METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE

RESULT CONTROL

Catalase Differentiates 1. Use a loop or sterile Positive: Enterococci Positive:

Test catalase-positive wooden stick to transfer Copious produce a Staphylococcus
micrococcal and a small amount of bubbles are peroxidase that aureus
staphylococcal colony growth to the produced slowly catalyzes (ATCC25923)
species from surface of a clean, dry the breakdown of
catalase-negative glass slide. Negative: H2 O2, and the Negative:
streptococcal 2. Place a drop of 30% No or few test may appear Streptococcus
species. hydrogen peroxide (H2 bubbles are weakly positive pyogenes
O2) onto the medium. produced (ATCC19615)
(3% can also be used
for most organisms.)
3. Observe for the
evolution of oxygen
bubbles
Coagulase Differentiate Slide Test - Place a Positive: Clot of Positive:
Test Staphylococcus drop of EDTA rabbit any size Staphylococcus
aureus (positive) plasma on a slide and aureus
from coagulase- emulsify the plasma with Negative: No (ATCC25923)
negative bacterial inoculum. Rock clot
staphylococci the slide and observe for Negative:
(negative) clumping. Staphylococcus
epidermidis
Tube Test – Emulsify (ATCC12228)
bacterial inoculum in a
tubes containing 0.5 mL
of rabbit plasma.
Incubate tube at 35°C to
37°C in ambient air for 1
to 4 hours.
Oxidase Detects presence of Cytochrome Moisten filter paper with Positive: Using a nickel- Positive:
test cytochrome oxidase is the substrate or use a Development of based wires Pseudomonas
oxidase essential in detected using commercial paper disk dark-purple containing aeruginosa
identification of the oxidation of impregnated with color within 10 chromium and
oxidase-negative substrate 1% substrate. Rub a seconds iron to inoculate Negative:
Enterobacteriaceae, tetramethyl-p- bacterial inoculum in the may cause false- Escherichia coli
differentiating them phenylenediam filter paper. Negative: positive result
from other Gram- ine Absence of
negative bacilli dihydrochlorid Observe for color color Microdase test
e to indophenol change within 10 – Modified
producing a seconds. oxidase test
dark-purple used to
product. differentiate
Micrococcus (+)

1
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

to
Staphylococcus
(-) species.
Detection of
oxidase enzyme

Nitrate and Used to determine Microorganisms Inoculate nitrite broth Positive: Nitrate reduction Positive:
nitrate test whether an capable of (usually Brain Heart (Nitrate → nitrite is supportive lest No gas + red-
organism can reducing nitrate Infusion) with inoculum. = no gas, red] for E.coli
reduce nitrates and and nitrite to Place an inverted Enterobacteriace
nitrites to gaseous nitrogen do not Durham tube inside the [Nitrate → ae up to genus Gas + red-
nitrogen or to other turn color and test tube. Incubate for nitrite → level. However, P. aeruginosa
compounds do produce gas 48hours. Examine nitrite nitrogen gas = requires
containing nitrogen. in the nitrite broth for nitrogen gas in red], confirmatory test
reduction test. the Durham tube. Add for final Negative:
Nitrogen nitrate reagents A [Nitrate → identification Acinetobacter
containing (sulfanilic acid) and B nitrogen gas = baumannii
compounds (alpha- gas, no color],
includes naphthylamine). Add
dioxohydrazine zinc dust to detect [Nitrate → other
(N2O2), nitrate (no reduction). nitrogen
ammonia (NH3) compounds = no Proteus mirabilis
or nitric oxide gas, no color can be used as a
(NO). change] positive control

Negative:
Nitrate = turns
red after
addition of zinc
Citrate Used to identify Bacteria that Inoculate bacteria in a Positive: Some organisms Positive:
utilization organisms capable can grow on this Simmon citrate agar Growth on the utilizes citrate Enterobacter
of using citrate as medium slant. Incubate for up to medium, with or even without aerogenes
sole source of produce an 7 days. without a color change. (growth + blue)
carbon and enzyme, citrate- change in the Growth is
ammonium salts permease, color of the considered as
as sole source of capable of indicator. Color positive.
nitrogen. Part of converting change from Negative control:
IMViC test. citrate to green to blue. Escherichia coli
pyruvate. (no growth +
Negative: color change)
Bacteria uses Absence of
citrate to growth
2
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

convert
ammonium ions
to ammonia,
changing the pH
of the media to
alkaline.
Simmon citrate
contains
bromthymol
blue as
indicator.
Acetamide Used to identify Acylamidase Inoculate bacteria in a Positive: Growth without Positive control,
utilization organisms capable enzyme acetamide agar slant. Deamination of color change Pseudomonas
of using acetamide deaminates Incubate for up to 4 acetamide may indicate a aeruginosa
as a sole source of acetamide to days. results to blue positive result. If (growth + blue)
carbon release color further incubation
ammonia, results in no Negative control:
changing the Negative: No color change, Escherichia coli
color of media color change repeat test with (no growth +
from green to less inoculum. green)
royal blue.

Growth at Differentiates The test is used Inoculate two Positive: Positive control:
42°C pyocyanogenic to determine the Trypticase soy agar Good growth at Pseudomonas
Pseudomonas from ability of the slants with both 35°C and aeruginosa
other Pseduomonas organism to Pseudomonas colony. 42°C
spp grow at 42°C. Incubate 1 tube at 35°C Negative control:
Several and the other 1 at 42°C. Negative: Pseudomonas
Pseudomonas Record presence of No growth at fluorescens
spp. Have been growth after 18-24 42°C but good
isolated in the hours. growth at 35°C
laboratory that
are capable of
growth at
elevated
temperature.

3
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

Indole Test Used to identify Tryptophanase Inoculate a drop of Positive: Can be also Positive
organisms that enzyme culture (tryptophan Pink to red- used to control:
produce the enzyme hydrolyzes broth) or stabbing a colored ring differentiate E. coli (Kovac’s),
tryptophanase. Part tryptophan to bacterial inoculum (SIM after addition of organisms under
of IMVIC, but can be indole. Addition medium). Incubate for reagent anaerobic Haemophilus
detected under SIM of Kovac’s 48hours. Add 0.5mL of conditions influenzae
medium. reagent (p- Kovac’s or Ehrlich’s Negative: (Ehrlich’s),
dimethylaminob reagent No color change
enzaldehyde Porphyromonas
and asaccharolytica
hydrochloride) (anaerobic)
reacts with
indole, Negative
producing red control:
color. Ehrlich’s Klebsiella
reagent pneumoniae
contains PDAB (Kovac’s),
and ethyl
alcohol, it Haemophilus
detects small parainfluenzae
amounts of (Ehrlich’s),
indole
Bacteroides
fragilis
(anaerobic)
Methyl Red Differentiales Determines the Inoculate broth culture to Positive: 48hours should Positive:
Voges- Enterobacteriaceae ability to MRVP broth. Incubate MR: bright red, be passed before MR + VP =
Proskauer family produce stable for 48hours. Separate reading because Escherichia coli
Tests acid products into two aliquots. Add 5- MR weakly red- some organisms
from glucose 6 drops of MR to the first orange; will not have Negative control:
fermentation tube. Read immediately. produced MR - VP =
(lactic, acetic, Add 6 drops of alpha- VP red color enough products Enterobacter
formic, naphthol and 2 drops aerogenes
succinic), and KOH to second tube.
ability of some Shake and observe for 5 Negative:
organisms to minutes. MR yellow color,
produce neutral VP: yellow color
end products MR is red (positive
(acetoin, result) at pH 4.4 OR 4.5
butanediol). and yellow (negative
Methyl red result) at pH 6.2.
detects mixed
acid pathway.

4
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

VP pathway
detects ability to
convert acid to
acetoin and
butanediol VP
pathway
organisms uses
small acid
therefore
producing a
negative MR.
VP uses alpha-
naphtol and
40% KOH,
Methyl Red In the first pathway, The pH must
Test mixed acid products drop to 4.4 or
(lactic, acetic, formic less for the MR
and succinic) result, indicator to take
leading to a on its acidic red
decrease in the pH color
of the medium and a
positive MR test.
Voges In the second In the presence
Proskauer pathway, of oxygen and
Test acetylmethyl 40% potassium
carbinol /acetoin is hydroxide,
an intermediate acetoin is
product tobutylene converted to the
glycol. diacetyl form,
which results in
It is the neutral a red color in
product detected in the presence of
the VP reaction. alpha-napthol.

5
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

Urease Test Determines an Urea is the Streak on a urea agar Positive: Urease negative Positive:
(Christense organism’s ability to product of slant or drop an inoculum Light orange to may appear as Proteus
n’s Meth produce the enzyme decarboxylation in a urea broth. Put a pink false positive due vulgaris,
urease, which of amino acids. loose cap and incubate to prolonged
hydrolyzes urea. Hydrolysis of for 48hours to 7 days incubation. weak+:
Proteus spp. May urea yields Negative: Check the Klebsiella
be presumptively ammonia and No color change incubation after pneumoniae
identify to rapidly CO2. Ammonia 24hours for
hydrolyze urea alkalinizes the detection of rapid Negative:
medium urease Escherichia coli
producing pink producers.
color. Rapid Rapid urease
urease-positive producers:
turns the Proteus and
medium to pink Morganella
with 24 hours
of incubation. Weak urease
Uses phenol producers:
red as indicator. Klebsiella
pneumoniae
and
Oxidative- Differentiates Non- Two lubes of OF medium Positive (in open Slow-growing Fermenter
Fermentati microorganisms fermentative is inoculated with deep tube): organisms may Escherichia coli
ve Test based on the ability bacteria are stabs. Overlay one of the yellow indicates not produce
to oxidize or ferment routinely tested tubes with mineral oil. oxidation of results for Oxidizer:
specific for their ability Incubate for up to 7 days carbohydrates several days Pseudomonas
carbohydrates to produce acid aeruginosa
from six Positive (in
carbohydrates mineral oil):
(glucose, yellow indicates
lactose, fermentation of
sucrose, carbohydrates
maltose, xylose,
mannitol). Two Negative (in
tubes are open tube):
inoculated, one no color change
is overlaid with (non-oxidative)
mineral oil for
anaerobic Negative (in
metabolism mineral oil)
no color change
(non-
fermentative)

6
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

OF medium Glucose
fermenter:
When acid
production is
detected on
both tubes since
fermentation
can occur with
or without
oxygen

Glucose
oxidizer: Acid is
detected by the
open aerobic
tube

On-utilizer:
Some bacteria
do not use
glucose as a
substrate)
Gelatin It is used as a Organisms that Organisms that Positive: Some organisms Positive.
Hydrolysis presumptive test for produces produces extracellular partial or total grows poorly or Bacillus subtilis
identification of extracellular proteolytic enzyme liquefaction at do not grow at all
various organisms, proteolytic (gelatinases) can liquefy the top at 4°C in this medium Negative:
including enzyme gelatin. Gelatin is liquid Escherichia coli
Staphylococcus (gelatinases) Negative: above 20°C,
spp., can liquefy complete therefore Uninoculated QC
Enterobacteriaceae gelatin. solidification of determination tube:
, and some Gram- the tube at 4°C of results must medium
positive bacilli be completed becomes solid
alter after
refrigeration refrigeration

7
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

Phenylalani Detects the ability of Phenylalanine Inoculate phenylalanine Positive: Positive:

ne the organism to deaminase slant with a drop of broth Green color Proteus
Deaminase oxidatively removes the culture. Incubate for 18- after addition of mirabilis
Test deaminate amine from 24 hours. Add 5 drops of ferric chloride'
phenylalanine to phenylalanine. 10% aqueous ferric Negative:
phenylpyruvic acid. chloride. Negative: Escherichia coli
The genera The reaction No color change
Morganella, Proteus results in
and Providencia can production of
be differentiated ammonia and
from the other phenylpyruvic
members of the acid.
Enterobacteriaceae.
The PPA is
detected by
adding few
drops of 10%
ferric chloride
forming a green
colored
complex.
Salt Determine the ability The salt Inoculate bacteria in BHI Positive: Positive:
Tolerance of an organism to tolerance test is broth. Incubate for 48 Visible turbidity Proteus
Test grow in high a selective and hours. in the broth, with mirabilis
concentrations in differential or without color
salt. Differentiates medium. change (purple Negative:
Enterococci from Enterococci are to yellow) Escherichia coli
nonenterocci resistant to high
salt Negative:
concentrations No turbidity and
6.5% NaCI no color change
(BHI) is used as
test medium
Broth contains
small amount of
glucose and
bromocresol
purple as
indicator

8
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

Motility These tests are Inoculum is Stab an inoculum Positive: Motile Positive:
Testing used to determine stabbed into the straight into an agar organism will Escherichia coli
whether an enteric center of the medium at a depth of spread out from
organism is motile. deep agar, 1/3-1/4 inch (preferably the site of Negative:
An organism must Bacterial motility SIM). Incubate and inoculation Staphylococcus
have flagella to be is evident by a examine daily for up to 7 aureus
motile. diffuse zone of days Negative:
growth from the remains at the
line of site of Inoculum
inoculation.
Some grows
throughout the
entire medium,
where some
appears as
nodules or
pellicle
Deoxyribon DNase differentiates DNase Inoculate DNase agar Positive: Positive:
ucleic acid organisms based on produced by the with the organism to be Green to Staphylococcus
Hydrolysis the production of organism tested and streak for colorless around aureus
hydrolyzes isolation. Incubate the streak.
deoxyribonuclease. DNA. The aerobically for 13- Negative:
It is used to medium is pale 24hours Negative: Escherichia coli
distinguish Serratia green because Remains green
spp from of DNA-methyl
Enterobacter spp, green complex.
Staphylococcus Fading of green
aureus from other to colorless
species; Moraxella Indicates DNA
catarrhalis from hydrolysis.
Neisseria spp
CAMP Test The purpose of Certain Streak S. aureus. down Positive: A small Positive:
Christie, Atkins and organism the center of the sheep Enhanced percentage of Streptococcus
Munch-Peterson is produces blood agar plate. Streak hemolysis group A agalactiae
to differentiate group extracellular test organism across the indicated by streptococci may
B streptococci from hemolytic plate perpendicular to arrow head- have a positive Negative:
other streptococcal protein that the S. aureus streak. shaped CAMP reaction Streptococcus
species. Listeria synergistically Incubate overnight. hemolysis at the pyogenes
monocytogenes also with beta-lysin juncture of the
exhibits positive of S. aureus on two organisms.
CAMP reaction sheep blood
agar. Organism
is streaked

9
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

perpendicular to Negative: Reverse CAMP

the S. aureus No test- used to
streak. Observe enhancement of differentiate
zone of hemolysis Clostridium
hemolysis near perfringens from
the proximity of other Clostridium
streak spp. Group B
strep is streaked
and Clostridium
spp is
perpendicularly
streaked to the
primary streak.
Observe for
double
arrowhead
hemolysis
Bile Differentiates Bile solution On a SBAP colonized Positive: Enzyme activity Positive control:
Solubility Streptococcus (sodium with bacteria, placing Disintegration of may be reduced Streptococcus
Test pneumoniae (+) deoxycholate) one to two drops of 10% colonies in old cultures. pneumoniae
from alpha- rapidly lyses sodium deoxycholate. Therefore,
hemolytic pneumococcal For a tube test, use a Negative: Intact negative results Negative control:
streptococci (-) colonies. 2% sodium colonies with colonies Enterococcus
Presence of deoxycholate. Incubate should be tested faecalis
autolytic for 30mins Examine for for further
enzyme lysis of colony. identification
amidase
initiates lysis but
bile salts lowers
adhesion of
bacterial cell to
the colony, thus
accelerating the
bacterial natural
autolytic
process

10
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

Bile Presumptive test for Gram (+) In a bile esculin slant, Positive: As a result of Positive control:
Esculin Enterococci and bacteria other inoculate one to two Growth and nutritional Enterococcus
Test than some colonies. Incubate for blackening requirements, faecalis
Streptococcus bovis streptococci and 48hrs some may or
group. (Enterococci enterococci are Negative: may not grow. Negative control:
and Group D from inhibited by bile With or without Streptococcus
non-group D salts. growth and no pyogenes
viridans Organisms that blackening
streptococci) grow on 4% bile
and able to
hydrolyze
esculin to
esculetin will
demonstrate
growth. Ferric
ammonium
citrate reacts
with esculetin to
form brown-
black
precipitate.

PYR test L-pyrrolidonyl The enzyme Moisten the reagent disk Moisten the Positive:
arylamidase test is hydrolyzes L- with reagent grade reagent disk Enterococcus
presumptive pyrrolidonyl- water or put 1mL with reagent faecalis
identification of Bnaphthylamide reagent water in a sterile grade water or
group A streptococci to produce B- test tube. Inoculate a put 1mL reagent Streptococcus
and Enterococci by naphthylamine. small amount of water in a sterile pyogenes
the presence of The product can colonies Incubate at test tube.
enzyme L- be detected in room temp for 2 Inoculate a Negative:
pyrrolidonyl presence of minutes. Add the small amount of Streptococcus
arylamidase. N,N- detector Observe for red colonies agalactiae
methylamino- color Incubate at
cinnamaldehyde room temp for 2
resulting in minutes. Add
production of the detector
red color Observe for red
color

11
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

ONPG test To determine the Two enzymes Broth method of testing, Positive: Cultures that
(O- presence of late or required for the organism is taken Development of naturally produce
nitrophenyl slow fermenting lactose from a medium a yellow a yellow pigment
-beta-D- strains. To detect the fermentation containing a high coloration cannot be tested
galactopyra late lactose Lactose concentration of lactose (presence of ẞ- with this media
noside) fermenting strains of permease: and is inoculated into galactosidase)
Escherichia coli. actively the ONPG Broth. If the Note: The fluid All organisms
transfers organism possesses and disc will turn tested must be
To distinguish some lactose into the bela-galactosidase, the any shade of inoculated from a
Citrobacter species bacterial cell enzyme will split the yellow if positive lactose-
and Arizonae Beta beta- galactoside bond, for containing
subspecies (ONPG galactosidase: releasing o-nitrophenol galactosidase medium (e.g.,
positive) from similar degrades which is a yellow- enzyme. TSI or
Salmonella lactose into colored compound. This MacConkey).
subspecies (ONPG glucose and indicates a positive test. Negative:
negative) To galactose No color
speciale Shigella, Disk method, the development
since Shigella Lactose organism to be tested is (absence of
sonnei is the only fermenters taken from a medium enzyme)
ONPG-positive possess both containing a high
Shigella species enzymes concentration of lactose.
A dense suspension
Slow or late (turbidity equivalent to a
lactose McFarland 3) is
fermenters no prepared An ONPG disk
permease only is added to 0.5ml of the
beta suspension. If the
galactosidase organism possesses
Non lactose bela-galactosidase, the
fermenters: lack enzyme wall
both enzymes
X and V Differentiate The test Prepare a suspension Positive Growth X growth: Positive:
Factor Haemophilus spp. organism is comparable to 0.5 around the disk Haemophilus Growth around
Haemophilus are usually streaked MacFarland and swab shows ducreyi the XV disk only
fastidious into BHI, tryptic the entire surface of the shows a
organisms. Some soy agar or media with the X and V growth: requirement for
require X factor nutrient agar. suspension. Place the Haemophilus both factors
(hemin), some Impregnated growth factor disks on influenzae and Growth around
require V factor disks with X, V the agar surface. Parainfluenzae the V disk, no
(nicotinamide and XV factors Incubate for 24 hours. growth around
adenine dinucleotide are placed the X disk, and
[NAD]) or both. directly on the light growth
inoculum, around the XV

12
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

allowing disk shows a V

diffusion of factor
growth factors requirement
The organism
will grow around
the disk that Negative:
provides Growth over the
appropriate entire surface of
growth factor the agar
indicates no
requirement for
either X or V
factor
Novobiocin Presumptive Novobiocin Streak colonies into Positive: Test should be Positive:
Susceptibili identification and interferes with BAP II MHA is used, Any zone of performed on Susceptible
ty differentiation of DNA during standardize a bacterial inhibition catalase-positive, Staphylococcus
coagulase negative replication by isolate in MacFarland >16mm indicate coagulase- epidermidis
Staphylococci binding with standard. Place a susceptible negative cocci
DNA gyrase and Novobiocin disk and Negative:
blocks ATPase gently lap the disk to Gives misleading Resistant
activity. allow adhesion. Negative: results on urinary Staphylococcus
Novobiocin Incubate for 18-24 Any zone of inocula saprophyticus
impregnated hours. inhibition
disks (5ug) <16mm It is produced by
inhibits the resistant Streptomyces
growth of niveus
susceptible
organisms
Bacitracin Presumptive Bacitracin Streak two to three Positive: Performance Positive:
Susceptibili identification and inhibits suspected colonies from Any zone of depends on the Streptococcus
ty differentiation of synthesis of a pure culture onto BAP inhibition integrity of the pyogenes,
bela-hemolytic bacterial cell Place a Bacitracin disk >10mm indicate disk. Disks must Micrococcus
group A streptococci walls TaxoA in the heaviest streak susceptible be properly luteus
(S. pyogenes) from disks (1st quadrant) Gently stored
other beta-hemolytic impregnated tap the disk to allow Negative: Negative:
streptococci Can be with small adhesion. Incubate for No zone of MR-VP+
also used to identify amounts of 18-24 hours preferably inhibition, Streptococcus
staphylococci Bacitracin (0.04 with 5- 10% carbon resistant agalactiae,
species from units) is placed dioxide Staphylococcus
Micrococci(inhibit on agar plate, aureus
allowing the
diffusion of Bacitracin is
antibiotic and produced by

13
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

inhibits the Bacillus

growth of licheniformis or
susceptible Bacillus subtilis
organisms. var Tracy
Optochin Optochin Optochin Streak two to three Positive: Any Equivocal Positive:
Susceptibili (ethylhydrocupreine interferes with suspected colonies from zone of interpretation: Streptococcus
ty hydrochloride) lyses the ATPase and a pure culture onto BAP inhibition <14mm zone of pneumoniae
pneumococci production of Place an Optochin disk >14mm indicate inhibition is
(susceptible) but ATP in in the media. Gently tap susceptible questionable for Negative:
alpha-hemolytic microorganisms, the disk to allow some strains Streptococcus
streptococci are Optochin- adhesion. Incubate for Negative: pneumococci pyogenes
resistant impregnaled 18-24 hours preferably No zone of and considered
disks (TaxoP) is with 5% carbon dioxide. inhibition, as susceptible,
diffused on BAP, Measure the zone of resistant can be confirmed
zone of inhibition by a positive bile
inhibition solubility test.
indicales
susceptibility of
the organism
Cetrimide This is test primarily This is used to Inoculate a cetrimide Positive: Some enteric Positive control:
Agar used to isolate and determine the agar slant with one drop Growth with bacteria exhibits Pseudomonas
purify Pseudomonas ability of an of BHI broth culture. variation in color a weak yellow aeruginosa
aeruginosa from organism to Incubate for up to 7 of colonies color which can
contaminated grow in the days. be distinguished Negative control:
specimens presence of Negative: with production Escherichia coli
cetrimide, a No growth of fluorescein.
toxic substance
that inhibits the
growth of many
bacteria by
causing the
release of
nitrogen and
phosphorus.

14
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

Decarboxyl This test is used to This detects the Standardize bacterial Positive: The fermentation Positive control
ation Tests differentiate enzymatic suspension in 0.5 Alkaline of dextrose in (yellow to
decarboxylase- ability of an MacFarland and (purple) color medium causes purple):
producing organism to inoculate each of the change. acid change,
Enterobacteriaceae decarboxylate three decarboxylase howeve it would Lysine =
from other gram- ar amino acid to broths (arginine, lysine, Negative: No not mask the Klebsiella
negative rods form an amine. ornithine). Add a 4mm color change. alkaline color pneumoniae,
Decarboxylation mineral oil in each tube. change broth by
or hydrolysis of Incubate and observe decarboxylation Ornithine =
an amino acid for 24, 48 72, 96 hours. reaction. Enterobacter
results in aerogenes;
alkaline pH and
a color change Arginine =
Pseudomonas
aeruginosa

Negative
control
(yellow):

Lysine =
Citrobacter
freundii;

Ornithine =
Proteus
vulgaris;

Arginine =
Escherichia coli
MUG Test 4-methylumbelliferyl- E. coli and other Wet the reagent disk Positive: Do not test Positive control:
B-d-glucuronide test Enterobacteriac with reagent grade Electric blue colonies isolated Escherichia coli
is used to eae produces water and rub a colony, fluorescence from media
presumptively enzyme B-d- preferably placed in a containing dyes. Negative control:
identify various glucuronidase test tube. Incubate for Only test on Klebsiella
genera of which up to 2 hrs. Observe the Negative: oxidase-positive pneumoniae
Enterobacteriaceae hydrolyzes B-d- disk under 366nm UV Lack of organisms
and verotoxin- glucopyranoside light. fluorescence because
producing -uronic negatives
Escherichia coli. derivatives to naturally
aglycons and d- fluoresce
glucuronic acid.
The MUG

15
METHODS PURPOSE PRINCIPLE METHOD EXPECTED LIMITATION QUALITY PICTURE
RESULT CONTROL

impregnated
into the disk is
hydrolyzed by
the enzyme to
yield 4-
methylumbellifer
yl moiety which
fluoresces blue
under long UV
wavelengths.
However,
verotoxin-
producing
strains of E. coli
(0157:H7) do
not produce
MUG and
negative results
indicates
presence of
clinically
important strain.

16