to form black iron sulfide precipitate (H2S positive)

reduction of salt did occur/positive

black precipitate forms

reduction of salt did not occur/negative

no black precipitate was formed

SIM + indole production

organisms that produce tryptophanase hydrolyze trytophane and produce indole

Kovac’s reagents are added after incubation and tests for indole presence

Salmonella enterica infection symptoms

diarrhea

abdominal cramping

subjective fever

muscle aches

bloody stools

Salmonella enterica infection transmission

food-borne illness (ex. salsa and guac)

lead to hospitalization and death

major of contamination:

• raw vegetables

• improper/poor storage

• contamination by food workers

Salmonella enterica infection diagnosis

patient stool samples were collected

• gram-negative enteric organism, was identified

• IMViC and differential media were inoculated

• Salmonella enterica serotype Typhimirium —> causing agent

Salmonella enterica serotype Typhimirium

causing agent of Salmonella enterica infections

MR-VP test

identifies gram-negative bacteria, members of Enterobacteriaceae specifically (E. coli and Enterobacter aerogenes)

differentiates between 2 pathways:

mixed acid fermentation

acetoin and 2,3butanediol production

use MR-VP medium which contain glucose (carbon source)

all enteric bacteria

metabolize glucose to form pyruvate

methyl red (MR) test

determines if bacterium is capable of mixed acid fermentation

done by adding a pH indicator —> methyl red

mixed acid fermentation

converts pyruvate to stable acid form (ex. lactic)

Proteus vulgaris and E. coli

bacteria that performs mixed acid fermentation

positive methyl red test

medium is red

large amounts of acid

pH below 4.4

uses lactic acid fermentation

negative methyl red test

medium turns yellow

neutral products produced

pH above 6.2

Voges-Proskauer (VP) test

identifies organisms that ferment sugars to produce alcohol end products acetoin and butaneidol

done by adding Barritt’s reagents (alpha naphthol + 40% KOH) to determine if these products were produced by the bacteria

positive VP test

red colored medium

butylene glycol pathway was used

negative VP test

yellow/brown/copper colored medium

E. coli

Enterobacter aerogenes

bacteria used for MR-VP test

MR-VP test procedure

1. uses loop to transfer the inoculum to MRVP tubes and place tubes in 37 degree Celsius incubator

2. add Kovac’s reagent in MR test tubes + add Barritt’s reagent in VP test tubes

inconclusive MR test

orange colored medium

butylene glycol pathway

converts pyruvate to neutral end products like acetoin and butanediol

citrate

utilized by some bacteria as a source of energy but only if they can transport it into their cell

used in citrate test

nitrogen

from source like ammonia (NH4+)

requirement for growing bacteria in citrate test

citrate agar

contains citrate (carbon source), ammonium phosphate (nitrogen source), and bromothymol blue (pH indicator)

tests organism’s ability to use these components

citrate test

utilization of citrate and nitrogen release alkaline by-products which raise the pH and result in a color change of the media from green to blue

positive citrate test

medium changes from green to blue

did release alkaline

pH is equal to or greater than 7.5

negative citrate result

medium stays green

did not release alkaline

neutral pH

E. coli

Klebsiella pneumoniae

Enterobacter aerogenes

bacteria used in citrate test

Enterobacter aerogenes

Klebsiella pneumoniae

bacteria that tests positive in citrate test

E. coli

bacteria that tests negative in citrate test

fermentation products

Eschericha

Acetobacter

Lactobacillus

Streptococcus

Clostridium

Saccharomyces

acetic acid and vinegar

what Eschericha and Acetobacter produce

lactic acid and cheese

what Lactobacillus and Streptococcus produce

isopropanol and rubbing alcohol

what Clostridium produces

ethanol, wine, and beer

what Saccharomyces produces

ammonium phosphate

bacteria growing on citrate agar use ______ and convert it to ammonia —> creates alkaline environment

triple sugar iron agar (TSA)

differential medium used to identify gram negative enteric bacteria

triple sugar iron agar (TSA)

tests for fermentation of certain carbohydrates and identifies H2S producing bacteria

triple sugar iron agar (TSA)

bacteria that use protein for energy source are detected using this medium

lactose, glucose (low amount), and sucrose

three sugars contains in TSA

phenol red

pH indicator in TSA

iron salt and nutrient base-peptone

2 sulfur sources in TSA

iron salt

detects H2S gas (results from using sulfur)

black in color

nutrient base-peptone

detects protein catabolism, alkaline pH

bright red

glucose

most organisms can utilize ______

once available _____ is depleted, organisms must find another nutrient source (lactose, sucrose, or protein)

alkaline was produced

shows red colored TSA

acid was produced

shows yellow colored TSA

gas was produced

bubbles were visible in TSA

hydrogen sulfide (H2S) was produced

black precipitate was shown in TSA

alkaline/acid TSA

glucose fermentation ± peptone catabolism

acid/acid TSA

glucose and lactose/sucrose fermentation

alkaline/alkaline TSA

protein catabolism

TSA procedure

medium is inoculated with a stab into the butt and streak down the slant using a needle to stab into the butt and streak the slant

Salmonella typhimurium

Shigella dysenteriae

Alcaligenes faecalis

E. coli

bacteria used in TSA