enterobacteriacea ii biochemical reaction 2بكتريا عملي
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Enterobacteriaceae (2)Biochemical reactions
ByDr. Nabil El Aila
Assistant Professor of Molecular MicrobiologyMedical Technology Department
Al -Aqsa University
Dr. Nabil El AilaDiagnostic Microbiology
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Dr. Nabil El AilaDiagnostic Microbiology
Characters of Enterobacteriaceae• All Enterobacteriaciae
– Gram-negative rods
– Ferment glucose with acid production
– Reduce nitrates into nitrites
– Oxidase negative
• Facultative anaerobic
• Motile exceptShigella and Klebsiella
• Non-capsulated except Klebsiella
• Non-fastidious
• Grow on bile containing media (MacConkey agar) Dr. Nabil El Aila
Diagnostic Microbiology
Enterobacteriaceae
• Some Enterobacteriaceae are true pathogens– Salmonella spp.– Shigella spp.– Yersinia spp.– Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)
• Most members of the Enterobacteriaceae are opportunisticor cause secondary infections of wounds, the urinary and respiratory tracts, and the circulatory system e.g. E. coli.
• Enterobacteriaceaedivided into TWO main groups according to action on LACTOSE– Lactose Fermenters (LF)
• E. coli, Citrobacter, Klbesiella, Enterobacter
– Lactose Non-Fermenters (LNF)• Salmonella, Shigella, Proteus, Yersinia Dr. Nabil El Aila
Diagnostic Microbiology
Identification of Enterobacteriaceae
• Gram stain– All Enterobacteriaceae are Gram-negative rods
– Arranged in single
Dr. Nabil El AilaDiagnostic Microbiology
Identification of Enterobacteriaceae
Biochemical reactions
• Oxidase test
– All members of Enterobacteriaceae are oxidase negative
– Pseudomonas is oxidase positive
• O/F test
– All members of Enterobacteriaceae are O+/F+
– Pseudomonas is O+/F-
• Nitrate reductase
– All members of Enterobacteriaceae are nitrate reductase positive
– Pseudomonas is nitrate reductase negative
Classification of Enterobacteriaceae
Enterobacteriaceae
Lactose fermentersE. coli, Citrobacter,
Klebsiella, Enterobacter
Non-lactose fermenterSalmonell, ShigellaProteus, Yersinia
�There are several selective and differential media used toisolate distinguishes between LF & LNF
�The most important media are:�MacConkey agar�Eosin Methylene Blue (EMB) agar�Salmonella Shigella (SS) agar�In addition to Triple Sugar Iron (TSI) agar
Differentiation between LF & NLF by Growth on MacConkey agar
MacConkey AgarContains
Bile salts Crystal violet Lactose Neutral red
�MacConkey agar is selective & differential medium for Enterobacteriaceae
Inhibit growth of G+ve bacteria
Cause of selectivity
Cause of differentialpH indicator
Acidic: Pink
Lactose feremnters
Pink colonies
Lactose non feremnters
colorless colonies
Classification of Enterobacteriaceae according to lactose
fermentation (growth on MacConkey Agar)
Enterobacteriaceae
Lactose Fermenters Lactose Non-Fermenters
Escherichia coliKlebsiella sppEnterobacter sppCitrobacter spp
Salmonella sppSchigella sppProteus sppYersinina spp
Pink colonies Colorless colonies
AcidNeutral red
No acid
Dr. Nabil El AilaDiagnostic Microbiology
Identification of Enterobacteriaceae
Differentiation between LF & NLF by Growth on MacConkey agar
• Method:
– MacConkey agar is inoculated with tested organism using
streak plate technique
– Incubate the plate in incubator at 37 C/24 hrs
• Results:
– LF organism appears as pink colonies (e.g. E. coli)
– NLF organism appears as colorless colonies (e.g. Shigella)
Flame & Cool
Flame & Cool
Flame & Cool
1 23
45
Growth of Enterobacteriaceae on
MacConkey agar
Uninoculated plate Lactose non feremtersSalmonella, Shigella,
Proteus
Lactose feremtersE. coli, Citrobacter
Klebsiella, Enterobacter
Colorless colonies Pink colonies
Dr. Nabil El AilaDiagnostic Microbiology
Reaction on Salmonella Shigella (SS) agar• SS agar is a selective & differential medium used for isolation of
Salmonella and Shigella• The selective agents are bile salts, and brilliant green dye, which inhibit
gram-positive organisms• The medium contains only lactoseas a differential agent and thus
differentiates on the basis of lactose fermentation• The formation of acid on fermentation of lactose causes the neutral red
indicator to make pink colonies• Non lactose fermenting organisms are colorless on the medium• SS agar contains sodium thiosulfateand ferric ammonium citrate
allows the differentiation of organisms that produce H2S– Lactose fermenters, such as E. coli, have colonies which are pink– Shigella appears transparent or amber– Salmonella appears transparent or amber with black centers due to
H2S production
LactoseLactose fermenter
AcidNeutral red
Pink colonies
Ferrous sulfideBlack precipitate
H2S + Ferric ammonium citrate
Identification of EnterobacteriaceaeDifferentiation between LF & NLF by Growth on SS agar
• Method:
– SS agar is inoculated with tested organism using
streak plate technique
– Incubate the plate in incubator at 37 C/24 hrs
Flame & Cool
Flame & Cool
Flame & Cool
1 2
3
4
5
Dr. Nabil El AilaDiagnostic Microbiology
A Klebsiella pneumoniaeB Escherichia coliC Salmonella spD Proteus mirabilisE Ps. aeruginosa
Both are lactose fermenters
Both Salmonella sp. & Proteus product H2S
Pseudomonas colonies are nearly colorless
Growth of Enterobacteriaceae on SS agar
Reaction on Triple Sugar Iron (TSI) Agar• TSI contains
– Enzymatic Digest of Casein, Enzymatic Digest of Animal Tissue,
and Yeast Enriched Peptone provide the nitrogen, carbon, and
vitamins required for organismgrowth.
– Three different types of sugars
• Glucose (1 part)
• Lactose (10 part)
• Sucrose (10 part)
– Phenol red (acidic: Yellow)
• TSI dispensed in tubes with equal butt & slant
Reaction on Triple Sugar Iron (TSI) Agar
• Principle
– To determine the ability of an organism to attack a specific
carbohydrateincorporated into a basal growth medium, with
or without the production of gas, along with the determination
of possible hydrogen sulphideproduction.
• When the carbohydrates are fermented, acid production is detectedby the Phenol Red pH indicator.
• SodiumThiosulfateis reduced to hydrogen sulfide, and hydrogensulfide reacts with an iron salt yielding the typical black iron sulfide.
• Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator. SodiumChloride maintainsthe osmotic balanceof the medium. Agar is the solidifying agent
Reaction on TSI
• Method:
– Inoculate TSI medium with an organism by
inoculating needle by stabbing the butt and
streaking the slant
– Incubate at 37°C for 24 hours
Dr. Nabil El AilaDiagnostic Microbiology
Example
Result
Reaction on TSI
H2SSlant
color
Butt
color
Non fermenter
e.g.
Pseudomonas
Alk/Alk/-
(No action on sugars)NegativeRedRed
LNF
e.g. ShigellaA/Alk/-
(Glucose fermented without H2S)
Negative
RedYellow
LNF
e.g. Salmonella &
Proteus
A/Alk/+ (Glucose fermented
with H2S)
Positive
black in
buttRedYellow
LF
e.g. E. coli,
Klebsiella,
Enterobacter
A/A/-
(three sugars are
fermented)
NegativeYellowYellow
Result
EMBSSMacConkey
O/FNitrate reductase
OxidaseGram stain
Metallic sheen
LFLFO+/F++ve-ve-ve rodE. coli
DarkLFLFO+/F++ve-ve-ve rodsCitrobacter
DarkLFLFO+/F++ve-ve-ve rodsKlebsiella
DarkLFLFO+/F++ve-ve-ve rodsEnterobacter
ColorlessNLF/H2S
NLFO+/F++ve-ve-ve rodsSalmonella
ColorlessNLFNLFO+/F++ve-ve-ve rodsShigella
ColorlessNLF/H2S
NLFO+/F++ve-ve-ve rodsProteus
Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae
MotilityUreaseCitrateVPMRIndoleTSI
Motile-ve-ve-ve+ve+veA/A/-E. coli
Motile-ve+ve-ve+ve+veA/A/-Citrobacter freundii
Non motile
+ve+ve+ve-ve-veA/A/-Klebsiella pneumoniae
Motile+ve+ve+ve-ve-veA/A/-Enterobacter cloacae
Motile-ve+ve-ve+ve-veA/Alk/+Salmonella typhi
Non motile
-ve-ve-ve+ve-veA/Alk/-Shigella boydii
MotileSwarwing
+ve+ve-ve+ve-veA/Alk/+Proteus mirabilis
Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae
Lysine Iron Agar (LIA)• Lysine iron agar (LIA) slants test organisms for the ability to
deaminate lysine or decarboxylate lysine.Lysine deaminationis an aerobicprocess which occurs on the slant of the media.Lysine decarboxylation is an anaerobicprocess which occurs in the buttof the media.
• LIA slants contain lysine, glucose, peptones, bromcresol purple (pH indicator), sodium thiosulfate and ferric ammonium citrate. If the organism has the ability to decarboxylate lysine, it produces an amine end-product which reacts with the pH indicator to give a purple color in the butt of the tube.
(Negative decarboxylation: yellow butt).
• If the organism has the ability to deaminate lysine, the ammonia produced will react with the ferric ammonium citrate to produce a dark red color on the slant of the tube. (Negative deamination: purple slant). Organisms which produce hydrogen sulfide gas will exhibit a black precipitate in the butt of the tube.
Lysine Iron Agar (LIA)• This agar is used as a diagnostic test for salmonellae.
• Salmonellae are the only group of Enterobacteriaceae that regularly
decarboxylate lysin(by lysine decarboxylase) and produce large
amounts of hydrogen sulphide.
• Bacteria that decarboxylate lysine cause an alkaline reaction (purple
colour) throughout the medium.
• Those that do not, produce an alkaline slant and an acid butt
(yellow) due to fermentation of glucose.
• Some bacteria like proteus speciesmay deaminate the lysine and
produce a red slant and acid butt.
• Production of hydrogen sulphidecauses a blackening in the
medium due to formation of ferrous sulphide. The indicator is
bromcresol purple.
Tube 1: Positive decarboxylation (butt), negative deamination (slant)Tube 2: Negative decarboxylation (butt), positive deamination (slant)
Dr. Nabil El AilaDiagnostic Microbiology
Uninoculated medium Alkaline/alkaline/H2S:
Salmonella.
3. Alkaline/yellow/H2S:
Citrobacter.
Lysine Iron Agar (LIA)
COLLECTION, TRANSPORT, AND EXAMINATIONOF FAECES ( STOOL SPECIMENS )
Possible pathogens
Gram Positive Gram Negative
• Clostridium perfringens Shigella species
type A and C Salmonella species
• Clostridium difficile Campylobacter species
Bacillus cereus ( toxin ) Escherichia coli
• Staphylococcus aureus(toxin) ( ETEC,EIEC,EPEC)
Vibrio cholerae 01
Other Vibrio species
Yersinia enterocolitica
• Viruses : mainly rotaviruses, adenoviruses, coxsackieviruses, echoviruses, and
polioviruses.
• Fungi : Candida albicans .Dr. Nabil El Aila
Diagnostic Microbiology
Parasites :Eggs : Amoebae :
Ascaris lumbricoides Entamoeba histolytica
Hookworm Flagellates:Trichuris trichiura Giardia lamblia
Schistosoma species Trichomonas hominis
Hymenolepis species Ciliates :Diphylobothrium latum Taenia species
Entrobius vermicularis Balantidium coli,
Larvae:Strongyloides stercoralisCysts :Entamoeba histolytica
Giardia lamblia
Balantidium coli
Isospora belli ( oocysts )
CommensalsThe normal microbial flora of the gastrointestinal tract is greatly influenced by diet. Microorganisms, which may form part of this normal flora, include:Gram Positive Gram NegativeEnterococci Escherichia coli*Anaerobic streptococci Proteus*Lactobacilli Enterobacter*Clostridia Hafnia *
Citrobacter*Providencia*
Morganella*Serratia*Klebsiella *Bacteroides speciesPseudomonas aeruginosa
• These genera belong to the family Enterobacteriaceae. Enterobacteria are often described as coliforms.
• Fungi: Candida species and yeasts.• Other: Mycoplasma and a variety of protozoa.
Collection of stool sample
• Stool specimens are usually collected in a clean, dry disinfectant free
bedpan or suitable wide necked container.
• The container need not be sterile, ask the patient to avoid
contaminating the stool with urine.
• Transfer a portion of the specimen especially which contains mucus,
pus or bloodinto a clean dry leak-proof container.
• A diarrhealstool usually gives good results.
• Label the specimen, on the container not lid, and send it with a request
form to reach the laboratory within 1 hour.
• Stool passed into the toilet bowel must not be used for culture.
• No toilet paper should be placed in the bed pan or specimen container,
which may contain bismuththat interferes with the laboratory tests.
Collection of stool sample
• If it is not possible to obtain stool specimen, a rectal swab may be used
to obtain the sample by inserting a cotton swab into the anus beyond
the anal sphincter for about 10 seconds, carefully rotate the swab and
withdraw.
• If delayover 18-24 hours is suspected, the specimen should be mixed
with an equal volume of buffered glycerol saline. Also you can insert
the swab in a container of sterile cary - Blair transport medium.
• Salmonella, Shigella , Vibrio and Yersenia species survive well in cary-
Blair medium for up to 48 hours but Campylobacter for up to 6 hours.
• If cholera is suspected transfer about 1ml of specimen into 10ml of
sterile alkaline peptone water , label and send to the microbiology
department within 8 hours of collection.Dr. Nabil El Aila
Diagnostic Microbiology
Suspected organisms
1- E-coli (infant &NLF)2- salmonella & shigella3- campylobactorjejuni most common human pathogen
indol&citrate (+)urease (- )oxidase (+) nalidixic acid (S) cephalo thin (R)
Dr. Nabil El AilaDiagnostic Microbiology
Suspected organisms
• 4- V.choleraeserogroup -01
catalse (+)oxidase (+)motile (+)lactose (NLF)sensitive to(dry – sunlight – acid pH)TCBS (thiosulfate citrate bile sucrose
Agar –yallow colonies)
Stool or Rectal swab culture*MacConky plate (NLF)
*XLD agar*HE
*selenite F broth/ GN broth
*TCBS (y.colores)*alkaline peptone water (APW)
*Selective media for campylobactor(skirrow&blaser with suplements)campylobactor microaerophilic
(42C°-O2 5%-Co210%-N2 85%-48hr)
Direct Culture SS agar
Stool/Rectal swab
GN/Selenite broth
Subculture Hecktoen
Check for suspected colonies
Incubate Overnight 37C°
Incubate Overnight 37C°
Check for suspected colonies
Confirmation using Biochemical and serological reaction
Detection of Salmonella & Shigella
Salmonella on SS-agar: Salmonella on Hektoen agar:
Salmonella on XLD Gram stain of Salmonella
Bichemical reactions of Salmonella on Triple Sugar Iron
Dr. Nabil El AilaDiagnostic Microbiology
Bichemical reactions of Salmonella on API20E
Salmonella on SS-agar: Salmonella on Hektoen agar:
Salmonella on XLD Gram stain of Salmonella
Bichemical reactions of Salmonella on Triple Sugar Iron
Dr. Nabil El AilaDiagnostic Microbiology
Shigella on SS-agar:Shigella on Hektoen agar:
Shigella on XLDGram stain of Shigella
Dr. Nabil El AilaDiagnostic Microbiology
Dr. Nabil El AilaDiagnostic Microbiology
API20E
Direct Blood agar
Stool/Rectal swab
Alkaline peptone water
Subculture TCBSCheck for suspected colonies
Incubate Overnight 37C°
Incubate Overnight 37C°
Check for suspected colonies
Confirmation using Biochemical and serological reaction
Detection of VibrioCholera
Direct TCBS
Dr. Nabil El AilaDiagnostic Microbiology
Vibrio cholerae TCBSDr. Nabil El Aila
Diagnostic Microbiology