Enterobacteriaceae , Enterobacter and their Biochemical Test

Enterobacteriaceae , Enterobacter and their Biochemical Test

1. ENTEROBACTERIACEAE

Enterobacteriaceae:
 The Enterobacteriaceae are a large family of C . that includes, along with many
harmless symbionts.
 many of the more familiar pathogens,
 such as Salmonella, Escherichia coli, Yersinia pestis, Klebsiella andShigella.
 Other disease-causing bacteria in this family include Proteus, Enterobacter, Serratia,
and Citrobacter.
Characteristics:
1. Members of the Enterobacteriaceae are rod-shaped,
2. and are typically 1-5 μm in length.
3. They appear as small grey colonies on blood agar.
4. Most have many flagella used to move about
5. And a few genera are nonmotile.
6. They are not spore-forming.
7. Many members of this family are a normal part of the gut flora found in the intestines of
humans and other animals.
8. Some are found in water and soil .
9. Some are are parasites. Caused infection in plants and animals.
Genera of Enterobacteriaceae:
 Citrobacter
 Enterobacter
 Escherichia, e.g. Escherichia coli
 Klebsiella, e.g. Klebsiella pneumoniae
 Pantoea
 Proteus, e.g. Proteus vulgaris
 Salmonella
 Serratia, e.g. Serratia marcescens
 Shigella

2. Enterobacter:
A. Enterobacter is a genus of common Gram-negative, facultatively anaerobic, rod-shaped,
non-spore-forming bacteria of the family Enterobacteriaceae.
B. Several strains of these bacteria are pathogenic and cause opportunistic
infections inimmunocompromised.
C. Caused urinary andrespiratory tracts infections.
D. Its a member of coliform group of bacteria.
E. Can growth at 44.5 °C in the presence of bile salts.
Scientific name: Enterobacter
Higher classification: Enterobacteriaceae
Rank: Genus

Species of Enterobacter:
aerogenes
E. amnigenus
E. agglomerans
E. arachidis
E. asburiae
E. cancerogenous
E. cloacae
E. cowanii
E. dissolvens
E. gergoviae
E. helveticus
E. hormaechei
E. intermedius
E. kobei
E. ludwigii
E. mori
E. nimipressuralis
E. oryzae
E. pulveris
E. pyrinus
E. radicincitans
E. taylorae
E. turicensis
E. sakazakii Enterobacter soli

3. Signs and symptoms:
Enterobacter infections do not have a clinical presentation that is specific enough to
differentiate them from other acute bacterial infections.
Bacteremia:
Signs of Enterobacter bacteremia include the following:
 Physical examination findings consistent with systemic inflammatory response syndrome
(SIRS): Including heart rate that exceeds 90 bpm, a respiratory rate greater than 20, and a
temperature above 38°C or below 36°C
 Fever: Occurring in more than 80% of children and adults with Enterobacterbacteremia
 Hypotension and shock: Occur in as many as one third of cases
 Septic shock: Manifested as disseminated intravascular coagulation, jaundice, acute
respiratory distress syndrome, and other complications of organ failure
 Purpura fulminans and hemorrhagic bullae
 Ecthyma gangrenosum
 Cyanosis and mottling: Frequently reported in children with Enterobacterbacteremia

Lower respiratory tract infections:
Enterobacter lower respiratory tract infections can manifest identically to those caused
by Streptococcus pneumoniae or other organisms. The physical examination findings may
include the following:
 Apprehension
 High fever or hypothermia
 Tachycardia
 Hypoxemia
 Tachypnea
 Cyanosis
Patients with pulmonary consolidation may present with crackling sounds, dullness to
percussion, tubular breath sounds, and egophony. Pleural effusion may manifest as dullness to
percussion and decreased breath sounds.
See Clinical Presentation for more detail

4.  what medium are use for Enterobacter?
Nutrient Agar is a best media for the growth of both positive and negative bacterial.
We prepare Nutrients Agar media and purring the plates, after Autoclaving we do purring of this media
in the plates. After solidification of these plates ,we sticking of sample on the plates and then we
incubated these plates in the incubator at 37 °C for 24 hours.
After 24 hours we observe the growth of bacteria in these plates. We observe the colonies of
bacteria, either its pink or red .
Then we doing Sub Culturing , and we pick a colony from master plate and then streak it on
another plate containing Nutrient Agar Media . After that place in incubator at 37°C for 24hr
and Then we perform Gram staining and different Biochemical Tests.

We can also use some specific medium for the isolation and identification of Enterobacter
Species.
The most important media are:
 Eosin Methylene Blue (EMB) agar
 MacConkey agar
 Salmonella Shigella (SS) agar
 Preparation of Nutrients Agar Media:
Procedure:
1. We take Flask, Graduate cylinder
2. We take 2.5 gram of nutrient agar in the flask and dissolved in 100 ml of distal water. Tightly
Shake the Flask to completely dissolved the nutrient agar in the flask.
3. The we put the flask along with 4 plates in the autoclave at 121 °C for 15 minutes to sterilized
the plates and dissolved the media in the plates.
4. After autoclaving we put the flask having prepared media in the LHF to become cool.
5. Then we purring these plates from this media and keep it for few minutes to become
solid.
6. After that we take a sample and streaking on the plates .
7. Then we put these plates in the incubator for 24hr at 37°C.
8. Then we will observe the growth of bacteria on the plates and also observe the colony
that either its pink colony or red colony.

5. 9. Then we start Gram staining and different Biochemical tests and then Microscopy.
Biochemical tests:
We perform different Biochemical tests for bacterial identification.
For Gram Negative Bacteria we perform the following Biochemical tests:
1. Indole Test
2. Citrate Utilization Test
3. Tripleb sugar iron Agar Test (TSI)
4. Catalase Test
5. Oxidase Test
6. Coagulase Test
7. Urease Test
 Indole test:
This article is about the biochemical test. For the detection of indoles such as LSD, see Ehrlich's
reagent.
The indole test is a biochemical test performed on bacterial species to determine the ability of
the organism to convert tryptophan into theindole. This division is performed by a chain of a
number of different intracellular enzymes, a system generally referred to as "tryptophanase."
 Performing Indole Test:
Like many biochemical tests on bacteria, results of an indole test are indicated by a change in
color following a reaction with an added reagent.
Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24–48 hours
before performing the test. Following incubation, add 5 drops of Kovac's reagent (isoamyl
alcohol, para-Dimethylaminobenzaldehyde, concentrated hydrochloric acid) to the culture
broth.
A positive result is shown by the presence of a red or red-violet color in the surface alcohol
layer of the broth. A negative result appears yellow. A variable result can also occur, showing an
orange color as a result. This is due to the presence of skatole, also known as methyl indole or
methylated indole, another possible product of tryptophan degradation.

6. The positive red color forms as a result of a series of reactions. The para-
Dimethylaminobenzaldehyde reacts with indole present in the medium to form a red rosindole
dye. The isoamyl alcohol forms acomplex with rosindole dye, which causes it to precipitate. The
remaining alcohol and the precipitate then rise to the surface of the medium.
A variation on this test using Ehrlich's reagent (using ethyl alcohol in place of isoamyl alcohol,
developed by Paul Ehrlich) is used when performing the test on nonfermenters and anaerobes.
 PROTOCOL:
o Inoculate the tube of tryptone broth with a small amount of a pure culture. Incubate at
35°C (+/- 2°C) for 24 to 48 hours.
o To test for indole production, add 5 drops of Kovács reagent directly to the tube (3, 5).
o A positive indole test is indicated by the formation of a pink to red color ("cherry-red
ring") in the reagent layer on top of the medium within seconds of adding the reagent.
o If a culture is indole negative, the reagent layer will remain yellow or be slightly cloudy .
(a) An uninoculated tube of tryptone broth.
(b) A positive indole test. The 48-hour Escherichia coli culture grown at 37°C tests positive for the
presence of indole as indicated by the red reagent layer after the addition of Kovács reagent.
(c) A negative indole test. The 48-hour Enterobacter aerogenes culture incubated at 37°C has not
broken down the tryptophan in the medium and thus no color change occurs upon addition of the
Kovács reagent. The reagent appears as a thin yellow layer on top of the culture medium.

(A) (B) (C)
 Reference
http://microbeonline.com/catalase-test-principle-uses-procedure-results/
http://www.vetbact.org/vetbact/?biochemtest=1