ACETAMIDE UTILIZATION
PRINCIPLE
This test is used to
determine the ability of an
organism to use acetamide
as the sole source of
carbon. Bacteria that can
grow on this medium
deaminate acetamide to
release ammonia. The
production of ammonia
results in a pH-driven color
change of the medium
from green to royal blue.
PRINCIPLE
This test is used to
determine if an organism
can use acetate as the sole
source of carbon. If so,
breakdown of the sodium
acetate causes the pH of
the medium to shift
toward the alkaline range,
turning the indicator from
green to blue.
PRINCIPLE
This test is used to
determine the effect of a
small amount of bacitracin
(0.04 U) on an organism.
Streptococcus pyogenes is
inhibited by the small
amount of bacitracin in the
disk; other beta-hemolytic
streptococci usually are
METHOD EXPECTED RESULTS QUALITY CONTROL
1. Inoculate acetamide slant Positive: Deamination of the acetamide resulting in a blue color
lightly with a needle using (+) Positive:
growth from an 18- to 24- Negative: No color change Pseudomonas
hour culture. Do not aeruginosa
inoculate from a broth
culture, because the growth
will be too heavy. (-) Negative:
Stenotrophomonas
2. Incubate at 35° C for up to
maltophilia
7 days.
ACETATE UTILIZATION
METHOD EXPECTED RESULTS QUALITY CONTROL
1. With a straight inoculating Positive: Medium becomes alkalinized (blue) because of the growth
needle, inoculate acetate of the organism (A). (+) Positive:
slant lightly from an 18- to Escherichia coli
24-hour culture. Do not Negative: No growth or growth with no indicator change to blue
inoculate from a broth ( B). (-) Negative:
culture, because the growth Shigella flexneri
will be too heavy.
2. Incubate at 35° C for up to
7 days.
BACITRACIN TEST
METHOD EXPECTED RESULTS QUALITY CONTROL
1. Using an inoculating loop, Positive: Any zone of inhibition around the disk (A).
streak two or three (+) Positive:
suspect colonies of a pure Negative: No zone of inhibition (B). Streptococcus
culture onto a blood agar pyogenes
plate.
2. Using heated forceps, (-) Negative:
place a bacitracin disk in
Streptococcus
the first quadrant (area of
agalactiae
heaviest growth). Gently
 tap the disk to ensure
adequate contact with the
agar surface.
3. Incubate the plate for 18
not.
to 24 hours at 35°C in
ambient air.
4. Look for zone of inhibition
around disk.
BILE ESCULIN AGAR
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
Gram-positive bacteria 1. Inoculate one to two Positive: Blackening of the agar slant (A).
other than some colonies from an 18- to 24- (+) Positive:
streptococci and hour culture onto the surface Negative: No blackening of medium (B). Enterococcus
enterococci are inhibited of the slant. faecalis
by the bile in this medium.
If streptococci and 2. Incubate at 35° C in (-) Negative:
enterococci can grow in ambient air for 48 hours.
Streptococcus mitis
the presence of 40% bile
and hydrolyze esculin, they
subsequently turn the
indicator, ferric
ammonium citrate, a dark
brown color. This dark
brown color results from
the combination of
esculetin (the end product
of esculin hydrolysis) with
ferric ions to form a
phenolic iron complex.
BILE SOLUBILITY TEST
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
The bile solubility test 1. Place 1 to 2 drops of 10% Positive: Colony disintegrates; an imprint of the lysed colony may
differentiates sodium desoxycholate to the remain within the zone (A). (+) Positive:
Streptococcus side of a young (13- to 24- Streptococcus
pneumoniae (positive) hour), well-isolated colony Negative: Intact colonies (B). pneumoniae
from alpha-hemolytic growing on 5% sheep blood
streptococci (negative). agar. NOTE: A tube test is (-) Negative:
Bile or a solution of a bile performed with 2% sodium
Enterococcus
salt, such as sodium desoxycholate.
desoxycholate rapidly lyses
pneumococcal colonies.
Lysis depends on the
presence of an
intracellular autolytic
enzyme. Bile salts lower
the surface tension
between the bacterial cell
membrane and the
medium, thus accelerating
the organism’s natural
autolytic process.
PRINCIPLE
The butyrate disk is a rapid
test for the detection of
the enzyme butyrate
esterase in identifying
Moraxella (Branhamella)
catarrhalis. If bromo-
chloro-indolyl butyrate
impregnated in the disks is
hydrolyzed by the enzyme,
a blue-colored indigo
compound is formed.
PRINCIPLE
Certain organisms
(including group B
streptococci) produce a
diffusible extracellular
protein (CAMP factor) that
acts synergistically with
the beta-lysin of
Staphylococcus aureus to
2. Gently wash liquid over faecalis
the colony, without
dislodging the colony from
the agar.
3. Incubate the plate at 35° C
in ambient air for 30
minutes. 4. Examine for lysis
of colony.
BUTYRATE DISK
METHOD EXPECTED RESULTS QUALITY CONTROL
1. Remove a disk from the Positive: Development of a blue color during 5-minute incubation
vial and place on a glass period (A). (+) Positive:
microscope slide. Moraxella
2. Add 1 drop of reagent Negative: No color change (B). catarrhalis
grade water. This should
leave a slight excess of water (-) Negative:
on the disk.
Neisseria
3. Using a wooden applicator
gonorrhoeae
stick, rub a small amount of
several colonies from an 18-
to 24-hour pure culture onto
the disk.
4. Incubate at room
temperature for up to 5
minutes.
CAMP TEST
METHOD EXPECTED RESULTS QUALITY CONTROL
1. Streak a beta-lysin– Positive: Enhanced hemolysis is indicated by an arrowhead-shaped
producing strain of S. zone of beta-hemolysis at the juncture of the two organisms (+) Positive:
aureus down the center (Figure 13-7, A). Streptococcus
of a sheep blood agar agalactiae
plate. A.) Positive, arrowhead zone of beta-hemolysis (at arrow) typical
2. Streak test organisms of group B streptococci. (-) Negative:
across the plate B.) Negative, no enhancement of hemolysis.
Streptococcus
perpendicular to the S.
 aureus streak. (Multiple pyogenes
organisms can be tested
cause enhanced lysis of on a single plate if they
red blood cells. are 3 to 4 mm apart.)
3. Incubate overnight at 35°
C in ambient air.
CATALASE TEST
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
The enzyme catalase 1. Use a loop or sterile Positive: Copious bubbles produced (A).
mediates the breakdown wooden stick to transfer a (+) Positive:
of hydrogen peroxide small amount of colony Negative: No or few bubbles produced (B). Staphylococcus
(H2O2) into oxygen and growth to the surface of a aureus
water. The presence of the clean, dry glass slide. NOTE: Some organisms (enterococci) produce a peroxidase that
enzyme in a bacterial slowly catalyzes the breakdown of (H2O2) and the test may appear (-) Negative:
isolate is evident when a 2. Place a drop of 30% weakly positive. This reaction is not a truly positive test.
Streptococcus
small inoculum is hydrogen peroxide (H2O2)
pyogenes
introduced into hydrogen onto the medium. 3. Observe
peroxide (30% for the slide for the evolution of oxygen
test), and the rapid bubbles (Figure 13-8).
elaboration of oxygen
bubbles occurs. The lack of
catalase is evident by a
lack of or weak bubble
production.
CETRIMIDE
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
1. Inoculate a cetrimide agar Positive: Growth (Figure 13-9, A). Negative: No growth (Figure 13-9,
slant with 1 drop of an 18- to B). (+) Positive:
24-hour culture in brain- Pseudomonas
This test is used to heart infusion broth.
determine the ability of an aeruginosa
organism to grow in the 2. Incubate at 35° C for up to
presence of cetrimide, a (-) Negative:
7 days. 3. Examine the slant
toxic substance that Escherichia coli
for bacterial growth.
inhibits the growth of
many bacteria.
 CITRATE UTILIZATION
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
1. Inoculate Simmons citrate Positive: Growth on the medium, with or without a change in the
agar lightly on the slant by color of the indicator. The color change of the indicator is due to
This test is used to
touching the tip of a needle acid or alkali production by the test organism as it grows on the
determine the ability of an
to a colony that is 18 to 24 medium. Growth usually results in the bromthymol blue indicator,
organism to utilize sodium (+) Positive:
hours old. NOTE: There is no turning from green to blue (A).
citrate as its only carbon
need to stab into the butt of Klebsiella
source and inorganic
the tube. Do not inoculate Negative: Absence of growth (B). pneumoniae
ammonium salts as its only
from a broth culture,
nitrogen source. Bacteria
because the inoculum will be (-) Negative:
that can grow on this
too heavy. 2. Incubate at 35° Escherichia coli
medium turn the
to 37° C for up to 7 days. 3.
bromthymol blue indicator
Observe for development of
from green to blue.
blue color, denoting
alkalinization.
COAGULASE TEST
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
This test is used to A. Slide test A. Slide test (+) Positive:
differentiate 1. Place a drop of coagulase Positive: Staphylococcus
Staphylococcus aureus plasma (preferably rabbit Macroscopic clumping in 10 seconds or less in coagulated plasma aureus
(positive) from coagulase plasma with EDTA) on a drop and no clumping in saline or water drop (left side).
negative staphylococci clean, dry glass slide. (-) Negative:
(negative). S. aureus 2. Place a drop of distilled Negative: No clumping in either drop.
Staphylococcus
produces two forms of water or saline next to the NOTE: All negative slide tests must be confirmed using the tube
epidermidis
coagulase: bound and free. drop of plasma as a control. test.
Bound coagulase, or 3. With a loop, straight wire, Equivocal: Clumping in both drops indicates that the organism auto
“clumping factor,” is or wooden stick, emulsify a agglutinates and is unsuitable for the slide coagulase test.
bound to the bacterial cell portion of the isolated colony
wall and reacts directly being tested in each drop, B. Tube test
with fibrinogen. This inoculating the water or Positive: Clot of any size (A, left side). Negative: No clot (B, right
results in an alteration of saline first. Try to create a side).
fibrinogen so that it smooth suspension.
precipitates on the 4. Mix well with a wooden
staphylococcal cell, applicator stick. 5. Rock the
causing the cells to clump slide gently for 5 to 10
when a bacterial seconds
suspension is mixed with
plasma. The presence of B. Tube test
bound coagulase
correlates well with free
coagulase, an extracellular
protein enzyme that
causes the formation of a
clot when S. aureus
colonies are incubated
with plasma. The clotting
mechanism involves
activation of a plasma
coagulase-reacting factor
(CRF), which is a modified
or derived thrombin
molecule, to form a
coagulase-CRF complex.
This complex in turn reacts
with fibrinogen to produce
the fibrin clot.
DECARBOXYLASE TESTS (MOELLER’S METHOD)
PRINCIPLE
This test measures the
enzymatic ability of an
organism to decarboxylate
(or hydrolyze) an amino
acid to form an amine.
Decarboxylation, or
hydrolysis, of the amino
acid results in an alkaline
pH change.
1. Emulsify several colonies
in 0.5 mL of rabbit plasma
(with EDTA) to give a milky
suspension.
2. Incubate tube at 35° C in
ambient air for 4 hours.
3. Check for clot formation.
NOTE: Tests can be positive
at 4 hours and then revert to
negative after 24 hours.
4. If negative at 4 hours,
incubate at room
temperature overnight and
check again for clot
formation.
METHOD EXPECTED RESULTS QUALITY CONTROL
A. Glucose nonfermenting Positive: Alkaline (purple) color change compared with the control
organisms tube (Figure 13-12, A). Negative: No color change or acid (yellow) (+) Positive:
1. Prepare a very heavy color in test and control tube. Growth in the control tube. NOTE: Lysine
suspension (≥McFarland No. The fermentation of dextrose in the medium causes the acid color Klebsiella
5 turbidity standard) in brain- change. It would not, however, mask the alkaline color change pneumoniae
heart infusion broth from brought about by a positive decarboxylation reaction (Figure 13-12, Ornithine
young bacteria (18 to 24 B). Uninoculated tube is shown in Figure 13-12, C. Enterobacter cloacae
hours old) growing on 5% Arginine
sheep blood agar. 2. Enterobacter cloacae
Inoculate each of the three Base —
decarboxylase broths
(arginine, lysine, and (-) Negative:
ornithine) and the control Lysine
broth (no amino acid) with 4 Enterobacter cloacae
drops of broth. Ornithine
3. Add a 4-mm layer of sterile Klebsiella
mineral oil to each tube. 4. pneumoniae
Incubate the cultures at 35° C Arginine
 in ambient air for up to 7 Klebsiella
days. pneumoniae
Base
B. Glucose-fermenting Klebsiella
organisms pneumoniae
1. Inoculate tubes with 1
drop of an 18- to 24-hour
brain-heart infusion broth
culture.
2. Add a 4-mm layer of sterile
mineral oil to each tube.
3. Incubate the cultures for 4
days at 35° C in ambient air.
DNA HYDROLYSIS
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
1. Inoculate the DNase agar Positive: When DNA is hydrolyzed, methyl green is released and
This test is used to with the organism to be combines with highly polymerized DNA at a pH of 7.5, turning the
determine the ability of an tested and streak for medium colorless around the test organism (Figure 13-13, A and B).
organism to hydrolyze isolation. Negative: If there is no degradation of DNA, the medium remains (+) Positive:
DNA. The medium is pale green Staphylococcus
green because of the 2. Incubate aerobically at 35° aureus
DNA–methyl green C for 13 to 24 hours.
complex. If the organism
(-) Negative:
growing on the medium
hydrolyses DNA, the green
Staphylococcus
color fades and the colony epidermidis
is surrounded by a
colorless zone.

PRINCIPLE
This test is used to
determine whether an
organism is able to
hydrolyze the glycoside
esculin.
ESCULIN HYDROLYSIS
METHOD
1. Inoculate the medium with
1 drop of a 24-hour broth
culture. 2. Incubate at 35° C
for up to 7 days. 3. Examine
the slants for blackening and
under the ultraviolet rays of
a Wood’s lamp for esculin
EXPECTED RESULTS QUALITY CONTROL
Positive: Blackened medium (Figure 13-14, A), which would also Positive: Klebsiella
show a loss of fluorescence under the Wood’s lamp. Negative: No pneumoniae
blackening and no loss of fluorescence under Wood’s lamp, or Negative: Shigella
slight blackening with no loss of fluorescence under Wood’s lamp. flexneri
Uninoculated tube is shown in Figure 13-14, B.

PRINCIPLE
The end products of
hydrolysis of hippuric acid
by hippuricase include
glycine and benzoic acid.
Glycine is deaminated by
the oxidizing agent
ninhydrin, which is
reduced during the
process. The end products
of the ninhydrin oxidation
react to form a purple-
colored product. The test
medium must contain only
hippurate, because
ninhydrin might react with
any free amino acids
present in growth media
or other broths.
PRINCIPLE
The test is used to
hydrolysis.
HIPPURATE TEST
METHOD EXPECTED RESULTS QUALITY CONTROL
1. Add 0.1 mL of sterile water Positive: Deep purple color (Figure 13-19, A). Negative: Colorless or Positive:
to a 12 × 75 mm plastic test slightly yellow pink color (Figure 13-19, B). Streptococcus
tube. 2. Make a heavy agalactiae Negative:
suspension of the organism Streptococcus
to be tested. 3. Using heated pyogenes
forceps, place a rapid
hippurate disk in the mixture.
4. Cap and incubate the tube
for 2 hours at 35° C; use of a
water bath is preferred. 5.
Add 0.2 mL ninhydrin
reagent and reincubate for
an additional 15 to 30
minutes. Observe the
solution for development of
a deep purple color.
INDOLE PRODUCTION
METHOD EXPECTED RESULTS QUALITY CONTROL
A. Enterobacteriaceae 1. Positive: Pink- to wine-colored ring after addition of appropriate A. Kovac’s method
determine the ability of an
organism to split
tryptophan to form the
compound indole
PRINCIPLE
The LAP test is used to
identify catalasenegative
gram-positive cocci. The
LAP disk is a rapid test for
the detection of the
enzyme leucine
aminopeptidase. Leucine-
β-naphthylamide
impregnated disks serve as
a substrate for the
detection of leucine
aminopeptidase. Following
hydrolysis of the substrate
by the enzyme, the
Inoculate tryptophane broth reagent (Figure 13-20, A). Negative: No color change after the Positive: Escherichia
with 1 drop from a 24-hour addition of the appropriate reagent (Figure 13-20, B). coli Negative:
brain-heart infusion broth Klebsiella
culture. 2. Incubate at 35° C pneumoniae B.
in ambient air for 48 hours. Ehrlich’s method
3. Add 0.5 mL of Kovac’s Positive:
reagent to the broth culture. Elizabethkingia
B. Other gram-negative meningoseptica
bacilli 1. Inoculate Negative: CDC group
tryptophane broth with 1 EO-2
drop of a 24-hour broth
culture. 2. Incubate at 35° C
in ambient air for 48 hours.
3. Add 1 mL of xylene to the
culture. 4. Shake mixture
vigorously to extract the
indole and allow to stand
until the xylene forms a layer
on top of the aqueous phase.
5. Add 0.5 mL of Ehrlich’s
reagent down the side of the
tube.
LAP TEST
METHOD EXPECTED RESULTS QUALITY CONTROL
1. Before incubation, slightly Positive: Development of a red color within 1 minute after adding Positive:
dampen the LAP disk with cinnamaldehyde reagent (Figure 13-21, A). Negative: No color Enterococcus faecalis
reagent grade water. Do not change or development of a slight yellow color (Figure 13-21, B). Negative:
supersaturate the disk. 2. Leuconostoc sp.
Using a wooden applicator
stick, rub a small amount of
several colonies of an 18- to
24-hour pure culture onto a
small area of the LAP disk. 3.
Incubate at room
temperature for 5 minutes.
4. After this incubation
period, add 1 drop of
cinnamaldehyde reagent.
resulting β-naphthylamine
produces a red color upon
the addition of
cinnamaldehyde reagent.
PRINCIPLE
This test is used to
determine an organism’s
ability to metabolize litmus
milk. Fermentation of
lactose is evidenced by the
litmus turning pink as a
result of acid production. If
sufficient acid is produced,
casein in the milk is
coagulated, solidifying the
milk. With some
organisms, the curd
shrinks and whey is
formed at the surface.
Some bacteria hydrolyze
casein, causing the milk to
become strawcolored and
resemble turbid serum.
Additionally, some
LITMUS MILK
METHOD EXPECTED RESULTS QUALITY CONTROL
1. Inoculate with 4 drops of a Alkaline: Alcaligenes
24-hour broth culture. 2. faecalis Acid:
Incubate at 35° to 37° C in Enterococcus faecium
ambient air. 3. Observe daily Peptonization:
for 7 days for alkaline Burkholderia cepacia
reaction (litmus turns blue),
acid reaction (litmus turns
pink), indicator reduction,
acid clot, rennet clot, and
peptonization. Multiple
changes can occur over the
observation period. 4. Record
all changes.
organisms reduce litmus,
in which case the medium
becomes colorless in the
bottom of the tube.
LYSIN IRON AGAR
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
This test is used to 1. With a straight inoculating 1. Alkaline slant/alkaline butt (K/K) = lysine decarboxylation and no Alkaline slant and
determine whether a needle, inoculate LIA (Figure fermentation of glucose (Figure 13-23, A). 2. Alkaline slant/acid butt butt, H2S positive:
gram-negative rod 13-23, E) by twice stabbing (K/A) = glucose fermentation (Figure 13-23, C). 3. NOTE: Patterns Salmonella
decarboxylates or through the center of the shown in 1 and 2 above can be accompanied by a black precipitate typhimurium Alkaline
deaminates lysine and medium to the bottom of the of ferrous sulfide (FeS), which indicates production of H2S (Figure slant, acid butt:
forms hydrogen sulfide tube and then streaking the 13-23, B). 4. Red slant/acid butt (R/A) = lysine deamination and Shigella flexneri Red
(H2S). Lysine iron agar slant. 2. Cap the tube tightly glucose fermentation (Figure 13-23, D). slant, acid butt:
(LIA) contains lysine, and incubate at 35° C in Proteus vulgaris
peptones, a small amount ambient air for 18 to 24 Lysine iron agar. A, Alkaline slant/alkaline butt (K/K). B, Alkaline
of glucose, ferric hours. slant/alkaline butt, H2S positive (K/K H2S+). C, Alkaline slant/acid
ammonium citrate, and butt (K/A). D, Red slant/acid butt (R/A). E, Uninoculated tube.
sodium thiosulfate. When
glucose is fermented, the
butt of the medium
becomes acidic (yellow). If
the organism produces
lysine decarboxylase,
cadaverine is formed.
Cadaverine neutralizes the
organic acids formed by
glucose fermentation, and
the butt of the medium
reverts to the alkaline
state (purple). If the
decarboxylase is not
produced, the butt
remains acidic (yellow). If
oxidative deamination of
lysine occurs, a compound
is formed that, in the
presence of ferric
ammonium citrate and a
coenzyme, flavin
mononucleotide, forms a
burgundy color on the
slant. If deamination does
not occur, the LIA slant
remains purple.
METHYL RED/VOGES - PROSKAUER (MRVP) TESTS

PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

MICRODASE TEST
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

M OTILITY TESTING
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

MRS BROTH
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

MUG TEST
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

NITRATE REDUCTION
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
 NITRITE REDUCTION
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

ONPG (O - NITROPHENYL - β - D - GALACTOPYRANOSIDE) TEST

PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

OPTOCHIN TEST
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

OXIDASE TEST (KOVAC ’ S M ETHOD )

PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

OXIDATION / FERMENTATION (OF) MEDIUM (CDC METHOD )

PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

PHENYLALANINE DEAMINASE
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

PYR TEST
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL
 PYRUVATE BROTH
PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

SALT TOLERANCE TEST

PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

SPOT INDOLE TEST

PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

TRIPLE SUGAR IRON AGAR (TSI)

PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

UREA HYDROLYSIS (CHRISTENSEN’S METHOD)

PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL

X AND V FACTOR TEST

PRINCIPLE METHOD EXPECTED RESULTS QUALITY CONTROL