MICROBIOLOGY LAB (MIDTERMS)

SUPPLEMENTAL HANDOUT
Dr. Mark Calban
BICHEMICAL & CULTURAL CHARACTERISTICS OF BACTERIA
Cultural Characteristics of Enterics
To isolate Shigella and Salmonella from fecal matter, USE SELECTIVE
MEDIA.
To selectively inhibit gram-positive organisms and separate enterics
in broad categories, USE DIFFERENTIAL MEDIA.
E. coli colonies on blood agar.
E coli colonies and other enteric
bacteria are generally circular,
convex, smooth with distinct
edges.
*Some strains of E. coli may
exhibit hemolysis on blood agar.
*Enterobacter: colonies are
similar but
Somewhat more mucoid.
Klebsiella oxytoca colonies on
MacConkey agar. Note that
Klebsiella colonies are generally
large, very mucoid, and tend to
coalesce with prolonged
incubation
November 2015
EMB (Eosin Methylene Blue) AGAR
 Selective and differential media for fecal coliform isolation
 Similar to MacConkey agar, except uses eosin and methylene
blue as dyes instead
o Eosin Y & Methylene blue are pH indicators  form dark
purple precipitate at LOW pH (ACIDIC); also inhibit Gram
(+) growth
 Used to test for lactose fermentation
o Sucrose and lactose as carbohydrate sources, as support
for growth of fecal coliforms, and as means of
differentiation.
o Vigorous fermenters  dark purple dye complex formed
o E. coli  green metallic sheen
o Slow fermenters  mucoid pink colonies
o Non-lactose/sucrose fermenters  clear/colorless (also
means bacteria is not a fecal coliform)
 Positive result: blue-black color (can have metallic sheen, as for
E. coli)
 Negative result: clear/colorless

Comparison of Enterobacter (LEFT) with Klebsiella (RIGHT) colonies.

Enterobacter colonies are less mucoid than Klebsiella.

EMB Agar. LEFT: differences in streak appearance of bacteria which

are non-lactose fermenters (clear) and lactose fermenters (purple).
RIGHT: green metallic sheen exhibited by E. coli

Comparison of E. coli, Salmonella spp., and Shigella spp. on
Salmonella-Shigella agar(SSA). Note that both Salmonella and
Shigella produce colonies similar to E.coli, but DO NOT FERMENT
LACTOSE whereas E. coli does (hence change in color of agar).
* E.coli – pink colonies; Salmonella – colorless with black centers due
to H2S gas; Shigella – colorless
MacConkey AGAR
 Selective for Gram (-) organisms
 Includes bile salts and crystal violet which inhibit Gram (+)
growth and the growth of some fastidious Gram (-) bacteria
(e.g., Haemophilus and Neisseria)
 Carbohydrate source: only lactose
 Positive result: pink or red colonies (lactose fermenters)
 Negative result: clear colonies (non-lactose fermenters)

Biochemical Tests and Agars for Identification

a) Indole Production – from tryptophan; used in rapid
identification
b) Voges Proskauer reaction – production of acetylmethylcarbinol
from dextrose; used less often
c) Growth Characteristics – culture on media that contain special
dyes and carbohydrates (EMB, Mac Conkey, Deoxycholate)
distinguishes lactose fermenting colonies (colored) from non-
lactose fermenting colonies (non-pigmented).

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MacConkey agar. Left side shows positive result (Positive for lactose GAS H2S GAS,H2S GAS H2S GAS,H2S
fermentation) while right side exhibits a negative result. Theoretical results for TSI. First letter corresponds to color of slant.
Second letter corresponds to color of butt.
TRIPLE SUGAR IRON (TSI) AGAR “A” for acidic (with color change). “K” for alkaline (No color change).
 Tests the ability of an organism to: Gas = formation of bubbles; splitting of medium; displacement of
a) Ferment glucose and/or lactose medium
b) Produce hydrogen sulfide (H2S)
 Used to differentiate salmonellae and shigellae from other
enteric Gram (-) rods in stools
 Contents:
▪ 0.1% glucose ▪ ferrous sulfate (to detect H2S)
▪ 1.0% sucrose ▪ tissue extracts (protein growth
substrate)
▪ 1.0 % lactose ▪ Phenol red (pH indicator)

 Principle of TSI:
o Sugar fermentation allows limited ATP production under
little/no O2 conditions. Acids and CO2 as byproduct
o For fermenters, glucose is the first sugar catabolized.
o IF glucose is used up or unavailable, two things can
happen:
1) Organism has enzyme for lactose  continues
fermenting, production of acids and/or CO2 A B C D
2) Organism does NOT have enzyme  protein Actual TSI Agar Results.
deamination, A: with gas, yellow slant & butt; B: no gas, yellow butt, red slant
production of NH3 C: (+) H2S, red slant; D: Red slant & butt

Enteric organisms will ferment glucose with the production

of acids which will change the color of the medium in the butt
and along the slant from red to yellow because of a reduction
in the pH (within the first few hours). However, since the
glucose is present in small amounts (0.1%), the supply is
soon exhausted and the organisms (if non-lactose/sucrose
fermenter) growing on the surface of the slant in the
presence of oxygen are forced to catabolize peptones and
amino acids for their energy supply. Alkaline end-products
are produced from these substances which revert the pH of
the slant to an alkaline pH and thus change the color of the
agar slant back to red.
Lactose/sucrose fermenters produce acid slants and acid
butts. Acid slants do not revert to an alkaline status because
lactose (1%) and sucrose (1%) are being fermented.
SUMMARY
Laboratory (LF: Lactose Fermenter; NLF: Non-Lactose Fermenter)
1. Growth on media
a. Eosin-Methylene Blue (EMB) Agar
Selective for Gr- Enteric Bacilli; Differential for lactose and
non-lactose fermenters
CHO: lactose
Lactose Fermenter (LF): pink to purple
Non Lactose Fermenter (NLF): colorless
Characteristic Colonies on EMB:
E. coli: LF; pink to purple colonies w/ GREEN METALLIC
SHEEN
Klebsiella: LF; pink to purple MUCOID colonies
Enterobacter: LF; pink to purple w/ dark center (FISH EYE
COLONIES)

b. MacConkey (MAC) Agar

 Types of result: Selective for Gr- Enteric Bacilli; differentiates lactose
Activity Slant Butt Gas Black H2S fermenters from non-lactose fermenters
Obligate aerobe Red Red - - Inhibitor for Gr+: crystal violet & bile salt
Glucose ferment Red Yellow - - CHO: lactose
pH indicator: neutral red
Lactose +/or LF: pink colonies
Sucrose Yellow Yellow - - NLF: colorless colonies
Fermenter
Gas producer + c. Triple Sugar Iron (TSI)
H2S + CHO: lactose (1%), sucrose (1%), glucose (0.1%)
pH indicator: phenol red
* Note that gas production and H2S production may occur with strict
H2S indicator: ferrous sulfate
glucose fermenters or lactose/sucrose fermenters.

Gas production: results from oxidation of fermentation products to

CO2 & H2O
Alkaline/Red Slant: results from oxidative decarboxylation of
proteins, leading to formation of amines TSI REACTIONS TYPICAL ORGANISMS
Black H2S: results from production of H2S which reacts with ferrous LF E – Escherichia coli
A/A
sulfate to form FeS. Gas (+) K – Klebsiella
Gas
H2S (-) E – Enterobacter
H2S-
**Salmonella and Shigella: typically produce alkaline slant and acid Non-LF Salmonella
butt K/A
Gas (+) Proteus
Gas
Other enteric bacteria: acid slant, acid and gas in butt H2S (+)
H2S+
NLF Shigella
K/A Gas (-)
H2S- H2S (-)

NLF Pseudomonas
K/K Non-GF Alcaligenes
H2S- Gas (-)
H2S (-)
LF: lactose fermenter SF: sucrose fermenter GF: glucose
fermenter
Gas (+): ALL AEROGENIC except SHIGELLA (most inert organism)
A/A A/A A/A A/A A/K K/A K/A K/A K/A K/K H2S (+): SPACEd

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 S – Salmonella
P – Proteus
A – Arizona
C – Citrobacter
Ed - Edwardsiella
2. Indole test
 Detects tryptophanase
 Medium: Sulfide Indole Motility (SIM)
 Indicator: Kovac’s or Ehrlich’s reagent – p-
dimethyaminobenzaldehyde (PDAB)
 (+) red ring; (-) yellow/brown ring

3. Methyl Red/Voges Proskauer Test

Medium: MRVP Broth
a. Methyl red
 Mixed acids fermentation pathway
 Organism that produce enough acid will overcome the
neutralizing effect of the buffer
 (+) pH<4.5 RED ; (-) yellow soln.

b. Voges Proskauer
 Butylene glycol pathway
 Detects acetoin (acetylmethylcarbinol)
 Reagents: KOH and α-naphthol
 (+) RED ; (-) yellow/copper-like

4. Citrate utilization
 Organism utilized citrate, producing ammonia and converted
to ammonium hydroxide. This alkaline compound raises the pH
of the medium and takes the blue color.
 Medium: Simmon Citrate Agar (SCA)
 pH indicator: bromthymol blue
 (+) Prussian blue color ; (-) green

5. Urease test
 Determine urease
 Medium: Christensen’s urea or Stuart’s urea Broth
 pH indicator: phenol red
 (+) MAGENTA/pink-red ; (-) no color change

Rapid Urease production (w/in 24°) PPM

o Proteus, Providencia, Morganella
*Note: Not all species of Providencia are (+) for urease.

Late/Slow Urease production (after 4°) CKEYS

o Citrobacter, Klebsiella, Enterobacter, Yersinia, Serratia

6. ONPG
 Rapid test to detect β-galactosidase
 Identification of late lactose fermenter (LLF)
 Substrate: ONPG (o-nitrophenyl-β-D-galactopyranoside)
 (+) YELLOW

7. Phenylalanine deaminase
 Enzyme that removes amino group (NH2) from the amino acid
 (+) GREEN SLANT AND FLUID

Escherichia IMViC ++--

TSI A/AG H2S-
 UTI – 90%
 Sepsis
 Meningitis – causes meningitis in infants
 Diarrheal diseases

Klebsiella IMViC --++

TSI A/AG H2S-
* K. oxytoca +-++
 Exhibit mucoid growth, large polyssacharide capsule
 K. pneumonia – Friedlander’s bacillus, encapsulated and
appears as mucoid colonies that tend to string
 K. ozaenae – purulent sinus infection
 K. rhinoscleromatis – granuloma of the nose and oropharynx

Enterboacter IMViC --++

TSI A/AG H2S-
 Opportunistic infections: UTI, RT and wound infections
 Most predominant isolate is E. cloacae
 E. sakazakii - produces yellow pigmentation and intensifies at
25°C

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