• The Enterobacteriaceae family contains a large number of genera that are
biochemically and genetically related to one another.
• They are commonly found in the intestine of animals, hence got the name
“enterobacteria” or “enteric bacteria
• Many of them are pathogenic to humans, plants, and animals and can develop
a fatal infection
Bacterial identification is important in many areas
• Public health
• Clinical diagnosis to correct and timely treat disease or
infection
• Appropriate treatment
• Environmental monitoring to identify biological threat agents
• Food safety monitoring
• Tracking and tracing disease spread and outbreaks
• Identifying new isolates, e.g. antibiotic-resistant isolates
Common Characteristics
• Gram-negative
• Rod-shaped (usually short bacilli; about 1 – 5 μm)
• Facultatively anaerobic
• Non-sporing
• Mostly motile with peritrichous flagella, ( most species in the genera Klebsiella and
Shigella are non-motile)
• Catalase positive, oxidase negative: used to differentiate from Non-Enterobacteria
• Both lactose fermenter and non-fermenter: MacConkey media is used to differentiate
• Usually, acid-producing
• Usually, nitrate-reducing
• Characteristic antigens are known as enterobacterial common antigens (ECA). The outer
membrane (O), flagella (H), and capsule (K) are antigens in most the enterobacteria.
Primary isolation Media
1) TSA
• To get Pure and Isolated Colonies
• From TSA record colony characteristics in your logbook
2) MacConkey:
• To differentiate between Lactose Fermenters and
Lactose non fermenters
• Record colony characteristics in your logbook
5. Serratia
6. Morganella 7. Providencia (are phylogenetically related and termed as Proteeae)
Biochemical Tests for the Identification of
Enterobacteriaceae Family
Members of the Enterobacteriaceae family are identified based on their biochemical
properties.
Commonly used biochemical tests are: IMViC, Urea test and TSI agar
1. Indole Test
2. Methyl Red (MR) Test
3. Test Voges -Proskauer (VP) Test
4. Citrate Test
5. Urease Test
6. Motility
7. Oxidase Test
1) Indole Test:
Procedure:
• Inoculate the Tryptophan broth or Sulfide – Indole – Motility (SIM) medium tube aseptically using given
culture (label the tube with your name, your assigned organism(s) and the name of the
test media.
• Incubate the tube at 37°C for 24-28 hours.
• Add 0.5 ml of Kovac’s reagent to the broth culture.
• Observe for the presence or absence of ring.
2) Motility: if entire tube turns turbid shows motility
Sulfide production: presence of Black ppt in any amount
shows Sulfur reduction and H2S production
3 & 4) MR-VP test
• The MR-VP test determines the possible types of glucose fermentation being conducted by a bacterial
species.
• Some bacterial species may ferment glucose a using mixed acid fermentation pathway while some other
bacterial species may ferment glucose via the butanediol fermentation pathway.
Methyl Red (MR) Test Voges -Proskauer (VP) Test
Methyl Red (MR) Test Detects Mixed Acid Voges-Proskauer (VP) Test Detects
Fermentation Butanediol Fermentation
• Obtain two MR-VP broth tube ( Label one tube as MR test and other tube as VP test)
• Also label the tube with your name, your assigned organism(s) and the name of the test
media.
• Aseptically transfer bacteria with a loop to inoculate the MR-VP broth.
• Incubate the inoculated tubes @ 35C for 24-48 hrs
• Add 10 drops of methyl red reagent to “MR” • First add 15 drops of VP reagent 1(alpha-
tube. napththol) to the tube and then add 5 drops
• Examine the color of the medium. of VP reagent 2 (40% KOH). Wait for 30 mins
• Record the results. or more
• Immediate color changes to Red in color in • Pink color ring will form at the top surface of
tube the media in the tube
5) Citrate Test
Detects the ability of organisms (bacteria) to utilize citrate as a sole source of energy.
• Take a Citate agar slant (label the tube with your name, your assigned
organism(s) and the name of the test media)
• Streak the slant in a Zig zag pattern using a well-isolated colony of a given culture
• Incubate at 35C for 24-48 hrs.
• Observe a color change from green to blue along the slant.
6) Urease Test
• Depends on bacteria to produce urease enzyme to utilise urea present in media
The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol
red from light orange to magenta (pink).
• Take a Urease agar slant (label the tube with your name, your assigned organism(s) and the name of the test
media)
• Streak the slant in a Zig zag pattern using a well-isolated colony of a given culture
• Incubate at 35C for 24-48 hrs.
• Observe a color change from light orange to Magenta pink.
Oxidase Test
• The oxidase test checks for the presence of an enzyme
called cytochrome oxidase in bacteria.
• This enzyme helps transfer electrons and causes a
special dye, called tetramethyl-p-phenylene-
diamine (TMPD), to turn purple.
