1. Gram-negative rods Enterobacteriacea Clinical Microbiology
MESHACK JUMA
MEDICAL MICROBIOLOGY DEPT (UON)
Msc. Med Micro (UON), PGD-RM(UON),
Bsc. Med Micro(JKUAT), Dip MLS, HND Dx Micro.

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

3. Classification of Bacteria

4. Characters of Enterobacteriaceae
All Enterobacteriaciae
Gram-negative rods
Ferment glucose with acid production
Reduce nitrates into nitrites
Oxidase negative
Catalase positive
Facultative anaerobic
All motile except Shigella, Klebsiel and Yersinia
Non-capsulated except Klebsiella
Non-fastidious
Grow on bile containing media (MacConkey agar)

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

6. 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

7. Identification of Enterobacteriaceae
Gram stain
All Enterobacteriaceae are Gram-negative rods
Arranged in single

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

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

10. Classification of Enterobacteriaceae
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 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
pH indicator
Acidic: Pink
Cause of differential
Cause of selectivity
Lactose non feremnters
colorless colonies
Lactose feremnters
Pink colonies

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

13. Incubate the plate in incubator at 37 C/24 hrs Results:
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 1 2 3 4 5

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

15. 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 fermenter
Neutral red
Lactose
Acid
Pink colonies
H2S + Ferric ammonium citrate
Ferrous sulfide
Black precipitate

16. Incubate the plate in incubator at 37 C/24 hrs
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 1 2 3 4 5

17. 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

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

19. Reaction on Triple Sugar Iron (TSI) Agar
TSI contains
Three different types of sugars
Glucose (0.1%)
Lactose (1%)
Sucrose (1%)
Phenol red (acidic: Yellow)
TSI dispensed in tubes with equal butt & slant
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.

20. 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

21. Results Example Result Reaction on TSI H2S Slant color Butt color LNF
e.g. Shigella
A/Alk/-
(Glucose fermented)
Negative
Red
Yellow
LNF e.g. Salmonella & Proteus
A/Alk/+
(Glucose fermented with H2S)
Positive
black in butt
LF e.g. E. coli, Klebsiella,Enterobacter
A/A/-
(three sugars are fermented)
Non fermenter e.g. Pseudomonas
Alk/Alk/-
(No action on sugars)

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