Microbiology Laboratory 7

Salmonella-Shigella Agar
MacConkey Agar
PURPOSE:
PURPOSE: MacConkey agar selects for gram negative bacteria and Salmonella-Shigella medium provides for inhibition of normal flora
also differentiates lactose fermenters (pink-red) from non-lactose coliforms and differentiation of stool pathogens
fermenters(colorless).
PRINCIPLE:
PRINCIPLE: Bile salts inhibit gram positive basteria, which allows for Bile salts inhibit gram positive bacteria, and brilliant green agar and
the isolation of gram negative bacteria. Neutral red and crystal violet bile salts inhibit the gram negative coliforms. Lacotse is the sole
further inhibit the gram positive bacteria. Lactose is the only carbohydrate source. Neutral red indicator is red in acidic conditions.
carbohydrate source. Neutral red indicator is brown in pH 6.8 to 8.0 Lactose fermenters appear pink-red, whereas non-lactose
and pink-red at pH less than 6.8 fermenters appear clear. To detect H2s production, sodium
thiosulfate serves as sulfur source. When H2S is formed, it combines
PROCEDURE: with ferric ammonium citrate to form ferric sulfide (FeS), which is
1. Streak agar for isolation represented by black centered colonies.
2. Incubate at 35-37 °C for 18-24 hours and observe for
PROCEDURE:
growth and color.
1. Streak agar for isolation.
3.
2. Incubate at 35-37 °C for 18-24 hours and observe for
INTERPRETATION: If lactose is fermented, the medium is acidified,
growth and color.
and bile salts are precipitated. The precipitated dye is absorbed,
resulting in a pink-to-red complex.
INTERPRETATION:
Normal flora coliforms = pink to red colonies.
Shigella = colorless colonies without balck centers.
MacConkey Agar – pink colonies
Salmonella = colorless colonies with black centers
(Enterobacter cloacae – rapid lactose
fermenter)

B
A

MacConkey agar – pink colonies

A. Salmonella-Shigella Agar (SSA)
(Escherichia coli – rapid lactose
B. SSA – Salmonella typhii
fermenter)
C. SSA – E. coli
C

MacConkey agar – colorless colonies

(Salmonella typhimurium – nonlactose
Xylose Lysine Deoxycholate (XLD) Agar
fermenter)
PURPOSE:
The XLD is used for the isolation and differentiation of stool
pathogens and inhibition of normal flora coliforms.

PRINCIPLE:
Sodium deoxycholate inhibits gram positive bacteria, partially inhibits
MacConkey Agar Classification according to use: Differential
the growth of E. coli, and inhibits the swarming of Proteus. Phenol
medium for gram negative bacilli
red indicator becomes yellow in acidic environments. Fermentation
of xylose results in yellow colonies. Most members of
INDICATOR: Neutral red
Enterobacteriacae are xylose positive, except Shigella. Most strains
of Shigella cannot ferment lactose and thus produce red colonies.
Lysine positive bacteria first produce yellow coloniesas xylose is
A. MAC showing LFO with pink or colored
fermented, followed by red colonies, indicating lysine
colonies
decarboxylation. H2S positive colonis have black centers due to
Organism used : E. coli
reaction of H2S with ferric ammonium citrate.

PROCEDURE:
1. Streak agar for isolation.
2. Incubate at 35-37 °C for 18-24 hours and observe for
B. MAC showing LFO with mucoid colines
growth and color.
Organism used : Klebsiella
pneumoniae
INTERPRETATION:
Salmonella = red colonies with black centers
Citrobacter and Proteus = yellow colonies with black centers

Differential capabilities
A of XLD agar for lactose-
C. MAC showing NLFO with colorless transluscent C fermenting, gram
negative bacilli (e.g E.
colonies
Organism used: Proteus vulgaris
B coli, arrow A), nonlactose
fermenters (e.g Shigella
spp., arrow B) and H2S
producers (e.g Salmonella
spp., arrow C)

D. MAC
Organism used: Enterobacter
Hektoen Enteric Agar

PURPOSE: Hektoen enteric medium selects for stool pathogens by

inhibiting the normal flora of the lower GI tract.
 PRINCIPLE: C. EMB showing NLFO with colorless
A high concentration of bile salts inhibits gram positive bacteria and transluscent colonies
gram negative coliforms. Lactose, sucrose, and salicin are Organism used: Proteus vulgaris
carbohydrate sources. Bromthymol blue indicator has the following
pH ranges:
> 7.6 – blue
6.0-7.6 – green
< 6.0 – yellow
Sodium thiosulfate is the sulfur source of H2S detection. H2S
combines with ferric ammonium citrate to form ferric sulfide (FeS),
which is represented by black-centered colonies. If one, two, or three UREASE TEST
of the carbohydrates are fermented, the colonies are orange in color.
Nonfermenters produce green colonies. INDICATOR: Phenol red

PROCEDURE: MEDIUM: Urea broth

1. Streak agar for isolation.
2. Incubate at 35-37 °C for 18-24 hours and observe for PRINCIPLE: Microorganism that posses the enzyme, urease,
growth and color. hydrolyze urea to ammonia and CO2. Phenol red turns fuschia in the
presence of alkaline end products.
INTERPRETATION:
Pathogens = green colonies or green colonies with black centers
Normal flora (except Yersinia enterocolitica, which produce yellow
colonies due to its fermentation of sucrose) = yellow colonies

Differential capabilities of A. Uninoculated : Salmon pink

HE agar for lactose-
fermenting, gram negative
bacilli (e.g E. coli, arrow A),
B nonlactose fermenters (e.g
Shigella spp., arrow B) and
C
H2S producers (e.g B. Positive result: Fuschia pink
Salmonella spp., arrow C)

Eosin Methylene Blue (EMB) Agar

PURPOSE: C. Negative result: Salmon pink

The EMB medium selects for gram negative bacteria and also
differentiates lactose fermenters (purple color to “green metallic
sheen”) from non-lactose fermenters (colorless)

PRINCIPLE:
Eosin and methylene blue are dyes that inhibit the gram positive Urease Reaction
bacteria. Lacotse is the only carbohydrate source in most
formulation. PURPOSE: Urease is an enzyme that splits urea into alkaline end
products. The reaction is useful in the identification of rapid urease
PROCEDURE: producers, such as Proteus and Morganella, as well as weak urease
Streak agar for isolation. producers, such as Klebsiella pneumoniae and some species of
1. Incubate at 35-37 °C for 18-24 hours and observe for Enterobacter.
growth and color.
PRINCIPLE: Urease splits the urea molecule into ammonia (NH3),
INTERPRETATION: If lactose is fermented, precipitated eosin and carbon dioxide (CO2), and water (H2O). Ammonia reacts in solution
methylene are absorbed resulting in a purple color medium. A to form an alkaline compound, ammonium carbonate, which results
classical “green metallic sheen” is produced by E. coli, which is a in an increased pH of the medium and a color change in the indicator
rapid lactose fermenter. Non-lactose fermenters produce colorless to pink-red.
colonies on EMB.
Urea + 2H2O Urease CO2 +H2O + 2 NH3
Eosin methylene blue agar –
greenish metallic sheen (E. coli –
rapid lactose fermenter)

(NH4)2CO3
Ammonium carbonate

Media and Reagents: Christensen’s (urea) agar tubes or Stuart

urea broth

Eosin Methylene Blue Agar Classification according to use: PROCEDURE:

Differential medium for gram negative bacilli 1. Using Stuart broth, dissolve one urea disk in 1 ml sterile,
distilled water.
INDICATOR: Eosin and methylene blue 2. Using single, isolated, 18-24 hour colony and streak slant of
agar tube or inoculate broth.
3. Replace cap loosely or parafilm broth.
owing LFO with greenish metallic sheen Incubate at 35°C for 18-24 hours.

used : Escherichia coli Urease Test (Christensen’s Agar)

A. Positive – rapid urease activity: red

throughout meadium
B. Positive – slow urease activity: red in
slant
B. EMB showing LFO with mucoid colines C. Negative – no urease activity: remains
Organism used : Klebsiella pneumoniae yellow
Urease Test (Stuart Urea Broth) INDOLE BROTH

1. Uninoculated control PURPOSE:

2. Positive – red color in medium Indole broth is used for distinguishing Enterobacteriaceae based on
3. Negative – no color change (buff to pale the ability to produce indole from tryptophan. The test is particularly
yellow) useful for the identification of lactose-fermenting member of
Enterobacteriaceae. Escherichia coli is indole positive, whereas
Enterobacter and Klebsiella are indole negative. Indole is also useful
in the inspection of Proteus. P. mirabilis is indole negative, P. vulgaris
is positive.

PRINCIPLE:
Tryptophan present in peptone is oxidized by sertain bacteria to
indole, skatole, and indole-acetic acid. The intracellular enzymes that
metabolize tryptophan are known as tryptophanse. Indole is detected
in broth cultures of bacteria with an alcoholic p-
dimethlyaminobenzaldehyde reagent. Indole reacts with the
INTERPRETATION: acetaldehyde to form a red product. Two reagents may be used to
detect indole, Kovac’s and Ehrlich’s. Ehrlich’s reagent is believed to
Stuart Broth be more sensitive than Kovac’s reagent and is recommended for
Positive: red color in medium indole detection in anaerobes and nonfermentative bacteria. Kovac’s
Negative: no color change (buff to pale yellow) reagent was initially used to classify members of the
Strong positive reactions are seen only with Proteus spp. Enterobacteriaceae and should be used with these organisms.
(Proteus and Morganella) and may be interpreted as
early as after 4 hours of incubation. Tryptophan Tryptophanase indole +
Weakly positive reactions (pink to orange) may be seen Pyruvic acid +
with K. pneumoniae and other slow urease ammonia
producers. Indole +
p-dimethylaminobenzaldehyde Red
Christensen’s agar
Positive: (rapid urease activity) red throughout medium REAGENTS AND MEDIA:
(seen only with Proteus and Morganella) Tryptophan (1%) broth
Positve: (slow urease activity) red in slant (K. Kovac’s reagent or Ehrlich’s reagent
pneumoniae) Xylene or chloroform for extraction if using Ehrlich’s reagent
Negative: (no urease activity) medium remains yellow
PROCEDURE:
QUALITY CONTROL: 1. Inoculate indole broth
Proteus vulgaris – positive: rapid urease producer 2. Replace cap loosely and incubate at 35°C for 18-24 hours.
K. pnemoniae – positive: slow urease producer 3. Add 5 drops of Kovac’s reagent directly to the broth culture.
E. coli – negative: no urease production Observe for red color in the upper alcohol layer.
4. If using Erlich’s reagent, first add 1 ml xylene or chloroform to
the broth culture. Shake gently and then add 5 drops of the
reagent.
Urease (+) Slow
INTERPRETATION:
Negative reaction: no color development
Positive reaction: red ring at the interface of the reagent and broth
(or reagent and xylene or chloroform)
Variable reaction: orange color, indicates production of skatole, a
methylated intermediate that may be a precursor to indole
production.
SIM Medium (Sulfur Reduction, Indole Production, Motility)
QUALITY CONTROL:
1. SULFUR REDUCTION
E. coli – positive control (red ring)
Enterobacter cloacae – negative control (no color development)
PURPOSE: This test is used to identify those bacteria capable of
reducing sulfur. This is particularly important in differentiating certain
3. MOTILITY (Test)
general enteric orhanisms.
PURPOSE:
PRINCIPLE: Hydrogen sulfide, H2S, can be formed by putrefaction
This procedure determines the motility of the bacteria through
or anaerobic respiration. The medium contains cysteine, an amino
semisolid media. Shigella and Klebsiella are the only nonmotile
acid containing sulfur, and sodium thiosulfate plus peptonized iron or
Enterobacteriaceae. Yersinia enterocolitica is nonmotile at 37°C but
ferrous sulfate. H2S will react with the iron or ferrous sulfate, forming
is motile at 22°C.
a black precipitate. If the black precipitate is present, the test is
positive for H2S production. No precipitate is a negative test.
PRINCIPLE:
The medium contains a small amount of agar, which allows motile
2. INDOLE PRODUCTION
bacteria to move out from the line of inoculation. Nonmotile
organisms grow only along the line of inoculation; 1%
PURPOSE: The indole test is used to identify bacteria capable of
triphenytetrazolium chloride may be added to medium to aid
producing indole using the enzyme tryptophanase. It is one
visualization of the reaction. Bacteria incorporate this colorless dye
component of the IMVIC tests for differentiating Enterobacteriaciae.
and reduce it to a red pigment. Thus reddening of the medium can
be used as an indication for the extent of bacterial growth.
PRINCIPLE: The enzyme tryptophanase can convert the amino acid,
tryptophan, to indole, ammonia and pyruvic acid. The by-product,
PROCEDURE:
idole, is the metabolite identified by this test. When Kovac’s reagent,
1. With a sterile inoculating needle, select one colony and stab
which contains hydrochloric acid and dimethylaminobenzaldehyde
the needle to the bottom area of the agar.
and amyl alcohol, a red layer will form when indole is present. No
2. incubate at 35°C for 18-24 hours, and examine for growth
color in this layer is a negative result.
around the line of inoculation.
Indole production. A. Postive – red ring
INTERPRETATION:
at the interface of the reagent and broth;
Motile – diffuse growth extending laterally from line of inoculation
B. Negative – no color development
Nonmotile – growth only along line of inoculation

QUALITY CONTROL:
Proteus mirabilis – motile
Klebsiella pneumoniae – nonmotile
 Motility test. 1 and 3 – Motile – diffuse growth E. Ornithine decarboxylation test: negative –
extending laterally from line of inoculation indicated yellow
by turbidity of the medium
2 – nonmotile – growth only along the line of F. Indole production test:
inoculation positive – red ring

G. Indole production test: negative – absence of

red rng

MOTILITY (Medium) NOTE:

1. Do not shake because the medium is semisolid.
PURPOSE: This medium can be stab-inoculated with an inoculating 2. Kovac’s reagent for indole test should be added after reading the
needle to indicate motility. motility and ornithine decarboxylation reaction.

PRINCIPLE: The lower agar concentration in the medium allows

limited movement of motile bacteria from the area of stab. Motility Lysine Iron Agar (LIA)
will be detectable as diffuse growth radiating from the stab line.
Purpose
LIA can be used to determine the ability of the organism to
SIM medium control deaminate lysine, decarboxylate lysine and produce H2S gas. It is
useful in the identification of Salmonella, Proteus, Providencia, and
Morganella. Members of the Proteus group (Proteus, Providencia
and Morganella) are the only members of the Enterobacteriaceae
that are deaminase positive.

Nonmotile with no H2S Principle

LIA contains a small amount of protein, glucose, lysine, sulfur, H2S
indicator, agar, and the pH indicator bromcresol purple. As glucose
fermentation occurs, the deep of the tube turns yellow. Lysine
decarboxylation produces alkaline cadaverine and leads to
reversion of the deep from yellow to purple.

Motile with H2S production Lysine deamination occurs in the presence of oxygen (on the slant)
and results in production of a red color. H2S production is noted by
a black precipitate in the deep as H2S gas reacts with ferric
ammonium citrate.

Media
LIA slants

A – positive indol red ring at the interface Procedure

B – negative indol; no color development 1. inoculate LIA by using straight wire to stab the dep (?)
and to streak the slant.
2. incubate at 35C for 18 to 24 hours, if necessary, incubate
for 48 hours.

Interpretation
• Lysine decarboxylase positive: purple/purple
• Lysine decarboxylase negative: purple/yellow
MOTILITY INDOLE ORNITHINE MEDIUM (MIO) • Deaminase positive: red/yellow
• H2S positive: blackening
Indicator: Bromcresol purple
Indicator: Bromcresol purple
Classification according to consitency: semisolid
• Acid: Yellow
Results to be observed: • Alkaline state: Purple
1. motility
2. indole production Results to be observed:
3. ornithine decarboxylation 1. deamination on the slant portion only:
• positive: red slant
A. Uninoculated: Cadaverine color • negative: purple slant
2. Lysine decarboxylation on the butt portion only:
• positive: purple butt
• negative: yellow slant
A. Uninoculated: Cadaverine color
B. Motility Test: Motile – showing a diffused B. K/K
growth or haziness in the medium
• alkaline slant (purple): negative deamination
• alkaline butt (purple): positive lysine decarboxylation

C. K/A
• alkaline slant (purple): negative deamination
• acid butt (yellow): negative lysine decarboxylation

C. Motility test: Nonmotile – showing growth D. R/Y or R/A

along the stabbing line • red slant: positive deamination
• acid butt (yellow): negative lysine decarboxylation

Lysine iron agar.

A. Alkaline slant/alkaline
butt (K/K).
D. Ornithine decarboxylation test: positive – B. Alkaline slant/alkaline
butt, H2S positive (K/K H2S
purple
+).
C. Alkaline slant/acid butt
(K/A).
D. Red slant/acid butt (R/A). Lactose fermentation requires two enzymes: lactose permease,
E. Uninoculated tube which actively transfers lactose into the bacterial cell, and beta-
Decarboxylase Reactions galactosidase, which degrades lactose into glucose and galactose.
Non-lactose fermenters lack both enzymes, and those known as
Purpose: slow or late lactose fermenters possess the beta-galactosidase but
Moeller decarboxylase medium is used for determining the lack the permease. Lactose fermenters possess the beta-
production of decarboxylase by bacteria. galactosidase but lack the permease. Lactose fermenters possess
both enzymes.
Priniciple:
The decarboxylases are enzymes that attack the carboxyl group of ONPG is useful in detecting late lactose fermenters because the
specific amino acids, forming amines and carbon dioxide. The ONPG molecule is structurally similar to lactose. ONPG can enter
amines formed are alkaline, and they alter the color of the pH the bacterial cell without a permease. In the presence of beta-
indicator. galactosidase, ONPG (colorless) is converted into galactose and O-
The amino acid to be tested is added to the Moeller base medium nitrophenyl, which is yellow chromogen and the alkaline end
in a 1% concentration. Each decarboxylase reactions is specific for product.
a particular amino acid. Tests for lysine decarboxylase, ornithine
decarboxylase, and arginine dihydrolase are generally performed Media and Reagents
on the enteric bacteria. Lysine is carboxylated to cadaverine; • ONPG tablets or disks
ornithine is decarboxylated to putrescine, and arginine undergoes a
• Sterile distilled water
dihydrolase reactions to form citrulline, which is then converted to
• Sterile 1.0 mL pipettes
ornithine in a decarboxylation.

Media and Reagents Procedure

Moeller decarboxylase broths containing 1. if using ONPG tablets, dissolve one tablet in 1.0 ml of
sterile distilled water, ONPG disks are dissolved in 0.5 ml
• 1% lysine
sterile distilled water.
• 1% ornithine 2. mix and allow tablet to dissolve (5 to 10 minutes)
• 1% arginine 3. inoculate with four or five colonies of an 18 to 24 hour
Sterile mineral oil culture. Use sterile needle to select colonies and mix
well
Procedure
1. inoculate test cultures into the tubes of decarboxylase 4. parafilm all tubes and incubate at 35oC for 4 to 6 hours.
media for each amino acid to be tested. Include a control
tube for each organism Interpretation
2. Overlay all tubes with 5-10 mm of sterile mineral oil Positive reaction: yellow color within 20 minutes to 24 hours
Replace cap. Negative reaction: colorless after 24 hours
3. Incubate at 35oC for 24 hours. Quality control
E.coli: positive-yellow
Interpretation Salmonella typhimurium: negative – no
Glucose fermentation indicates the organism is viable and the color change
medium turns yellow.
Decarboxylation is indicated by a blue-purple color in the medium
ONPG (O-nitrophenyl-beta-
Incubate all tubes negative for decarboxylation for another 24 Dgalactopyranoside) test
hours and read again.
A- negative – no color change or colorless
Quality control after 24 hours
All should be read at 24 hours. B- positive – yellow color w/in 20 min to 24 hrs
Arginine
• enterobacter cloacae: positive (purple); alkaline Methyl Red-Voges Proskauer (MR-VP) Tests
• Klebsiella pneumoniae: negative (yellow), acidic
Purpose
Lysine: MR-VP broth is a dextrose broth medium buffered with peptone.
Glucose is fermented to pyruvic acid by one of two pathways,
• Klebsiella pneumoniae: positive (purple), alkaline
which results in either a positive MR or a positive VP test. The tests
• Enterobacter cloacae: negative (yellow), acidic
are particularly useful for the lactose-fermenting
Enterobacteriaceae. Escherichia coli is MR positive and VP
Ornithine: negative, whereas most members of the Klebsiella-Enterobacter-
• Enterobacter cloacae: positive (purple), alkaline Serratia-Hafnia group are VP positive.
• Klebsiella pneumoniae: negative (yellow), acidic
Principle
Control: inoculate the control with the same organisms that are In the first pathway, mixed acid products (lactic, acetic, formic and
tested in the amino aicd tubes. All reactions for the control should succinic) result, leading to a decrease in the pH of the medium and
be negative (yellow), acidic. a positive MR test. The pH must drop to 4.4 or less for the MR
indicator to take on its acidic red color.

Decarboxylase In the second pathway, acetylmethyl carbinol (acetoin) is an

reactions. intermediate product to butylenes glycol. Acetoin is the neutral
product detected in the VP reaction. The broth should be heavily
Positive – purple inoculated, with a small volume of broth used for the VP test to
Negative – yellow obtain favorable results at 24 to 48 hours of incubation.

In the presence of oxygen and 40% potassium hydroxide (KOH),

acetoin is converted to diacetyl form, which results in a red color
in the presence of alpha-napthol.

Metabolism of glucose using MR and VP pathways

ONPG Reaction Glucose

Purpose
The ONPG (o-nitrophenyl-beta-D-galactopyranoside) reaction
determines the presence of late or slow fermenting strains. The Acetoin Pyruvic acid Mixed acid
fermentation
test is useful in detecting late lactose fermenting strains of
Escherichia coli and distinguishing some Citrobacter species and
arizonae subspecies, which are ONPG positive, from similar KOH + air pH < 4.4 (red)
Salmonella subspecies, which are ONPG negative. It is also useful in
speciation of Shigella, since S. sonei is the only ONPG-positive
SHigella species.
Diacetyl + methyl red
Principle

Napthol + creatine
Pink-red complex
Positive
 The medium contains protein sources (beef extract, peptone, yeast
extract, proteose peptone) that permits the growth of most
bacterial strains. Lactose, sucrose, and glucose are present as well
as phenol red indicator. Glucose is in a concentration one-tenth
Media and Reagents that of the other carbohydrates. Ferrous sulfate is present as an
MR-VP broth; glucose base indicator of hydrogen sulfide production.
MR pH indicator
5% alpha-napthol in absolute methyl alcohol The TSI is a two reaction chamber with an aerobic slant portion and
40% KOH containing 0.3% creatine an anaerobic deep portion. The slant of the tube is exposed to
atmospheric oxygen and will become alkaline due to oxidative
Procedure decarboxylation of peptides and amino acids. The slant tends to
1. inoculate medium with a heavy suspension of an 18 to 24 become and remain alkaline (red). Amino acid degradation is
hour culture. minimal in the deep (anaerobic) portion, and thus a small quantity
2. Incubate for 48 hours or until sufficient growth occurs in of acid produced can be detected because few amines are being
broth. formed from amino acids.
3. After 48 hours, split broth by pipetting half into a clean
test tube. Bacteria that ferment glucose, but not lactose or sucrose, only
4. Perform the MR test on one tube: produce small quantities of acid and cannot counteract the
a. Add five drops of MR indicator to the aliquot degradation of amino acids at the slant, which results in an alkaline
with a Pasteur pipettle pH due to oxidative decarboxylation. Such organisms
b. Interpret the color result immediately characteristically produce an alkaline slant over an acid deep (K/A)
5. Perform the VP test on the second aliquot
a. Add 0.6 ml (6 drops) of alpha-napthol reagent Organisms that ferment both glucose and lactose and/or sucrose
to VP aliquot and shake well produce large quantities of acid, which overcome the alkaline
b. Add 0.2 ml (2 drops) of 40% KOH reagent to reaction of the slant, yielding an acid slant over an acid deep
aliquot (A/A).
c. Gently shake tubes for 30 seconds to 1 minute
to expose reaction to atmospheric oxygen. This An organism incapable of fermenting glucose produces no change in
oxidizes acetoin to obtain a color reaction. the indicator and is characterized by an alkaline slant over an
d. Allow tubes to staind at least 10 to 15 minutes alkaline deep (K/K).
before attempting to interpret color results,
although the reaction is often immediate. A sulfur source, sodium thiosulfate, provides sulfur atoms to detect
the production of H2S, H2S reacts with iron salts (ferrous sulfate or
Note ferric ammonium citrate) to produce the black precipitate of
The order of adding reagents is very important. A reversal of the ferrous sulfide.
order may lead to false-negative results
The production of gas during fermentation is indicated by the
Interpretation presence of cracks in the medium or the “pulling away” of the
• positive MR test: distinct red color at surface of the medium from the walls of the test tube.
medium
• Negative MR test: yellow color at surface of the Materials
medium Selected members of Enterobacteriaceae
• Delayed reaction: orange color. Continue incubation Nonfermentative gram-negative bacilli
and repeat test in 4 days. No attempt should be TSI slants
made to interpret an MR test before 48 hours’
incubation, since false-positive results may occur Procedure
• Positive VP test: pink-red color at surface of the 1. use single, isolated 18 to 24 hour colony
2. select colony with sterile needle and stab within ½ inch
medium
of the bottom of the agar
• Negative VP test: yellow color at the surface of the
3. streak colony up slant
medium
• A copper-like color is interpreted as negative, since 4. leave cap on loosely and incubate at 35 to 37oC for 18 to
this is caused by the action of the reagents when 24 hours.
mixed. 5. Read and interpret results

Quality Control Quality Control

• E. coli: MR positive- red, VP negative – no pink-red • Salmonella typhimurium: alkaline/alkaline
color (purple/purple) – H2S positive
• Enterobacter cloacae: MR negative- no color, VP
positive pink-red color • Shigella flexneri: alkalin/acidic (purple/yellow) – H2S
negative
Methyl red test. • Proteus vulgaris: red/yellow

A – positive – distinct red color at Notes

surface of the medium 1. H2S producing strains of Proteus may not blacken this
media
B – negative – yellow color at
2. Morganella morganii does not consistently produce a red
surface of the medium
color after 24 hours incubation

Indicators: Phenol red

Voges Proskauer test
H2S indicators:
ammonium iron citrate, sodium thiosulfate
A – Positive – pink red color at
surface of the medium • acid state – yellow
• alkaline state – red
B – Negative – yellow color at
surface of the medium Sugars present: glucose or dextrose, lactose and sucrose

Results to be observed
1. fermentation of sugar
Triple Sugar Iron Agar (TSIA) 2. production of gas
3. production of hydrogen sulfide
Purpose
TSI agar can be considered an initial step in the identification of A. Uninoculated: Orange butt & slant
the Enterobacteriaceae.

Principle B. A/A G
• acid slant: yellow
 • acid butt: yellow Lactose or sucrose not Serratia, Anaerogenic
• gas production: positive fermented Escherichia coli
o bubbles or displacement of the medium K/K H2S - Glucose not fermented Pseudomonas
• hydrogen sulfide production: negative
o no blackening of the butt Lactose or sucrose not
fermented Alkaligenes
• sugars fermented: glucose/dextrose, lactose and
A/@H2S + Glucose fermented with gas; Citrobacter freundii
sucrose (LFO)
lactose or sucrose fermented
Note: those species of Proteus that ferment sucrose may produce
C. K/A G w/ H2S an acidic slant.
• Alkaline slant: red *A-acid; @- acid and gas; K-alkaline (no change)
• Acid butt: yellow
• Gas production: positive Triple sugar iron agar.
o bubbles or displacement of the
medium A – acid slant/acid butt with gas no
• Hydrogen sulfide production: H2S (A/@)
negative B – alkaline slant/acid butt, no gas,
o no blackening of the butt H2S positive (K/A H2S +)
C – Alkaline slant/no change butt, no
• Sugar/s fermented: glucose/dextrose
gas, no H2S (K/NC)
only (NLFO) D – Uninoculated tube
D. K/K or K/N
• alkaline slant: red
• alkaline or neutral butt: red or orange Simmons Citrate Reaction
• gas production : negative
o no bubbles & no displacement of Purpose
medium The citrate reaction is used to determine if a member of the
• hydrogen sulfide production: negative enterobacteriaceae is capable of utilizing citrate as the sole source
o no blackening of the butt of carbon. No other protein or carbohydrate that might provide
• sugars fermented: none (NLFO) another carbon source must be present in the medium. The
reaction is useful in identification of the lactose fermenting
E. K/A G+ Enterobacteriaceae. Escherichia coli is a citrate negative, whereas
• alkaline slant: red Enterobacter and Klebsiella are positive.
• acid butt: yellow
Principle
• gas production: positive Simmons citrate agar contains sodium citrate, which serves as the
o bubbles or displacement of only carbon source. If the organism can utilize citrate, the sodium
medium citrate is converted to ammonia, which is then converted to
• hydrogen sulfide production: ammonium hydroxide. The alkalinity of indicator takes on its
positive alkaline color, which is blue
o blackening of the butt
• sugars fermented: Medium
glucose/dextrose only (NLFO) Simmons citrate agar

F. A/A G+ Procedure
• acid slant: yellow 1. Use single, well-isolated 18 to 24 hour colony
• acid butt: yellow 2. select colony with sterile needle and streak citrate slant
• gas production: positive lightly
o bubbles or displacement of 3. leave cap on loosely and incubate at 35oC for 18 to 24
the medium hours
• hydrogen sulfide production:
positive Interpretation
o blackening of the butt A positive test is indicated by growth with an intense blue color on
• sugar/s fermented: the slant or solely the presence of growth. Compare to a an
glucose/dextrose, lactose and uninoculated tube for correct interpretation. A negative test is
sucrose (LFO) indicated by the absence of growth and no color change in the
medium (remains green). False positive results may occur with an
inoculum that is too heavy.
G. K/A
• alkaline slant: red Quality Control
• acid butt: yellow Klebsiella pneumoniae: blue with growth – positive
• gas production: negative E. coli: no growth without color change
o no bubbles/no displacement of
the medium Indicator
• hydrogen sulfide production: negative Bromthymol Blue
o no blackening of the butt
Test to be observed:
• sugar/s fermented: glucose only (NLFO)
Citrate utilization test
Quality Control Principle
• Proteus mirabilis: motile – growth extending Utilization of citrate as the sole source of carbon
laterally from line of inoculation
• Klebsiella pneumoniae: non-motile – growth only
along line of inoculation

Summary of TSI Reactions A. Uninoculated: green

Reactions Carbohydrate Fermented Typical Organisms
A/@ H2S Glucose with acid and gas Escherichia,
B. Positive Reaction: Prussian blue
Klebsiella
(left tube)
Lactose and/or sucrose with
acid and gas Enterobacter
K/@ H2S Glucose with acid and gas Salmonella, Proteus

Lactose or sucrose not

fermented Citrobacter
K/A H2S - Glucose with acid; no gas Shigella, Providencia
 C. Negative Reaction: Green

Deaminase Reactions

Purpose
Deaminase activity can be determined using the amino acids
phenylalanine or tryptophan. Only Proteus, Providencia and
Morganella species possess the deaminase enzyme.

Principle
Deamination of the amino acid results in a colored compound with
the addition of 10% ferric chloride (FeCl3):

Phenylalanine Phenylpyruvic acid + 10% FeCl3 (green)

Phenylalanine
deaminase

Tryptophan Indole-pyruvic acid + 10% FeCl3 (brown)

Tryptophan
deaminase

Media and Reagents

Phenylalanine or tryptophan agar or tablets
10% FeCl3
Procedure
1. inoculate the agar slant (or tablet dissolved into 1 ml
sterile distilled water) with a few pure colonies of the
test organism. Replace cap
2. Incubate 24 hours at 35oC
3. Add four to five drops of 10% FeCl3 to the agar surface or
tube. Rotate the tube and mix gently to provide contact
for reagent and media

Interpretation
• appearance of an intense green color indicates a
positive deamination for Phenylalanine
• Appearance of a brown color indicates a positive
deamination for tryptophan.

Quality Control
• Proteus vulgaris: positive (green or brown color with
FeCl3)
• Escherichia coli: negative (no color development
with FeCl3)

Phenylalanine deamination

A – negative – slant remains

colorless

B – positive – appearance of an
intense green color

- fin -

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thanks, leigh, for getting half of the work done!

Hi to 2nd yr vbelles, roch, aina and andree!