.

Gram-negative rodsGlucose is fermented with strong acid

formation and often gasCytochrome oxidase activity is negativeNitrate is reduced to nitrite

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

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

Flood surface of smear with crystal violet solution

After 1 min thoroughly rinse with cold tap waterFlood smear with Gram’s iodine for 1 minRinse smear with acetone-alcohol decolorizer

until no more crystal violet in rinse effluentRinse with cold tap waterFlood smear with safranin (regular Gram’s

stain) or basic fuchsin (enhanced Gram’s stain)Rinse with cold tap waterDry smear in slide rackMicroscopically examine stained smear using

oil-immersion light microscopy

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)

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

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

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

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)

EscherichiaShigellaSalmonella EdwardsiellaCitrobacter YersiniaKlebsiellaEnterobacterSerratiaProteusMorganella Providencia

Gram-negative and rod shaped (bacilli)Ferment rather than oxidize D-glucose

with acid and (often) gas productionReduce nitrate to nitriteGrow readily on 5% sheep blood or

chocolate agar in carbon dioxide or ambient air

Grow anaerobically (facultative anaerobes)

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

Environmental sites (soil, water, and plants)

Intestines of humans and animals

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 patientsInsect (flea) vector (unique for Yersinia

pestis)

Intestinal (diarrheal) infectionExtraintestinal infection

Urinary tract (primarily cystitis)Respiratory (nosocomial

pneumonia)Wound (surgical wound infection)Bloodstream (gram-negative bacteremia)Central nervous system (neonatal

meningitis)

Urinary tract infection: Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus mirabilis

Pneumonia: Enterobacter spp., Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis

Wound Infection: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis

Bacteremia: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis

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 Disease Control and Prevention

Shigella sonnei (serogroup D)Salmonella serotype EnteritidisSalmonella serotype TyphimuriumShigella flexneri (serogroup B)Escherichia coli O157:H7Yersinia enterocolitica

Yeast extract 0.3% (% = grams/100 mL)

Beef extract 0.3%Peptone 1.5%Proteose peptone 0.5%

Total Protein = 2.6%Lactose 1.0%Sucrose1 1.0%Glucose 0.1%

Carbohydrate = 2.1%1Absent in Kligler Iron Agar

Ferrous sulfateSodium thiosulfateSodium chlorideAgar (1.2%)Phenol redpH = 7.4

Yellow deep, purple slant: acid deep due to glucose fermentation , no lactose or sucrose fermentation with alkaline slant due to production of amine’s from protein

Black deep, purple slant: acid deep due to glucose fermentation with H2S production, no lactose or sucrose fermentation

Yellow deep and slant: acid deep and slant due to glucose as well as lactose and/or sucrose fermentation

Black deep and yellow or black slant: acid deep and slant with glucose and lactose and/or sucrose fermentation with H2S production

Fracturing or lifting of agar from base of culture tube: CO2 production

A/A + g = acid/acid plus gas (CO2)A/A = acid/acidA/A + g, H2S = acid/acid plus gas, H2SAlk/A = alkaline/acidAlk/A + g = alkaline/acid plus gasAlk/A + g, H2S = alkaline/acid plus

gas, H2SAlk/A + g, H2S (w) = alkaline/acid plus

gas, H2S (weak)

Escherichia coliKlebsiella pneumoniaeKlebsiella oxytocaEnterobacter aerogenesEnterobacter cloacaeSerratia marcescens1, 2

1Non-lactose, sucrose fermenter255% + g

Serratia marcescens1, 2

Yersinia enterocolitica2

145% of strains2Non-lactose, sucrose fermenter

Citrobacter freundiiProteus vulgaris1

1Non-lactose, sucrose fermenter

ShigellaProvidencia

Salmonella serotype Paratyphi A

Salmonella (most serotypes)Proteus mirabilisEdwardsiella tarda

Salmonella serotype Typhi

Peptone 1.7% Polypeptone 0.3% Lactose1 1.0% Bile salts2 0.15% Crystal violet2

Neutral red3

Sodium chloride 0.5% Agar 1.35% pH=7.1

1Differential medium for lactose fermentation

2Inhibit gram positives and fastidious gram-negatives; MAC agar selective for

gram-negatives

3Red color at pH < 6.8

Peptone 1.0% Lactose1 0.5% Eosin y2

Methylene blue2

Agar pH = 7.2

1Modified formula also contains sucrose (0.5%)

2Inhibit gram-positives and fastidious gram-negatives; selective

for gram-negatives. Eosin y and methylene blue form a precipitate at acid pH; differential for lactose

fermentation

Presence of β-galactoside permease: Transport of β-galactoside (lactose) across the bacterial cell wall

Presence of β-galactosidase: Hydrolysis of β-galactoside bond (lactoseglucose + galactose)

ONPG: Orthonitrophenyl-β-D-galacto-pyranoside

Escherichia coli Red colonies,(A/A, ONPG+) pitted

Klebsiella1 Red colonies,(A/A, ONPG+) mucoid

Enterobacter Red colonies(A/A, ONPG+)

Citrobacter2 Red or colorless(A/A or Alk/A, ONPG+) colonies

Serratia Colorless colonies(A/A, ONPG+)

1K. pneumoniae, indole –, K. oxytoca, indole +2C. freundii, indole – and H2S +, C. koseri, indole + and

H2S –

Shigella Colorless Colonies(Alk/A; ONPG – A, B, and C1; ONPG + D1)

Salmonella Colorless Colonies(Alk/A + H2S; ONPG –)

Proteus Colorless Colonies(Alk/A + H2S2; ONPG –)

Edwardsiella tarda Colorless Colonies(Alk/A + H2S; ONPG–)

Yersinia Colorless Colonies(A/A, ONPG +)

1Shigella A, B, and C, ornithine –; Shigella D, ornithine +2Proteus mirabilis. P. vulgaris sucrose + with A/A + H2S on TSI

Escherichia coli Colonies with metallic green sheen

Klebsiella Colonies with precipitate (ppt)

and mucoid appearance

Enterobacter Colonies with pptCitrobacter Colonies with/without

pptSerratia Colonies without pptShigella Colonies without pptSalmonella Colonies without pptProteus Colonies without pptYersinia Colonies without ppt

ONPG Lac

Escherichia coli + +Shigella sonnei + –Citrobacter +

+/–Yersinia enterocolitica + –Klebsiella + +Serratia marcescens + –

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 thiosulfateFerric ammonium citrateAgar 1.35%Phenol redpH = 7.4

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).

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.

Escherichia coli and Klebsiella pneumoniae are lysine-positive coliforms that are also lactoseand sucrose fermenters. The high lactose and sucrose concentrations result in strong acid production, which quenches amines produced by lysine decarboxylation. Colonies and agar appear bright yellow. Neither Escherichia coli nor Klebsiella pneumoniae produce H2S.

Shigella species do not ferment xylose, lactose, and sucrose, do not decarboxylate lysine, and do not produce H2S. Colonies appear colorless.

Proteus mirabilis ferments xylose, and thereby provides H+ for H2S production. Colonies appear black on an agar unchanged in color (Proteus deaminates rather than decarboxylates amino acids). Proteus vulgaris ferments sucrose, and colonies appear black on a yellow agar.

Peptone 1.2%Yeast extract 0.3%Bile salts 0.9%Lactose 1.2%Sucrose 1.2%Salicin 0.2%Sodium chloride 0.5%Ferric ammonium citrateAcid fuchsinThymol blueAgar 1.4%pH = 7.6

High bile salt concentration inhibits growth of gram-positive and gram-negative intestinal commensals, and thereby selects for pathogenic Salmonella (bile-resistant growth) present in fecal specimens.

Salmonella species as non-lactose and non-sucrose fermenters that produce H2S form colorless colonies with black centers.

Shigella species (non-lactose and non-sucrose fermenters, no H2S production) form colorless colonies.

Lactose and sucrose fermenters (E. coli, K. pneumoniae) form orange to yellow colonies due to acid production.

Beef extract 0.5%Peptone 0.5%Bile salts 0.85%Sodium citrate 0.85%Brilliant green dye TraceLactose 1.0%Sodium thiosulfate 0.85%Ferric citrate 0.1%Neutral redAgar 1.4%pH = 7.4

Bile salts, citrates, and brilliant green dye inhibit gram-positives and most gram-negative coliforms

Lactose the sole carbohydrateSodium thiosulfate a source of sulfur for

H2S productionSalmonella forms transparent colonies with

black centersShigella forms transparent colonies without

blackeningLactose fermentative Enterobacteriaceae

produce pink to red colonies with bile precipitate for strong lactose fermenters

Feces1: MAC or EMB + XLD &/or SS or HE2

Sputum and Urine1: MAC or EMB Wound3:MAC or EMB Peritoneal and pleural fluid4: MAC or EMB Subculture of blood positive for gram-negative’s in broth

culture4: MAC or EMB CSF, pericardial fluid, synovial fluid, bone marrow5: Not

required

1Heavy population of commensal bacteria2Utilized with enrichment broth containing selenite or

mannitol to differentially inhibit enteric commensals3Commensal bacteria (skin) and frequent polymicrobial

etiology 4Possible polymicrobial etiology (normally sterile fluids)5Normally sterile, unimicrobial etiology predominant

HE or SS agar (absence of lactose fermentation1,2, H2S production1)

XLD agar (absence of lactose fermentation1,2, H2S production1, lysine decarboxylation1)

MAC or EMB agar (absence of lactose fermentation1,2)

TSI agar (glucose fermentation1,2, absence of lactose fermentation1,2, H2S production1)

Descending Order of Selectivity for Salmonella and Shigella

Winn, W., Jr., Allen, S., Janda, W., Koneman, E., Procop, G., Schrenckenberger, P., Woods, G.Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Sixth Edition, Lippincott Williams & Wilkins, 2006:Chapter 5. Medical Bacteriology: Taxonomy,

Morphology, Physiology, and Virulence.Chapter 6. The Enterobacteriaceae.

Murray, P., Baron, E., Jorgensen, J., Landry, M., Pfaller, M.Manual of Clinical Microbiology, 9th Edition, ASM Press, 2007: Farmer, J.J., III, Boatwright, K.D., and Janda

J.M. Chapter 42. Enterobacteriaceae: Introduction and Identification