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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
http
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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?
QuickTime and aTIFF (Uncompressed) decompressor
are needed to see this picture.