Professional Documents
Culture Documents
Basic Properties
Dr. John R. Warren
Department of Pathology
Northwestern University
Feinberg School of Medicine
June 2007
Characteristics of the
Enterobacteriaceae
• Gram-negative rods
• Glucose is fermented with strong acid
formation and often gas
• Cytochrome oxidase activity is
negative
• Nitrate is reduced to nitrite
Gram’s Stain for Bacterial
Morphology
• Crystal violet binds to cell wall
peptidoglycan with Gram’s iodine as a
mordant
• Safranin or basic fuchsin counterstains
bacterial cells decolorized by alcohol-
acetone
Gram’s Stain for Bacterial
Morphology
• Thick cell-wall peptidoglycan layer of gram-
positive bacteria strongly binds crystal violet
and resists decolorization by alcohol-acetone
• Thin cell-wall peptidoglycan layer of gram-
negative bacteria located beneath a thick
lipid-rich outer membrane weakly binds
crystal violet that is readily removed by
alcohol-acetone decolorization
Gram’s Stain Procedure
• Flood surface of smear with crystal violet solution
• After 1 min thoroughly rinse with cold tap water
• Flood smear with Gram’s iodine for 1 min
• Rinse smear with acetone-alcohol decolorizer until
no more crystal violet in rinse effluent
• Rinse with cold tap water
• Flood smear with safranin (regular Gram’s stain) or
basic fuchsin (enhanced Gram’s stain)
• Rinse with cold tap water
• Dry smear in slide rack
• Microscopically examine stained smear using oil-
immersion light microscopy
Glucose Fermentation
• Oxidation-reduction of glucose in the absence of molecular oxygen
(anaerobic glycolysis)
• Energy from hydrolysis of chemical bonds in anaerobic glycolysis
captured as high energy phosphate bonds of adenosine triphosphate
(ATP)
• NAD is reduced to NADH2 by accepting electrons during glycolytic
conversion of glucose to pyruvate
• NADH2 in turn reduces pyruvate with oxidation of NADH2 to NAD which
supports continued anaerobic glycolysis, and generation from
pyruvate of alcohols, carboxylic acids, and CO2 gas
• End products of glucose fermentation: organic acids and CO2 gas
• Fermentation detected by acidification of glucose-containing broth
(color change in broth or agar medium containing pH indicators), and
(for aerogenic species) production of gas (fractures in agar, gas
bubbles in inverted Durham tube)
• pH indicators: phenol red (yellow at acid pH), methyl red (red at acid
pH), neutral red (red at acid pH), bromcresol purple (yellow at acid pH)
Spot Cytochrome Oxidase Test
• The spot cytochrome oxidase test is
the first test performed with gram-
negative bacteria recovered in culture
• The optimal plate medium for a spot
cytochrome oxidase test is a trypticase
soy agar (TSA) containing 5% sheep
blood
• Bacterial colonies should be 18 to 24 hr
old
Spot Cytochrome Oxidase Test
• In a positive test, bacterial cytochrome
oxidase oxidizes the colorless reduced
substrate tetramethyl-p-phenylenediamine
dihydrochloride (TPDD) forming a dark
purple oxidized indophenol product
• Streak a small portion of bacterial colony to
filter paper soaked with a 1% solution of
TPDD
• If the streak mark turns purple in 10 sec or
less, the spot oxidase test is interpreted as
positive
Nitrate Reduction
• Enterobacteriaceae extract oxygen from
nitrate (NO3) producing nitrite (NO2)
• NO2 detected by reaction with α-
naphthylamine and sulfanilic acid producing
a red colored complex
• Absence of red color indicates either no
reduction of NO3 or reduction to products
other than NO2 (denitrification)
• Confirmation of true negative test: addition of
zinc ions which reduce NO3 to NO2 producing
a red color in the presence of α-
naphthylamine and sulfanilic acid
Enterobacteriaceae: Genetic
Properties
• Chromosomal DNA has 39-59%
guanine-plus-cytosine (G+C) content
• Escherichia coli is the type genus and
species of the Enterobacteriaceae
• Species of Enterobacteriaceae more
closely related by evolutionary distance
to Escherichia coli than to organisms
of other families (Pseudomonadaceae,
Aeromonadaceae)
Enterobacteriaceae: Major
Genera
• Escherichia
• Shigella
• Salmonella
• Edwardsiella
• Citrobacter
• Yersinia
• Klebsiella
• Enterobacter
• Serratia
• Proteus
• Morganella
• Providencia
Enterobacteriaceae:
Microbiological Properties
• Gram-negative and rod shaped (bacilli)
• Ferment rather than oxidize D-glucose
with acid and (often) gas production
• Reduce nitrate to nitrite
• Grow readily on 5% sheep blood or
chocolate agar in carbon dioxide or
ambient air
• Grow anaerobically (facultative
anaerobes)
Enterobacteriaceae:
Microbiological Properties
• Catalase positive and cytochrome oxidase
negative
• Grow readily on MacConkey (MAC) and eosin
methylene blue (EMB) agars
• Grow readily at 35oC except Yersinia (25o-
30oC)
• Motile by peritrichous flagella except
Shigella and Klebsiella which are non-motile
• Do not form spores
Enterobacteriaceae: Natural
Habitats
• Environmental sites (soil, water, and
plants)
• Intestines of humans and animals
Enterobacteriaceae: Modes of
Infection
• Contaminated food and water (Salmonella
spp., Shigella spp., Yersinia enterocolitica,
Escherichia coli O157:H7)
• Endogenous (urinary tract infection, primary
bacterial peritonitis, abdominal abscess)
• Abnormal host colonization (nosocomial
pneumonia)
• Transfer between debilitated patients
• Insect (flea) vector (unique for Yersinia
pestis)
Enterobacteriaceae: Types of
Infectious Disease
• Intestinal (diarrheal) infection
• Extraintestinal infection
Urinary tract (primarily cystitis)
Respiratory (nosocomial pneumonia)
Wound (surgical wound infection)
Bloodstream (gram-negative
bacteremia)
Central nervous system (neonatal
meningitis)
Enterobacteriaceae: Urinary
Tract Infection, Pneumonia
• Urinary tract infection: Escherichia coli,
Klebsiella pneumoniae, Enterobacter
spp., and Proteus mirabilis
• Pneumonia: Enterobacter spp.,
Klebsiella pneumoniae, Escherichia
coli, and Proteus mirabilis
Enterobacteriaceae: Wound
Infection, Bacteremia
• Wound Infection: Escherichia coli,
Enterobacter spp., Klebsiella
pneumoniae, and Proteus mirabilis
• Bacteremia: Escherichia coli,
Enterobacter spp., Klebsiella
pneumoniae, and Proteus mirabilis
Enterobacteriaceae: Nosocomial
Infections in the United States
1986-1989 and 1990-19961
• Escherichia coli 27,871 (13.7%)
• Enterobacter spp. 12,757 (6.2%)
• Klebsiella pneumoniae 11,015 (5.4%)
• Proteus mirabilis 4,662 (2.3%)
• Serratia marcescens 3,010 (1.5%)
• Citrobacter spp. 2,912 (1.4%)
1Enteric Reference Laboratory, Centers for
Inhibit gram positives and fastidious gram-negatives; MAC agar selective for
2
gram-negatives
TSI
Differential Reactions of the
Enterobacteriaceae by EMB
• Escherichia coli Colonies with metallic green
sheen
• Klebsiella Colonies with precipitate (ppt)
and mucoid appearance
• Enterobacter Colonies with ppt
• Citrobacter Colonies with/without ppt
• Serratia Colonies without ppt
• Shigella Colonies without ppt
• Salmonella Colonies without ppt
• Proteus Colonies without ppt
• Yersinia Colonies without ppt
ONPG Reaction and Lactose
Fermentation (Lac)
ONPG Lac
Escherichia coli + +
Shigella sonnei + –
Citrobacter + +/–
Yersinia enterocolitica + –
Klebsiella + +
Serratia marcescens + –
Xylose Lysine Deoxycholate
(XLD) Agar: Composition
• Xylose 0.35%
• Lysine 0.5%
• Lactose 0.75%
• Sucrose 0.75%
• Sodium chloride 0.5%
• Yeast extract 0.3%
• Sodium deoxycholate 0.25%
• Sodium thiosulfate
• Ferric ammonium citrate
• Agar 1.35%
• Phenol red
• pH = 7.4
XLD Agar: Growth of
Salmonella
• Salmonella selective due to bile salt.
• Xylose fermentation (except Salmonella
serotype Paratyphi A) acidifies agar
activating lysine decarboxylase. With xylose
depletion fermentation ceases, and colonies
of Salmonella (except S. Paratyphi A)
alkalinize the agar due to amines from lysine
decarboxylation.
• Xylose fermentation provides H+ for H2S
production (except S. Paratyphi A).
XLD Agar: Appearance of
Salmonella
• Ferric ammonium citrate present in
XLD agar reacts with H2S gas and
forms black precipitates within
colonies of Salmonella.
• Agar becomes red-purple due to
alkaline pH produced by amines.
• Back colonies growing on red-purple
agar-presumptive for Salmonella.
XLD Agar: Growth of Escherichia
coli and Klebsiella pneumoniae