This document provides information on biochemical reactions of the Enterobacteriaceae family of bacteria. It discusses that members of Enterobacteriaceae are gram-negative rods that ferment glucose with acid production and reduce nitrates. It describes several selective media used to isolate and distinguish lactose fermenters from non-fermenters, including MacConkey agar which produces pink colonies for fermenters and colorless for non-fermenters. Biochemical tests like TSI, lysine iron agar, and oxidase are also summarized to differentiate bacterial species within the Enterobacteriaceae family.

1. Enterobacteriaceae (2)
Biochemical reactions
By
Dr. Nabil El Aila
Assistant Professor of Molecular Microbiology
Medical Technology Department
Al -Aqsa University
Dr. Nabil El Aila
DiagnosticMicrobiology

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Dr. Nabil El Aila
DiagnosticMicrobiology

3. Characters of Enterobacteriaceae
• All Enterobacteriaciae
– Gram-negative rods
– Ferment glucose with acid production
– Reduce nitrates into nitrites
– Oxidase negative
• Facultative anaerobic
• Motile except Shigella and Klebsiella
• Non-capsulated except Klebsiella
• Non-fastidious
• Grow on bile containing media (MacConkey agar)
Dr. Nabil El Aila
DiagnosticMicrobiology

4. 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
opportunistic or cause secondary infections of wounds,
the urinary and respiratory tracts, and the circulatory
system e.g. E. coli.
• Enterobacteriaceae divided 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
DiagnosticMicrobiology

5. Identification of Enterobacteriaceae
• Gram stain
– All Enterobacteriaceae are Gram-negative rods
– Arranged in single
Dr. Nabil El Aila
DiagnosticMicrobiology

6. 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

7. Classification of Enterobacteriaceae
Enterobacteriaceae
Lactose fermenters
E. coli, Citrobacter,
Klebsiella, Enterobacter
Non-lactose fermenter
Salmonell, Shigella
Proteus, Yersinia
There are several selective and differential media used to
isolate 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

8. Differentiation between LF NLF by Growth on MacConkey agar
MacConkey agar is selective differential medium for Enterobacteriaceae
MacConkey Agar
Contains
Bile salts Crystal violet Lactose Neutral red
Inhibit growth of G+ve bacteria
Cause of selectivity
Cause of differential
pH indicator
Acidic: Pink
Lactose feremnters
Pink colonies
Lactose non feremnters
colorless colonies

9. Classification of Enterobacteriaceae according to lactose
fermentation (growth on MacConkey Agar)
Enterobacteriaceae
Lactose Fermenters Lactose Non-Fermenters
Pink colonies Colorless colonies
Escherichia coli
Klebsiella spp
Enterobacter spp
Citrobacter spp
Salmonella spp
Schigella spp
Proteus spp
Yersinina spp
Acid
Neutral red
No acid
Dr. Nabil El Aila
DiagnosticMicrobiology

10. 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 2
3
4
5

11. Growth of Enterobacteriaceae on
MacConkey agar
Colorless colonies Pink colonies
Uninoculated plate Lactose non feremters
Salmonella, Shigella,
Proteus
Lactose feremters
E. coli, Citrobacter
Klebsiella, Enterobacter
Dr. Nabil El Aila
DiagnosticMicrobiology

12. 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 lactose as 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 thiosulfate and 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
Lactose
Lactose fermenter
Acid
Neutral red
Pink colonies
Ferrous sulfide
Black precipitate
H2S + Ferric ammonium citrate

13. Identification of Enterobacteriaceae
Differentiation 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 Aila
DiagnosticMicrobiology

14. Growth of Enterobacteriaceae on SS agar
A Klebsiella pneumoniae
B Escherichia coli
C Salmonella sp
D Proteus mirabilis
E Ps. aeruginosa
Both are lactose fermenters
Both Salmonella sp. Proteus product H2S
Pseudomonas colonies are nearly colorless

15. 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 organism growth.
– 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

16. Reaction on Triple Sugar Iron (TSI) Agar
• Principle
– To determine the ability of an organism to attack a specific
carbohydrate incorporated into a basal growth medium, with
or without the production of gas, along with the determination
of possible hydrogen sulphide production.
• When the carbohydrates are fermented, acid production is detected
by the Phenol Red pH indicator.
• Sodium Thiosulfate is reduced to hydrogen sulfide, and hydrogen
sulfide reacts with an iron salt yielding the typical black iron sulfide.
• Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator.
Sodium Chloride maintains the osmotic balance of the medium.
Agar is the solidifying agent

17. 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 Aila
DiagnosticMicrobiology

18. Example
Result
Reaction on TSI
H2S
Slant
color
Butt
color
Non fermenter
e.g.
Pseudomonas
Alk/Alk/-
(No action on sugars)
Red Red Negative
LNF
e.g. Shigella
A/Alk/-
(Glucose fermented
without H2S)
Negative
Yellow Red
LNF
e.g. Salmonella
Proteus
A/Alk/+
(Glucose fermented
with H2S)
Positive
black in
Yellow Red butt
LF
e.g. E. coli,
Klebsiella,
Enterobacter
A/A/-
(three sugars are
fermented)
Negative
Yellow Yellow
Result

19. Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
MacCon SS EMB
key
Nitrate O/F
reductase
Gram Oxidase
stain
Metallic
sheen
E. coli -ve rod -ve +ve O+/F+ LF LF
Citrobacter -ve rods -ve +ve O+/F+ LF LF Dark
Klebsiella -ve rods -ve +ve O+/F+ LF LF Dark
Enterobacter -ve rods -ve +ve O+/F+ LF LF Dark
NLF/ Colorless
H2S
Salmonella -ve rods -ve +ve O+/F+ NLF
Shigella -ve rods -ve +ve O+/F+ NLF NLF Colorless
NLF/ Colorless
H2S
Proteus -ve rods -ve +ve O+/F+ NLF

20. Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
TSI Indole MR VP Citrate Urease Motility
E. coli A/A/- +ve +ve -ve -ve -ve Motile
Citrobacter A/A/- +ve +ve -ve +ve -ve Motile
freundii
Klebsiella A/A/- -ve -ve +ve +ve +ve
Non
pneumoniae
motile
Enterobacter A/A/- -ve -ve +ve +ve +ve Motile
cloacae
Salmonella A/Alk/+ -ve +ve -ve +ve -ve Motile
typhi
Non
motile
Shigella A/Alk/- -ve +ve -ve -ve -ve
boydii
Motile
Swarwing
Proteus A/Alk/+ -ve +ve -ve +ve +ve
mirabilis

21. Lysine Iron Agar (LIA)
• Lysine iron agar (LIA) slants test organisms for the ability to
deaminate lysine or decarboxylate lysine. Lysine deamination is an
aerobic process which occurs on the slant of the media. Lysine
decarboxylation is an anaerobic process which occurs in the butt
of 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.

22. 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 species may deaminate the lysine and
produce a red slant and acid butt.
• Production of hydrogen sulphide causes a blackening in the
medium due to formation of ferrous sulphide. The indicator is
bromcresol purple.

23. Tube 1: Positive decarboxylation (butt), negative deamination (slant)
Tube 2: Negative decarboxylation (butt), positive deamination (slant)
Dr. Nabil El Aila
DiagnosticMicrobiology

24. Lysine Iron Agar (LIA)
Uninoculated medium Alkaline/alkaline/H2S:
Salmonella.
3. Alkaline/yellow/H2S:
Citrobacter.

25. COLLECTION, TRANSPORT, AND EXAMINATION
OF 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
DiagnosticMicrobiology

26. 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 stercoralis
Cysts :
Entamoeba histolytica
Giardia lamblia
Balantidium coli
Isospora belli ( oocysts )

27. Commensals
The 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 Negative
Enterococci Escherichia coli*
Anaerobic streptococci Proteus*
Lactobacilli Enterobacter*
Clostridia Hafnia *
Citrobacter*
Providencia*
Morganella*
Serratia*
Klebsiella *
Bacteroides species
Pseudomonas 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.

28. 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 blood into a clean dry leak-proof container.
• A diarrheal stool 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 bismuth that interferes with the laboratory tests.

29. 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 delay over 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
DiagnosticMicrobiology

30. Suspected organisms
1- E-coli (infant NLF)
2- salmonella shigella
3- campylobactor
jejuni most common human pathogen
indolcitrate (+)
urease (- )
oxidase (+)
nalidixic acid (S)
cephalo thin (R)
Dr. Nabil El Aila
DiagnosticMicrobiology

31. Suspected organisms
• 4- V.cholerae
serogroup -01
catalse (+)
oxidase (+)
motile (+)
lactose (NLF)
sensitive to (dry – sunlight – acid pH)
TCBS (thiosulfate citrate bile sucrose
Agar – yallow colonies)

32. 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)

33. Detection of
Salmonella Shigella
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

34. Salmonella on SS-agar: Salmonella on Hektoen agar:
Salmonella on XLD Gram stain of Salmonella

35. Bichemical reactions of
Salmonella on Triple Sugar Iron
Dr. Nabil El Aila
DiagnosticMicrobiology
Bichemical reactions of
Salmonella on API20E

36. Salmonella on SS-agar: Salmonella on Hektoen agar:
Salmonella on XLD Gram stain of Salmonella

37. Bichemical reactions of
Salmonella on Triple Sugar Iron
Dr. Nabil El Aila
DiagnosticMicrobiology

38. Shigella on SS-agar:
Shigella on Hektoen agar:
Shigella on XLD
Gram stain of Shigella
Dr. Nabil El Aila
DiagnosticMicrobiology

39. Dr. Nabil El Aila
DiagnosticMicrobiology
API20E

40. Detection of Vibrio
Direct Blood agar
Stool/Rectal swab
Alkaline peptone water
Check for suspected colonies Subculture TCBS
Incubate Overnight 37C°
Incubate Overnight 37C°
Check for suspected colonies
Confirmation using Biochemical and serological reaction
Cholera
Direct TCBS
Dr. Nabil El Aila
DiagnosticMicrobiology

41. Vibrio cholerae TCBS
Dr. Nabil El Aila
DiagnosticMicrobiology