Gram negative rods

It is useful to medical students

1. Gram-negative rodsGram-negative rods
EnterobacteriaceaEnterobacteriacea
Clinical MicrobiologyClinical Microbiology
MESHACK JUMAMESHACK JUMA
MEDICAL MICROBIOLOGY DEPT (UONMEDICAL MICROBIOLOGY DEPT (UON((
Msc. Med Micro (UON), PGD-RM(UONMsc. Med Micro (UON), PGD-RM(UON(, (,
Bsc. Med Micro(JKUAT), Dip MLS, HND Dx MicroBsc. Med Micro(JKUAT), Dip MLS, HND Dx Micro..

2. Family Family EnterobacteriaceaeEnterobacteriaceae
often referred to as often referred to as
“enterics“enterics””
..

3. Bacteria
Gram positive Gram negative
Cocci Bacilli Cocci Rods

4. Characters of Characters of EnterobacteriaceaeEnterobacteriaceae
All All EnterobacteriaciaeEnterobacteriaciae
–Gram-negative rodsGram-negative rods
–Ferment glucose with acid production Ferment glucose with acid production
–Reduce nitrates into nitritesReduce nitrates into nitrites
–Oxidase negativeOxidase negative
–Catalase positiveCatalase positive
Facultative anaerobicFacultative anaerobic
All motile All motile exceptexcept Shigella, Klebsiel Shigella, Klebsiel and and YersiniaYersinia
Non-capsulated except Non-capsulated except KlebsiellaKlebsiella
Non-fastidiousNon-fastidious
Grow on bile containing media (MacConkey agar( Grow on bile containing media (MacConkey agar(

5. Microscopic and Colony Microscopic and Colony
MorphologyMorphology
Gram negative bacilli or coccobacilliGram negative bacilli or coccobacilli
Non-spore formingNon-spore forming
Colony morphology on BA or MH of little Colony morphology on BA or MH of little
value, as they look the same, except for value, as they look the same, except for
KlebsiellaKlebsiella
Selective and differential media are used Selective and differential media are used
for initial colony evaluation (ex. for initial colony evaluation (ex.
MacConkey, Hektoen Enteric (HE), MacConkey, Hektoen Enteric (HE),
Xylose lysine deoxycholate (Xylose lysine deoxycholate (XLDXLD(( agars agars((

6. EnterobacteriaceaeEnterobacteriaceae
Some Some Enterobacteriaceae Enterobacteriaceae are true pathogensare true pathogens
–SalmonellaSalmonella spp. spp.
–ShigellaShigella spp. spp.
–Yersinia Yersinia spp. spp.
–Certain strains of Certain strains of E. coli E. coli (ETEC, EPEC, EIEC, EHEC( (ETEC, EPEC, EIEC, EHEC(
Most members of the Most members of the Enterobacteriaceae Enterobacteriaceae are are
opportunistic or cause secondary infections of opportunistic or cause secondary infections of
wounds, the urinary and respiratory tracts, and wounds, the urinary and respiratory tracts, and
the circulatory system e.g. the circulatory system e.g. E. coliE. coli..
Enterobacteriaceae divided into TWO main Enterobacteriaceae divided into TWO main
groups according to action on LACTOSEgroups according to action on LACTOSE
–Lactose Fermenters (LF(Lactose Fermenters (LF(
E. coli, Citrobacter, Klbesiella, EnterobacterE. coli, Citrobacter, Klbesiella, Enterobacter
–Lactose Non-Fermenters (LNF(Lactose Non-Fermenters (LNF(
Salmonella, Shigella, Proteus, YersiniaSalmonella, Shigella, Proteus, Yersinia

7. Identification of Identification of EnterobacteriaceaeEnterobacteriaceae
Gram stainGram stain
–All All EnterobacteriaceaeEnterobacteriaceae are Gram-negative rods are Gram-negative rods
–Arranged in singleArranged in single

8. Identification of Identification of EnterobacteriaceaeEnterobacteriaceae
Based on Biochemical reactionsBased on Biochemical reactions
Can be grouped into threeCan be grouped into three:-:-
A) Ability to ferment carbohydrates to acid or A) Ability to ferment carbohydrates to acid or
Acid and gasAcid and gas..
B) Ability to utilize a substrate as the sole B) Ability to utilize a substrate as the sole
source of carbonsource of carbon
C) Ability to enzymaticaly degrade a C) Ability to enzymaticaly degrade a
compoundcompound

9. Identification of Identification of EnterobacteriaceaeEnterobacteriaceae
Biochemical reactionsBiochemical reactions
Oxidase testOxidase test
–All members of All members of EnterobacteriaceaeEnterobacteriaceae are oxidase negative are oxidase negative
–PseudomonasPseudomonas is oxidase positive is oxidase positive
Oxidative/Fermentation testOxidative/Fermentation test
–All members of All members of EnterobacteriaceaeEnterobacteriaceae are O+/F+ are O+/F+
–PseudomonasPseudomonas is Oxidative+ / Fermentation - is Oxidative+ / Fermentation -
Nitrate reductaseNitrate reductase
–All members of Enterobacteriaceae are nitrate reductase positiveAll members of Enterobacteriaceae are nitrate reductase positive
–PseudomonasPseudomonas is nitrate reductase negative is nitrate reductase negative
See & compare these tests for See & compare these tests for Pseudomonas spp Pseudomonas spp in the Labin the Lab

10. Classification of Classification of EnterobacteriaceaeEnterobacteriaceae
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

11. Differentiation between LF & NLF byDifferentiation between LF & NLF by Growth on MacConkey agarGrowth on MacConkey agar
MacConkey Agar
Contains
Bile salts Crystal violet Lactose Neutral red
 MacConkey agar is selective & differential medium for MacConkey agar is selective & differential medium for EnterobacteriaceaeEnterobacteriaceae
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

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

13. Identification of Identification of EnterobacteriaceaeEnterobacteriaceae
Differentiation between LF & NLF byDifferentiation between LF & NLF by Growth on MacConkey agarGrowth on MacConkey agar
Method:Method:
–MacConkey agar is inoculated with tested organismMacConkey agar is inoculated with tested organism
using streak plate techniqueusing streak plate technique
–Incubate the plate in incubator at 37 C/24 hrsIncubate the plate in incubator at 37 C/24 hrs
Results:Results:
–LF organism appears as pink colonies (e.g. LF organism appears as pink colonies (e.g. E. coliE. coli))
–NLF organism appears as colorless colonies (e.g. NLF organism appears as colorless colonies (e.g.
ShigellaShigella))
Flame & Cool
Flame & Cool
Flame & Cool
1 2
3
4
5

14. Growth of Enterobacteriaceae on
MacConkey agar
Uninoculated plate
Lactose non feremters
Salmonella, Shigella,
Proteus
Lactose feremters
E. coli, Citrobacter
Klebsiella, Enterobacter
Colorless coloniesPink colonies

15. Reaction on Salmonella Shigella (SS) agarReaction on Salmonella Shigella (SS) agar
SS agar is a selective & differential medium used for isolation of SS agar is a selective & differential medium used for isolation of
SalmonellaSalmonella and and ShigellaShigella
The selective agents are The selective agents are bile saltsbile salts, and , and brilliant green dyebrilliant green dye, which , which
inhibit gram-positive organismsinhibit gram-positive organisms
The medium contains only The medium contains only lactoselactose as a differential agent and thus as a differential agent and thus
differentiates on the basis of lactose fermentationdifferentiates on the basis of lactose fermentation
The formation of acid on fermentation of lactose causes the neutral The formation of acid on fermentation of lactose causes the neutral
red indicator to make pink coloniesred indicator to make pink colonies
Non lactose fermenting organisms are colorless on the mediumNon lactose fermenting organisms are colorless on the medium
SS agar contains SS agar contains sodium thiosulfatesodium thiosulfate and and ferric ammonium ferric ammonium
citratecitrate allows the differentiation of organisms that produce H2S allows the differentiation of organisms that produce H2S
–Lactose fermenters, such as Lactose fermenters, such as E. coliE. coli, have colonies which are , have colonies which are
pinkpink
–ShigellaShigella appears transparent or amber appears transparent or amber
–SalmonellaSalmonella appears transparent or amber with black centers appears transparent or amber with black centers
due to H2S productiondue to H2S production
Lactose
Lactose fermenter
Acid
Neutral red
Pink colonies
Ferrous sulfide
Black precipitate
H2S + Ferric ammonium citrate

16. Identification of Identification of EnterobacteriaceaeEnterobacteriaceae
Differentiation between LF & NLF byDifferentiation between LF & NLF by Growth on SS agarGrowth on SS agar
Method:Method:
–SS agar is inoculated with tested organismSS agar is inoculated with tested organism using using
streak plate techniquestreak plate technique
–Incubate the plate in incubator at 37 C/24 hrsIncubate the plate in incubator at 37 C/24 hrs
Flame & Cool
Flame & Cool
Flame & Cool
1
2
3
4
5

17. AA.. Klebsiella pneumoniaeKlebsiella pneumoniae
BB.. Escherichia coliEscherichia coli
CC: : Salmonella spSalmonella sp..
DD: : Proteus mirabilisProteus mirabilis
EE: : Ps. aeruginosaPs. aeruginosa
. .
BothBoth are lactose fermentersare lactose fermenters
BothBoth Salmonella sp. & Proteus Salmonella sp. & Proteus product H2Sproduct H2S
PseudomonasPseudomonas colonies are nearly colorlesscolonies are nearly colorless
Growth of Enterobacteriaceae on SS agar

18. Growth of Growth of EnterobacteriaceaeEnterobacteriaceae on on
EMB agarEMB agar
Coli-type colonies are very dark,
almost black e.g. E. coli

19. Reaction on Triple Sugar Iron (TSI) AgarReaction on Triple Sugar Iron (TSI) Agar
TSI containsTSI contains
–Three different types of sugarsThree different types of sugars
Glucose (0.1%)Glucose (0.1%)
Lactose (1%)Lactose (1%)
Sucrose (1%)Sucrose (1%)
–Phenol red (acidic: Yellow)Phenol red (acidic: Yellow)
TSI dispensed in tubes with equal butt & slantTSI dispensed in tubes with equal butt & slant
PrinciplePrinciple
–To determine the ability of an organism to attack a specific To determine the ability of an organism to attack a specific
carbohydrate incorporated into a basal growth medium, with or carbohydrate incorporated into a basal growth medium, with or
without the production of gas, along with the determination of without the production of gas, along with the determination of
possible hydrogen sulphide production. possible hydrogen sulphide production.

20. Reaction on TSIReaction on TSI
Method:Method:
–Inoculate TSI medium with an organism by Inoculate TSI medium with an organism by
inoculating needle by stabbing the butt and inoculating needle by stabbing the butt and
streaking the slantstreaking the slant
–Incubate at 37°C for 24 hoursIncubate at 37°C for 24 hours

21. Reaction on TSI
Result
Example
Butt colorSlant colorH
2
S
Yellow Red
Negative A/Alk/-
(Glucose fermented)
LNF
e.g. Shigella
Yellow Red
Positive
black in butt
A/Alk/+
(Glucose fermented with H
2
S)
LNF e.g. Salmonella &
Proteus
Yellow Yellow
Negative
A/A/-
(three sugars are fermented)
LF e.g. E. coli,
Klebsiella,Enterobacter
Red Red
Negative
Alk/Alk/-
(No action on sugars)
Non fermenter e.g.
Pseudomonas
Result
s

22. Practicle WorkPracticle Work
Gram stainGram stain
Oxidase testOxidase test
Indole testIndole test
O/F test *O/F test *
Nitrate reductase test*Nitrate reductase test*
Growth on MacConkey’s agarGrowth on MacConkey’s agar
Growth on EMB agar*Growth on EMB agar*
Growth on SS agarGrowth on SS agar
Reaction on TSIReaction on TSI