Enteric Bacteria

EnterobacteriaceaeGram negative bacilli

ferment glucoseoxidase (-)

Normal Flora vs. Pathogens

Escherichia Enterobacter Klebsiella Serratia Citrobacter

Proteus (usually nonpathogenic)

Shigella Salmonella

COLIFORMS NON-COLIFORMS

Whos a friendly enteric? (I.e., whos normal flora?)

Escherichia Enterobacter Klebsiella Serratia Citrobacter

Proteus (usually nonpathogenic)

COLIFORMS NON-COLIFORMS

Obligate Enteric Pathogens

http://www.mpg.de/news02/news0209.htm

Shigella dysenteriae

http://www.co.boulder.co.us/health/hpe/FactSheets/salmonella.htm

Salmonella typhimurium

Escherichia coli O157:H7

http://www.jfha.or.jp/saikin/shashin/o157.html

O157:H7

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Enterobacteriaceae: Coliform or non-coliform?

Lactose Fermentation?

normal flora pathogen

yes no

Eosin Methylene Blue (EMB) Agar

http://www.cat.cc.md.us/courses/bio141/labmanua/lab3/embeacolony.htmlE. aerogenes

S. TyphimuriumP. aeruginosa

E. coli

EMB: Table of resultsTABLE

4.5 EMB RESULTS AND INTERPRETATIONS

RESULT INTERPRETATION RECORD PRESUMPTIVE ID

Poor/nogrowth

Organism inhibited byE & MB NG Gram positive

Good growth Organism not inhibitedby E & MB + growth Gram negative

Growth is pink,mucoid

Organism fermentslactose with little acidproduction

pink Possible coliform

Growth ispurple toblack, +/-metallic sheen

Organism fermentslactose +/or sucrosewith much acidproduction

green/metallic/black

Probable coliform

Growth isnormal colot

Organism does notferment lactose orsucrose

NR Noncoliform

Microbes on MacConkeylactose

Enterobacter

http://www.austin.cc.tx.us/microbugz/24mac.html

E. coli

S. aureus

Salmonella

MacConkey: Table of resultsTABLE

4.7 MACCONKEY AGAR RESULTS AND INTERPRETATIONS

RESULT INTERPRETATION RECORD PRESUMPTIVE ID

Poor/nogrowth

Organism inhibited byCrystal violet &/or bile NG Gram positive

Good growth Organism not inhibitedby crystal violet or bile + growth Gram negative

Growth is pinkor red

Organism produces acidfrom lactosefermentation

pink/red Lactose fermenter

Growth isnormal color

Organism does notferment lactose NR Lactose nonfermenter

Black, J.G (2002) Microbiology: Principles and Explorations 5th Ed. Fig. 5.21

O2

(Aerobic respiration)

Electrons from carbos(or other food source)

Electron carriers

(Anaerobic respiration)

(Fermentation)

Pyruvic acidNO3-, SO42-,

CO2

Fermentation Pathways

Bla

ck, J

.G (2

002)

Mic

robi

olog

y: P

rinci

ples

and

Exp

lora

tions

5th

Ed.

Fig

. 5.1

1

Lactic acid

Glucose(or other sugar)

Pyruvic acid

Butyric acid, butanol,isopropyl alcohol,

acetone & CO2

Ethyl alcohol& CO2

Butanediol& CO2

Acetic aid,succinic acid,ethyl alcohol,

CO2 & H2

Propionic acid,acetic acid,

& CO2

Homolactic acidfermentation

Alcoholic fermentation

Butyric-butylicfermentation

Butanediolfermentation

Mixed-acidfermentation

Propionicfermentation

OF Glucose Deeps

Alkaline-neg. for O & F

+ O, -F +F,

O unknown

http://www.jlindquist.net/generalmicro/dfnewglucoseofpage.html

Starch Hydrolysis

Test for mixed fermentation & acid products

Tube 1: add 5 drops Methyl red

stays red = positive (pH6)

Test for Acetoin (neutral)

Tube 2: add 12 drops rgnt #1

Add 3 drops rgnt #2

Cover, shake or manually vortex

Wait 15-60 min.

Red= positive

No change or brown= negative

Methyl Red-Voges Proskauer Tests

http://biology.uwsp.edu/faculty/TBarta/biochem.html

++acid from glucose ferm.=+MR test (red)

Neutral products=-MR test (yellow)

Acetoin (neutral)= +VP test (red)

No Acetoin=-VP (brown)

Phenol Red Broth (a.k.a. Durham tubes

sugar fermented product acidic

peptone deaminated product alkaline

phenol redpH indicator

control

TABLE 5.2 PR BROTH RESULTS AND INTERPRETATIONS

RESULT INTERPRETATION RECORD

Yellow broth, bubble in tube Fermentation with acid & gas end products A/G

Yellow broth, no bubble Fermentation with acid, but no gas end product A/

Red broth, no bubble No fermentation /

Pink broth, no bubble Degradation of peptone; alkaline end products K

A/G A/ / K

Simmons Citrate

control

sodium citrate sole source

Citrate Test: Table of results

TABLE5.8 CITRATE TEST RESULTS AND INTERPRETATIONS

RESULT INTERPRETATION RECORD

Blue (ANY) Citrate is utilized +

No color change; growth Citrate is utilized +

No color change, no growth Citrate is not utilized

If negative today- put back and read again on Wednesday

If positive, record and discard tube

MIO-Motility, Indole, Ornithine

H2O +

tryptophanN

(indole)tryptophanase

indole+

Kovacs reagent

red color

MIO-Motility, Indole, Ornithine

H2O +

ORNITHINEPutriscine

Ornithine decarboxylase

Decarboxylationleaves an alkaline amine and makes the media purple

Escherichiacoli

Enterobacter

aerogenesKlebsiellaoxytoca

typical examples

+++glucose fermentation (acid rx.)

++indole production (red color with Kovacs reagent)

++decarboxylation of ornithine (anaerobicalkaline rx.)

++motility (cloudiness disregarding stab line)

+++deamination of amino acids (aerobic alkaline rx.)

321corresponding tube no. above (tubes arranged in pairs with Kovacs reagent added to right tube in each pair)

Urea Digestion

http://www.austin.cc.tx.us/microbugz/35urease.html

H2N NH2C

O(urea)

2 NH3 CO2(ammonia)

+urease

Urease Test: Table of resultsTABLE5.15 UREASE TEST RESULTS AND INTERPRETATIONSRESULT

24 hours 24 hours to 6days

INTERPRETATION RECORD

All pink All pink Rapid urea hydrolysis; strongurease production +

Partially pink Partially pink Slow urea hydrolysis; weakurease production weak +

Orange oryellow Partially pink

Slow urea hydrolysis; weakurease production weak +

Orange oryellow

Orange oryellow

No urea hydrolysis; ureaseabsent

SIM: Sulfur Reaction

(cysteine)

H2S + H2N CH COOHCH3

H2N CH COOH

CH2 SH

alanine(hydrogen sulfide)

cysteine desulfurase

Fe++

FeS(iron sulfide)

(black ppt)http://www.cat.cc.md.us/courses/bio141/labmanua/lab8/simh2s.html

SIM: Indole ReactionH2O +

NH3

H2N

CH COOHCH2N (tryptophan) N

(indole)

CH3C = OCOOH

(ammonia)

(pyruvicacid)

+

+

tryptophanase

indole+

Kovacs reagent

red color

SIM: Motility

+-

http://www.cat.cc.md.us/courses/bio141/labmanua/lab7/motil.html

SIM Test: Table of resultsTABLE

5.20 SIM-MEDIUM RESULTS AND INTERPRETATIONS

MOTILITYRESULT INTERPRETATION RECORD

Growth radiating from stab line Motility +

No radiating growth Nonmotile

SULFUR REDUCTIONRESULT INTERPRETATION RECORD

Black in medium Sulfur reduction (H2S production) +

No black in medium Sulfur not reduced

INDOLE PRODUCTIONRESULT INTERPRETATION RECORD

Red color when Kovacs added Tryptophan metabolized to indole& pyruvic acid +

Color of Kovacs unchanged Tryptophan not metabolized toindole & pyruvic acid

Why did we inoculate withPseudomonas aeruginosa?

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