Overview of Bacterial Identification Methods and Strategies

(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

Acetamide Differentiate Bacteria capable of 1. Inoculate acetamide slant Positive: Deamination of the acetamide, resulting Positive:
Utilization microorganisms growth on this medium with a needle using growth in a blue color Pseudomonas
based on the produce the enzyme from an 18- to 24-hour culture. Negative: No color change aeruginosa
ability to use acylamidase, which Do not inoculate from a broth (ATCC 27853)—
acetamide as the deaminates acetamide culture, because the growth will growth; blue
sole source of to release ammonia. be too heavy. color
carbon The production of 2. Incubate aerobically at 35°C Negative:
ammonia results in an to 37°C for up to 4 days. If Escherichia coli
alkaline pH, causing the equivocal, the slant may be (ATCC 25922)—
medium to change color reincubated for 2 additional no growth; green
from green to royal days. color
blue.

Acetate Differentiate This test is used to 1. With a straight inoculating Positive: Medium becomes alkalinized (blue) as a Positive:
Utilization organisms based differentiate an needle, inoculate acetate slant result of the growth and use of acetate Negative: Escherichia coli -
on ability to use organism capable of lightly from an 18- to 24-hour No growth or growth with no indicator change to -(growth; blue
acetate as the sole using acetate as the sole culture. Do not inoculate from a blue Negative:
source of carbon. source of carbon. broth culture, because the Shigella sonnei
Generally used to Organisms capable of growth will be too heavy. —small amount
differentiate using sodium acetate 2. Incubate at 35°C to 37°C for of growth; green
Shigella spp. from grow on the medium, up to 7 days.
Escherichia coli. resulting in an alkaline Limitations:
pH, turning the Some strains of
indicator from green to E. coli may use
blue. acetate at a very
slow rate or not
at all, resulting in
a false negative
reaction in the
identification
process.
l-Alanine-7- This test is used in The compound l- 1. Inoculate a pure colony of *Aerobic, gram-negative rods and coccobacilli will Positive:
amido-4- conjunction with Alanine-7-amido-4- overnight growth (16 to 18 appear fluorescent or blue (POSITVE). Escherichia coli -
methylcourmarin the Gram stain to methylcourmarin is hours *Aerobic, gram-positive rods and coccobacilli will (blue
(Gram-Sure) distinguish impregnated in a after initial culture) to 0.25 mL appear colorless (NEGATIVE). fluorescence
aerobic gram- commercially prepared of demineralized water in a Negative:
positive rods or disk (Remel-Thermo clean 12 by 75 mm test tube. Staphylococcus
coccobacilli that Fisher Scientific, 2. Place a Gram-Sure disk in the
may appear gram- Lenexa, KS). Gram- emulsion. Limitations:
negative or gram- negative organisms 3. Incubate at room Obligate
variable. produce an temperature for 5 to 10 anaerobic
aminopeptidase that is minutes. organisms may
capable of hydrolyzing 4. Observe blue fluorescence by fail to give
the reagent in the disk, placing the tube under expected results.
forming a blue long-wave ultraviolet light.
fluorescent compound
that is visible under
long-wave UV light.
Bacitracin This test is used The antibiotic bacitracin 1. Using an inoculating loop, streak two or Positive: Any zone of inhibition .Positive:
Susceptibility for presumptive inhibits the synthesis of three suspect greater than 10 mm; susceptible Streptococcus
identification and bacterial cell walls. A colonies of a pure culture onto a blood Negative: No zone of inhibition; pyogenes—
differentiation of disk (TaxoA) agar plate. resistant susceptible
beta-hemolytic impregnated with a 2. Using heated forceps, place a bacitracin Micrococcus luteus
group A small amount of disk in the first quadrant (area of heaviest —susceptible
streptococci bacitracin (0.04 units) is growth). Gently tap the disk to ensure Negative:
(Streptococcus placed on an agar plate, adequate contact with the agar surface. Streptococcus
pyogenes– allowing the antibiotic 3. Incubate the plate for 18 to 24 hours at agalactiae—
susceptible) from to diffuse into the 35°C to 37°C in ambient air for resistant
other beta- medium and inhibit the staphylococci and in 5% to 10% carbon Staphylococcus
hemolytic growth of susceptible dioxide (CO2) for streptococci aureus—resistant
streptococci. It is organisms. After differentiation.
also used to incubation, the 4. Look for a zone of inhibition around the Limitations:
distinguish inoculated plates are disk. Performance
staphylococci examined for zones of depends on the
species (resistant) inhibition surrounding integrity of the disk.
from micrococci the disks. Proper storage and
(susceptible). expiration dates
should be
maintained
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

Bile Esculin This test is used Gram-positive bacteria 1. Inoculate one to two colonies Positive: Growth and blackening of the agar slant Positive:
for the other than some from an 18- to 24-hour culture Negative: Growth and no blackening of medium Enterococcus
presumptive streptococci and onto the surface of the slant. faecalis—
identification of enterococci are 2. Incubate at 35°C to 37°C in growth; black
enterococci and inhibited by the bile ambient air for 48 hours. precipitate
organisms in the salts in this medium. Negative:
Streptococcus Organisms capable of Escherichia
bovis group. The growth in the presence coli—growth; no
test differentiates of 4% bile and able to color change
enterococci and hydrolyze esculin to Streptococcus
group D esculetin will pyogenes—no
streptococci from demonstrate growth. In growth; no color
non–group D addition, esculetin Change
viridans reacts with Fe31 and
streptococci. forms a dark brown to
black precipitate.

Bile Solubility This test Bile or a solution of a 1. After 12 to 24 hours of Positive: Colony disintegrates; an imprint of the Positive:
Test differentiates bile salt (e.g., sodium incubation on 5% sheep blood lysed colony may remain in the zone Streptococcus
Streptococcus deoxycholate) rapidly agar, place one to two drops of Negative: Intact colonies pneumoniae—
pneumoniae lyses pneumococcal 10% sodium deoxycholate on a bile soluble
(positive; soluble) colonies. Lysis depends well-isolated colony. Note: A Negative:
from alpha- on the presence of an tube test is performed with 2% Enterococcus
hemolytic intracellular autolytic sodium deoxycholate. faecalis—bile
streptococci enzyme, amidase. Bile 2. Gently wash liquid over the insoluble
(negative; salts lower the surface colony without dislodging the
insoluble). tension between the colony from the agar. Limitations:
bacterial cell 3. Incubate the plate at 35°C to Enzyme activity
membrane and the 37°C in ambient air for 30 may be reduced
medium, thus minutes. in old cultures.
accelerating the 4. Examine for lysis of colony. Therefore
organism’s natural negative results
autolytic process with colonies
resembling S.
pneumoniae
should be
further tested
for identification
with alternate
methods.
Butyrate Disk This is a rapid test Organisms capable of 1. Remove a disk from the vial Positive: Development of a blue color during the Positive:
(Catarrhalis to detect the producing butyrate and place on a glass microscope 5-minute incubation period Negative: No color Moraxella
Test) enzyme butyrate esterase hydrolyze slide. change catarrhalis—
esterase, to aid bromo-chlor-indolyl 2. Add one drop of reagent- formation of
identification of butyrate. Hydrolysis of grade water. This should leave a blue color
Moraxella the substrate in the slight excess of water on the Negative:
catarrhalis presence of butyrate disk. Neisseria
esterase releases 3. Using a wooden applicator gonorrhoeae—
indoxyl, which in the stick, rub a small amount of no color change
presence of oxygen several colonies from an 18- to Limitations:
spontaneously forms 24-hour pure culture onto the Incubation longer
indigo, a blue to blue- disk. than 5 minutes
violet color 4. Incubate at room may result in a
false-positive
temperature for up to 5 reaction.
minutes. False-negative
reactions may
occur if the
inoculum is too
small. If the
organism is
negative, repeat
with a larger
inoculum and
follow up with
additional
methods.
CAMP Test The Christie, Certain organisms 1. Streak a beta-lysin–producing Positive: Enhanced hemolysis is indicated by an Positive:
Atkins, and (including group B strain of S. aureus down the arrowheadshaped zone of beta-hemolysis at the Streptococcus
Munch-Peterson streptococci) produce a center of a sheep blood agar juncture of the two organisms agalactiae—
(CAMP) test is diffusible extracellular plate. Negative: No enhancement of hemolysis enhanced
used to hemolytic protein 2. Streak test organisms across arrowhead
differentiate (CAMP factor) that acts the plate perpendicular to the hemolysis
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

group B synergistically with the S. aureus streak within 2 mm. Negative:
streptococci beta-lysin of (Multiple organisms can be Streptococcus
(Streptococcus Staphylococcus aureus tested on a single plate.) pyogenes—beta-
agalactiae– to cause enhanced lysis 3.Incubate overnight at 35° to hemolysis
positive) from of red blood cells. The 37°C in ambient air. without
other group B streptococci are enhanced
streptococcal streaked perpendicular arrowhead
species. Listeria to a streak of S. aureus formation
monocytogenes on sheep blood agar. A
also produces a positive reaction
positive CAMP appears as an Limitation: A
reaction. arrowhead zone of small percentage
hemolysis adjacent to of group A
the place where the two streptococci may
streak lines come into have a positive
proximity. CAMP reaction.
The test should
be limited to
colonies with the
characteristic
group B
streptococci
morphology and
narrow-zone
beta-hemolysis
on sheep blood
agar
Catalase Test This test Aerobic and facultative anaerobic 1. Use a loop or Positive: Copious bubbles are produced Positive:
differentiates organisms produce two toxins during sterile wooden Negative: No or few bubbles are produced Staphylococcus
catalase-positive normal metabolism, hydrogen stick to transfer a aureus
micrococcal and peroxide (H2O2) and superoxide small amount Negative:
staphylococcal radical (O22). These bacteria have two of colony growth Streptococcus
species from enzymes that detoxify the products of to the surface of pyogenes
catalase-negative normal metabolism. One of these a clean, dry glass
streptococcal enzymes, catalase, is capable of slide. Limitations
species. converting hydrogen peroxide to 2. Place a drop of Some organisms
water and oxygen. The presence of the 30% hydrogen (enterococci)
enzyme in a bacterial isolate is peroxide (H2O2) produce a
evidenced when a small inoculum onto the peroxidase that
introduced into hydrogen peroxide medium. (3% can slowly catalyzes
(30% for the slide test) causes rapid also be used for the breakdown
elaboration of oxygen bubbles. The most organisms.) of H2O2, and the
lack of catalase is evident by a lack of 3. Observe for test may appear
or weak bubble production. the evolution of weakly positive.
oxygen bubbles This reaction is
not a truly
positive test.
False positives
may occur if the
sample is
contaminated
with blood agar
Cetrimide Agar This test is The test is used to 1. Inoculate a cetrimide agar Positive: Growth, variation in color of colonies Positive:
primarily used to determine the ability of slant with one drop of an 18- to Negative: No growth Pseudomonas
isolate and purify an organism to grow in 24-hour brain-heart infusion aeruginosa—
Pseudomonas the presence of broth culture. growth and color
aeruginosa from cetrimide, a toxic 2. Incubate at 35°C to 37°C for change; yellow-
contaminated substance that inhibits up to 7 days. green to blue-
specimens the growth of many 3. Examine the slant for
green colonies
bacteria by causing the bacterial growth.
Negative:
release of nitrogen and
phosphorous, which Escherichia
slows or kills the coli—no growth
organism. P. aeruginosa and no color
is resistant to cetrimide. change
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

Citrate The purpose of Bacteria that can grow 1. Inoculate Simmons citrate Positive: Growth on the medium, with or without Positive:
Utilization this test is to on this medium agar lightly on the slant by a change in the color of the indicator. Growth Enterobacter
identify organisms produce an enzyme, touching typically results in the bromothymol blue aerogenes—
capable of using citrate-permease, the tip of a needle to a colony indicator turning from green to blue growth; blue
sodium citrate as capable of converting that is 18 to 24 hours old. Do Negative: Absence of growth color
the sole carbon citrate to pyruvate. not inoculate from a broth Negative:
source and Pyruvate can then enter culture, because the inoculum Escherichia
inorganic the organism’s will coli—little to no
ammonium salts metabolic cycle for the be too heavy. growth; no color
as the sole production of energy. 2. Incubate at 35°C to 37°C for change
nitrogen source. Bacteria capable of up to 7 days.
The test is part of growth in this medium 3. Observe for growth and the
a series referred use the citrate and development of blue color, Limitations:
to as IMViC convert ammonium denoting alkalinization. Some organisms
(indole, methyl phosphate to ammonia are capable of
red, Voges- and ammonium growth on
Proskauer, and hydroxide, creating an citrate and do
citrate), which is alkaline pH. The pH not produce a
used to change turns the color change.
differentiate bromothymol blue Growth is
Enterobacteriacea indicator from green to considered a
e from other blue. positive citrate
gram-negative utilization test,
rods. even in the
absence of a
color change.

Coagulase Test The test is used S. aureus produces two forms of
to differentiate coagulase, bound and free. Bound
Staphylococcus coagulase, or “clumping factor,” is
aureus (positive) bound to the bacterial cell wall and
from coagulase- reacts directly with fibrinogen. This
negative results in precipitation of fibrinogen on
staphylococci the staphylococcal cell, causing the cells
(negative). to clump when a bacterial suspension is
mixed with plasma. The presence of
bound coagulase correlates 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.
A. Slide Test (Detection of Bound Coagulase Positive: Staphylococcus
or Clumping Factor) aureus
1. Place a drop of coagulase plasma (preferably rabbit
plasma with ethylenediaminetetraacetic acid [EDTA]) Negative: Staphylococcus
reagent on the reaction provided by the manufacturer. epidermidis
2. Place a drop of distilled water or saline next to the drop
of plasma in an adjacent reaction well as a negative control.
3. Place a drop of coagulase plasma reagent in a third Limitations:
adjacent reaction well as a positive control. Slide Test
4. With a loop, straight wire, or wooden stick, emulsify a Equivocal: Clumping in both
portion of the isolated colony being tested in the rabbit the rabbit plasma reagent
plasma reagent. Try to create a smooth suspension. and water or saline control
5. Mix well with a wooden applicator stick. drops indicate that the
6. With a loop, straight wire, or wooden stick, emulsify a organism autoagglutinates
known Staphylococcus aureus in the positive and negative and is unsuitable for the
control wells. slide coagulase test.
7. Mix all samples well with a new wooden applicator stick Tube Test
for each sample. 1. Test results can be
8. Rock the slide gently for 5 to 10 seconds. positive at 1 to 4 hours and
then revert to
Expected Results negative after 24 hours.
Positive: Macroscopic clumping in 10 seconds or less in 2. If negative at 4 hours,
coagulated plasma drop positive control, unknown clinical incubate at room
isolate along with no clumping in saline or water drop temperature overnight
Negative: No clumping in the unknown clinical isolate well and check again for clot
as long as the positive and negative controls demonstrate formation.
appropriate reactions as described.
Note: All negative slide tests must be confirmed using the
tube test

B. Tube Test (Detection of Free Coagulase)

1. Emulsify several colonies of the unknown clinical isolate
in 0.5 mL of rabbit plasma (with EDTA) to give a milky
suspension.
2. Repeat the same process with a known positive and
negative control organism.
2. Incubate tube at 35°C to 37°C in ambient air for 1 to 4
hours.
3. Check for clot formation.

Expected Results:
Positive: Clot of any size
Negative: No clot
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

Decarboxylase This test is used to This test measures A. Glucose-Nonfermenting Positive: Alkaline (purple) color change Positive:
Tests (Moeller’s differentiate the enzymatic ability Organisms compared with the control tube Lysine—Klebsiella
Method) decarboxylase- (decarboxylase) of an 1. Prepare a suspension Negative: No color change or acid pneumoniae—yellow to
producing organism to (McFarland No. 5 turbidity (yellow) color in test and control tube. purple
Enterobacteriacea decarboxylate (or standard) in brain-heart infusion Growth in the control tube. Ornithine—Enterobacter
e from other hydrolyze) an amino broth from an overnight culture aerogenes —yellow to
gram-negative acid to form an (18 to 24 hours old) growing on purple
rods. amine. 5% sheep blood agar. Arginine—Pseudomonas
Decarboxylation, or 2. Inoculate each of the three aeruginosa —yellow to
hydrolysis, of the decarboxylase broths (arginine, purple
amino acid results in lysine, and ornithine) and the
an alkaline pH and a control broth (no amino acid) Base Control:
color change from with four drops of broth. Positive Glucose Fermenters
orange to purple. 3. Add a 4-mm layer of sterile Klebsiella pneumoniae—
mineral oil to each tube. yellow
4. Incubate the cultures at 35°C to Enterobacter aerogenes—
37°C in ambient air. Examine the yellow
tubes at 24, 48, 72, and 96 hours.
B. Glucose-Fermenting Negative:
Organisms Lysine—Citrobacter freundii
1. Inoculate tubes with one drop —yellow Ornithine—Proteus
of an 18- to 24-hour brain-heart vulgaris —yellow Arginine—
infusion broth culture. Escherichia coli—yellow
2. Add a 4-mm layer of sterile
mineral oil to each tube. Limitations: The
3. Incubate the cultures for 4 days fermentation of dextrose in
at 35°C to 37°C in ambient air. the medium causes the acid
Examine the tubes at 24, 48, 72, color change. However, it
and 96 hours. would not mask the alkaline
color change brought
about by a positive
decarboxylation reaction
Deoxyribonucleic This test is used to The test is used to 1. Inoculate the DNase agar Positive: When DNA is hydrolyzed, methyl green is Positive:
Acid Hydrolysis differentiate determine the ability of with the organism to be tested released and combines with highly polymerized Staphylococcus
(DNase Test organisms based an organism to and streak for isolation. DNA at a pH of 7.5, turning the medium colorless aureus
Agar) on the production hydrolyze 2. Incubate aerobically at 35°C around the test organism Negative:
of deoxyribonucleic acid to 37°C for 13 to 24 hours. Negative: If no degradation of DNA occurs, the Escherichia coli
deoxyribonucleas (DNA). The medium is
medium remains green
e. It is used to pale green because of Note: Several variations of this
distinguish the DNA–methyl green media are available that include Limitations: Agar
Serratia spp. complex. If the agarose slants and toluene blue must be
(positive) from organism growing on (positive result appears a deep inoculated with a
Enterobacter spp., the medium hydrolyzes pink) or precipitation of the suspension of a
Staphylococcus DNA, the green color DNA by flooding with young broth
aureus (positive) fades and the colony is hydrochloric acid resulting in a culture (4 hours
from other surrounded by a visible precipitation of the old) or an 18- to
species, and colorless zone. polymerized DNA in the 24-hour
Moraxella absence of a dye. overnight colony
catarrhalis in 1 to 2 mL of
(positive) from saline.
Neisseria spp.
Esculin This test is used This test is used to 1. Inoculate the medium with Positive: Blackened medium which would also Positive:
Hydrolysis for the determine whether an one drop of a 24-hour broth show a loss of fluorescence under the Wood’s Enterococcus
presumptive organism is able to culture. lamp. faecalis
identification and hydrolyze the glycoside 2. Incubate at 35°C to 37°C for Negative: No blackening and no loss of Negative:
differentiation of esculin. Esculin is up to 7 days. fluorescence under the Wood’s lamp, or slight Escherichia coli
Enterobacteriaceae. hydrolyzed to esculetin, 3. Examine the slants for blackening with no loss of fluorescence under the Limitations: This
which reacts with Fe31 blackening and, under the Wood’s lamp. medium is a
and forms a dark brown ultraviolet rays of a Wood’s nonselective
to black precipitate. lamp, for esculin hydrolysis. agar. The bile
esculin
hydrolysis test
presented in
Procedure 12-5
is a selective
differential
method.
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

Fermentation Fermentation Carbohydrate A. Peptone Medium with Andrade’s Indicator A. Peptone
Media media are used to fermentation is the (for Enterics and Coryneforms) Medium with
differentiate process 1. Inoculate each tube with one drop of an 18- to 24-hourbrain-heart infusion broth Andrade’s
organisms based microorganisms use to culture. Indicator
on their ability to produce energy. Most 2. Incubate at 35°C to 37°C for up to 7 days in ambient air. Dextrose:
ferment microorganisms Note: Tubes are held only 4 days for organisms belonging to the Enterobacteriaceae Positive, with
carbohydrates convert glucose to family. gas: Escherichia
incorporated into pyruvate during 3. Examine the tubes for acid (indicated by a pink color) and gas production. coli
the basal glycolysis; however, 4. Tubes must show growth for the test to be valid. If no growth in the fermentation Positive, no gas:
medium. some organisms use tubes or control is seen after 24 hours of incubation, add one to two drops of sterile Shigella flexneri
Andrade’s alternate pathways. A rabbit serum per 5 mL of fermentation broth to each tube. B. Brain-Heart
formula is used to fermentation medium Expected Results Infusion Broth
differentiate consists of a basal Positive: Indicator change to pink with or without gas formation in Durham tube with
enteric bacteria medium containing a Negative: Growth, but no change in color. Medium remains clear to straw colored Bromocresol
from single carbohydrate B. Broth (Brain-Heart Infusion Purple Indicator
coryneforms, and (glucose, lactose, or Broth May Be Dextrose:
bromocresol sucrose) for Substituted) with Bromocresol Positive, with
purple is used to fermentation. Purple Indicator gas: Escherichia
distinguish However, the medium (for Streptococci and coli
enterococci from may contain various Enterococci) Negative, no
streptococci. color indicators, such as 1. Inoculate each tube with two gas: Moraxella
Andrade’s indicator, drops of an 18- to 24-hour osloensis
bromocresol, or others. brain-heart infusion broth culture. Limitations :
In addition to a color 2. Incubate 4 days at 35°C to 37°C in Readings after
indicator to detect the ambient air. 24 hours may
production of acid from 3. Observe daily for a change of the not be reliable if
fermentation, a bromocresol purple indicator no acid is
Durham tube is placed from purple to yellow (acid). produced. No
in each tube to capture Expected Results color change or a
gas produced by Positive: Indicator change to yellow result indicating
metabolism. Negative: Growth, but no change in color. Medium remains purple alkalinity may
occur if the
organism
deaminates the
peptone,
masking the
evidence of
carbohydrate
fermentation.
Flagella Stain This technique is Flagella are too thin to 1. Grow the organism to be stained at Observe the slide and note the Peritrichous:
(Wet Mount used to visualize be visualized using a room temperature on blood agar for 16 to following: Escherichia coli
Technique) the presence and bright field 24 hours. 1. Presence or absence of flagella Polar:
arrangement of microscope with 2. Add a small drop of water to a 2. Number of flagella per cell Pseudomonas
flagella for the ordinary stains, such microscope slide. 3. Location of flagella per cell aeruginosa
presumptive as the Gram stain, or a 3. Dip a sterile inoculating loop into sterile a. Peritrichous Negative:
identification of simple stain. A wet water. b. Lophotrichous Klebsiella
motile bacterial mount technique is 4. Touch the loopful of water to the colony c. Polar or monotrichous pneumoniae
species. used for staining margin briefly (this allows motile cells to d. Amphitrichous
bacterial flagella, and swim into the droplet of water). 4. Amplitude of wavelength
it is simple and useful 5. Touch the loopful of motile cells to the a. Short Limitations: Even
when the number and drop of water on the slide. Note: Agitating b. Long with a specific
arrangement of the loop in the droplet of water on the 5. Whether or not “tufted” stain,
flagella are critical to slide causes the flagella to shear off the visualization of
the identification of cell. flagella requires
species of motile 6. Cover the faintly turbid drop of water an experienced
bacteria. The staining on the slide with a cover slip. A proper wet laboratory
procedures require mount has barely enough liquid to fill the scientist and is
the use of a mordant space under a cover slip. Small air spaces not considered
so that the stain around the edge are preferable. an entry-level
adheres in layers to 7. Examine the slide immediately under technique.
the flagella, allowing 403 to 503 magnification for motile cells. If
visualization. motile cells are not seen, do not proceed
with the stain.
8. If motile cells are seen, leave the slide at
room temperature for 5 to 10 minutes.
This allows the bacterial cells time to
adhere either to the glass slide or to the
cover slip.
9. Gently apply two drops of Ryu flagella
stain (available from multiple
manufacturers) to the edge of the cover
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

slip. The stain will flow by capillary action
and mix with the cell suspension. Small air
pockets around the edge of the wet
mount are useful in aiding the capillary
action.
10. After 5 to 10 minutes at room
temperature, examine the cells for
flagella.
11. Cells with flagella may be observed at
1003 (oil) magnification in the zone of
optimum stain concentration, about
halfway from the edge of the cover slip to
the center of the mount.
12. Focusing the microscope on the cells
attached to the cover slip rather than on
the cells attached to the slide
facilitates visualization of the flagella. The
precipitate from the stain is primarily on
the slide rather than the cover slip.
Gelatin The production of This test is used to 1. Inoculate the gelatin deep with four to five drops of a 24-hour Positive: Bacillus
Hydrolysis gelatinases determine the ability of broth culture. subtilis
capable of an organism to produce 2. Incubate at 35°C to 37°C in ambient air for up to 14 days. Negative:
hydrolyzing extracellular proteolytic Note: Incubate the medium at 25°C if the organism grows better at 25°C than at 35°C. Escherichia coli
gelatin is used as a enzymes (gelatinases) 3. Alternatively, inoculate the gelatin deep from a 24-hour-old colony by stabbing Uninoculated
presumptive test that liquefy gelatin, a four or five times, 0.5 inch into the medium. control tube:
for the component of 4. Remove the gelatin tube daily from the incubator and place at 4°C to check for medium
identification of vertebrate connective liquefaction. Do not invert or tip the tube, becomes solid
various tissue. because sometimes the only discernible liquefaction occurs at after
organisms, Nutrient gelatin the top of the deep where inoculation occurred. refrigeration.
including medium differs from 5. Refrigerate an uninoculated control along with the inoculated
Staphylococcus traditional microbiology tube. Liquefaction is determined Limitations:
spp., media in that the only after the control has hardened Some organisms
Enterobacteriacea solidifying agent (agar) (gelled). may grow poorly
e, and some gram- is replaced with gelatin. or not at all in
Expected Results:
positive bacilli. When an organism this medium.
Positive: Partial or total liquefaction
produces gelatinase, Gelatin is liquid
of the inoculated tube (the control
the enzyme liquefies above 20°C;
the growth medium. tube must be completely solidified) therefore
Negative: Complete solidification of determination of
the tube at 4°C results must be
completed after
refrigeration.

Growth at 42°C This test is used to The test is used to 1. Inoculate two Positive: Good growth at both 35°C and 42°C Positive:
differentiate a determine the ability of tubes of trypticase Negative: No growth at 42°, but good growth at 35°C. Pseudomonas
pyocyanogenic an organism to grow at soy agar (TSA) with aeruginosa
pseudomonad 42°C. Several a light inoculum by
from other Pseudomonas spp. have lightly touching a Negative:
Pseudomonas been isolated in the needle to the top of Pseudomonas
spp. clinical laboratory that a single 13- to 24- fluorescens
are capable of growth hour-old colony
at elevated and streaking the
temperatures. slant.
2. Immediately
incubate one tube
at 35°C and one at
42°C.
3. Record the
presence of growth
on each slant after
18 to 24 hours.
Hippurate Production of the The end products of 1. Add 0.1 mL of sterile Positive: Deep purple color Positive:
Hydrolysis enzyme hydrolysis of hippuric water to a 12 by 75 Negative: Colorless or slightly yellow pink color Streptococcus
hippuricase is acid by hippuricase mm plastic test tube. agalactiae
used for the include glycine and 2. Make a heavy Negative:
presumptive benzoic acid. Glycine is suspension of the Streptococcus
identification of a deaminated by the organism to be tested. pyogenes
variety of oxidizing agent 3. Using heated
microorganisms. ninhydrin, which is forceps, place a rapid Limitations:
reduced during the hippurate disk in the A false-positive
process. The end mixture. result may occur
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

products of the 4. Cap and incubate if incubation
ninhydrin oxidation the tube for 2 hours at with ninhydrin
react to form a purple- 35°C; use of a water exceeds 30
colored product. The bath is preferred. minutes.
test medium must 5. Add 0.2 mL
contain only hippurate, ninhydrin reagent and
because ninhydrin reincubate for an
might react with any additional 15 to 30
free amino acids minutes. Observe the
present in growth solution for the
media or other broths. development of a deep
purple color.

Indole This test is used to The test is used to A. Enterobacteriaceae Positive: Pink- to wine-colored ring after A. Kovac’s
Production identify organisms determine an 1. Inoculate tryptophane broth with addition of appropriate reagent (A) Method
that produce the organism’s ability to one drop from a 24-hour brain-heart Negative: No color change after addition of Positive:
enzyme hydrolyze tryptophan to infusion broth culture. the appropriate reagent (B) Escherichia coli
tryptophanase. form the compound 2. Incubate at 35°C to 37°C in Negative:
indole. Tryptophan is ambient air for 48 hours. Klebsiella
present in casein and 3. Add 0.5 mL of Kovac’s reagent to pneumoniae
animal protein. Bacteria the broth culture. B. Ehrlich’s
with tryptophanase are B. Other Gram-Negative Bacilli Method
capable of hydrolyzing 1. Inoculate tryptophane broth with Positive:
tryptophan to pyruvate, one drop of a 24-hour broth culture. Haemophilus
ammonia, and indole. 2. Incubate at 35°C to 37°C in influenzae
Kovac’s reagent ambient air for 48 hours. Negative:
(dimethylamine- 3. Add 1 mL of xylene to the culture. Haemophilus
benzaldehyde and 4. Shake the mixture vigorously to parainfluenza
hydrochloride), when extract the indole and allow it C. Ehrlich’s
added to the broth to stand until the xylene forms a Method
culture, reacts with the layer on top of the aqueous (Anaerobic)
indole, producing a red phase. Positive:
color. An alternative 5. Add 0.5 mL of Ehrlich’s reagent Porphyromonas
method uses Ehrlich’s down the side of the tube. asaccharolytica
reagent. Ehrlich’s Negative:
reagent has the same Bacteroides
chemicals as the Kovac fragilis
preparation, but it also
contains absolute ethyl Limitations:
alcohol, making it Ehrlich’s method
flammable. Ehrlich’s may also be used
reagent is more to differentiate
sensitive for detecting organisms under
small amounts of anaerobic
indole. conditions.
Leucine The leucine The LAP disk is a rapid test 1. Before incubation, Positive: Development of a red color within 1 Positive: Enterococcus
Aminopeptidase aminopeptidase for the detection of the slightly dampen the minute after adding cinnamaldehyde reagent (A) faecalis—red color
(LAP) Test (LAP) test is used enzyme leucine LAP disk with Negative: No color change or development of a Negative: Aerococcus
for the aminopeptidase. Leucine- reagentgrade water. slight yellow color (B) viridans —no color
presumptive beta-naphthylamide– Do not supersaturate change
identification of impregnated disks serve as the disk. Limitations: The test
catalase-negative a substrate for the 2. Using a wooden result depends on the
gram-positive detection of leucine applicator stick, rub a integrity of the
cocci. aminopeptidase. After small amount of substrate impregnated
hydrolysis of the substrate several colonies of an disk.
by the enzyme, the 18- to 24-hour pure
resulting beta- culture onto a small
naphthylamine produces a area of the LAP disk.
red color upon addition of 3. Incubate at room
cinnamaldehyde reagent. temperature for 5
minutes.
4. After this
incubation period,
add one drop of
cinnamaldehyde
reagent.
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

Litmus Milk This test This test is used to 1. Inoculate Fermentation:
Medium differentiates determine an with four drops Clostridium
microorganisms organism’s ability to of a 24-hour perfringens—gas
based on various metabolize litmus milk. broth culture. production
metabolic Fermentation of lactose 2. Incubate at Acid:
reactions in litmus is demonstrated when 35°C to 37°C in Lactobacillus
milk, including the litmus turns pink as ambient air. acidophilus—clot
fermentation, a result of acid 3. Observe daily formation
reduction, clot production. If sufficient for 7 days for Peptonization:
formation, acid is produced, casein alkaline reaction Pseudomonas
digestion, and the in the milk is (litmus turns aeruginosa—
formation of gas. coagulated, solidifying blue), acid clearing
Litmus milk is also the milk. With some reaction (litmus Appearance of
used to grow organisms, the curd turns pink), indicator (litmus
lactic acid shrinks and whey is indicator dye)
bacteria. formed at the surface. reduction, acid
Some bacteria clot, rennet clot, Limitations :
hydrolyze casein, and Litmus media
causing the milk to peptonization. reactions are not
become straw colored Multiple specific and
and resemble turbid changes can should be
serum. In addition, occur over the followed up with
some organisms reduce observation additional tests
litmus, in which case period. for definitive
the medium becomes 4. Record all identification of
colorless in the bottom changes. microorganisms.
of the tube.

Lysine Iron Agar This test is Lysine iron agar contains 1. With a Alkaline slant/alkaline butt (K/K)—lysine Alkaline slant and butt: H 2S positive:
(LIA) used to lysine, peptones, a small straight decarboxylation and no fermentation of Citrobacter freundii
differentiate amount of glucose, ferric inoculating glucose Alkaline slant and butt: Escherichia coli
gram- ammonium citrate, and needle, Alkaline slant/acid butt (K/A)—glucose Alkaline slant and butt: H 2S positive:
negative sodium thiosulfate. The inoculate LIA fermentation Salmonella typhimurium
bacilli based medium has an aerobic slant by twice Note: Patterns shown in Figure 12-24, A Red slant, acid butt: Proteus mirabilis
on and an anaerobic butt. stabbing and C, can be accompanied by a black
decarboxylati When glucose is fermented, through the precipitate of ferrous sulfide (FeS), which Limitations: Proteus spp. that produce
on or the butt of the medium center of the indicates production of H2S hydrogen sulfide will not blacken the
deamination becomes acidic (yellow). If medium to Red slant/acid butt (R/A)—lysine medium. Additional testing, such as triple
of lysine and the organism produces the bottom deamination and glucose fermentation sugar iron agar, should be used as a follow-
the formation lysine decarboxylase, of the tube up identification method.
of hydrogen cadaverine is formed. and then
sulfide (H2S). Cadaverine neutralizes the streaking the
organic acids formed by slant.
glucose fermentation, and 2. Cap the
the butt of the medium tube tightly
reverts to the alkaline state and incubate
(purple). If the at 35°C to
decarboxylase is not 37°C in
produced, the butt remains ambient
acidic (yellow). If oxidative air for 18 to
deamination of lysine 24 hours
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. Bromocresol purple,
the pH indicator, is yellow at
or below pH 5.2 and purple
at or above pH 6.8.
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

Methyl Red and The combination This test is used to determine the 1. Inoculate MRVP broth with one drop from a 24-hour brainheart infusion MR positive/VP
Voges- test methyl red ability of an organism to produce broth culture. negative:
Proskauer Tests (MR) and Voges- and maintain stable acid end 2. Incubate at 35°C to 37°C for a minimum of 48 hours in ambient air. Tests Escherichia coli
Proskauer (VP) products from glucose should not be made with cultures incubated less than 48 hours, because
differentiates fermentation, to overcome the the end products build up to detectable levels over time. If results are MR
members of the buffering capacity of the system, equivocal at 48 hours, repeat the tests with cultures incubated at 35°C to negative/VP
Enterobacteriacea and to determine the ability of 37°C for 4 to 5 days in ambient air; in such instances, duplicate tests should positive:
e family. some organisms to produce be incubated at 25°C. Enterobacter
neutral end products (e.g., 2,3- 3. Split broth into aliquots for MR test and VP test. aerogenes
butanediol or acetoin) from A. Methyl Red (MR) Test
glucose fermentation. The methyl 1. Add five or six drops of methyl red reagent per 5 mL of broth. Limitations: The
red detects mixed acid 2. Read reaction immediately. MR test should
fermentation that lowers the pH of Expected Results not be read
the broth. The MR indicator is Positive: Bright red color, indicative of mixed acid fermentation. before 48 hours,
added after incubation. MR is red Weakly positive: Red-orange color. Negative: Yellow color because some
at pH 4.4 and yellow at pH 6.2. A B. Voges-Proskauer (VP) Test (Barritt’s Method) for organisms will
clear red is a positive result; yellow Gram-Negative Rods not have
is a negative result; and various 1. Add 0.6 mL (6 drops) of solution A (alpha-naphthol) and 0.2 mL (2 drops) produced
shades of orange are negative or of solution B (KOH) to 1 mL of MRVP broth. enough
inconclusive. The VP detects the 2. Shake well after addition of each products from
organism’s ability to convert the reagent. the
acid products to acetoin and 2,3- 3. Observe for 5 minutes. fermentation of
butanediol. Organisms capable of Expected Results glucose.MR-
using the VP pathway produce a Positive: Red color, indicative of negative
smaller amount of acid during acetoin production organisms may
glucose fermentation and Negative: Yellow color also not have
therefore do not produce a color had sufficient
C. Voges-Proskauer (VP) Test
change when the MR indicator is time to convert
(Coblentz Method) for
added. A secondary reagent is those products
streptococci
added, alpha-naphthol, followed and will appear
1. Use 24-hour growth from a blood
by potassium hydroxide (KOH); a MR positive.
agar plate to heavily
positive test result is indicated by a MRVP testing
inoculate 2 mL of MRVP broth.
red color complex. should be used
2. After 6 hours of incubation at 35°C
in conjunction
in ambient air, add 1.2 Ml (12 drops)
with other
of solution A (alpha naphthol) and
confirmatory
0.4 mL (four drops) solution B (40%
tests to
KOH with creatine).
differentiate
3. Shake the tube and incubate at
organisms
room temperature for 30 minutes.
among the
Enterobacteriac
eae.

Microdase Test This test is used to The microdase test is a rapid
(Modified differentiate gram- method to differentiate
Oxidase) positive, catalase- Staphylococcus from
positive cocci Micrococcus spp. by detection
(micrococci from of the enzyme oxidase. In the
staphylococci). presence of atmospheric
oxyen, the oxidase enzyme
reacts with the oxidase reagent
and cytochrome C to form the
colored compound,
indophenol.
Motility Testing These tests are The inoculum is stabbed
used to determine into the center of a
whether an semisolid agar deep.
enteric organism Bacterial motility is evident
is motile. An by a diffuse zone of growth
organism must extending out from the line
have flagella to be of inoculation. Some
motile. organisms grow throughout
the entire medium, whereas
others show small areas or
nodules that grow out from
the line of inoculation.
1. Using a wooden applicator stick, rub a small amount of several colonies of Positive:
an 18- to 24-hour pure culture grown on Micrococcus
blood agar onto a small area of the microdase disk. Note: Do luteus
not rehydrate the disk before use. Negative:
2. Incubate at room temperature for Staphylococcus
2 minutes. aureus
Limitations:
Positive: Development of Staphylococci
blue to purple-blue color should yield a
(A) negative color
Negative: No color change change, except
(B) for S. sciuri, S.
lentus, and S.
vitulus.
1. . Touch a straight Positive: Motile organisms will spread out into Positive: Escherichia
needle to a colony of a the medium from the site of inoculation (A) coli Negative:
young (18- to Negative: Nonmotile organisms remain at the Staphylococcus
24-hour) culture site of inoculation (B) aureus
growing on agar
medium. Limitations: Some
2. Stab once to a depth organisms will not
of only 1/3 to 1/4 inch in display sufficient
the middle of growth in this
the tube. medium to make an
3. Incubate at 35°C to accurate
37°C and examine daily determination, and
for up to 7 days. additional follow-up
testing is required.
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality Control

Lactobacillus This test is used to The MRS broth contains 1. Inoculate MRS broth with an Positive: Leuconostoc spp.—growth; gas Positive:
MRS Broth determine sources of carbon, 18- to 24-hour culture from production indicated by a bubble in the Durham Lactobacillus
whether an nitrogen, and vitamins agar tube lactis
organism forms to support the growth or broth. Negative:
gas during glucose of lactobacilli and other 2. Incubate 24 to 48 hours at Positive: Lactobacillus spp.—growth; no gas Escherichia coli
fermentation. organisms. It is a 35°C to 37°C in ambient air. production
Some selective medium that
Lactobacillus spp. uses sodium acetate Negative: No growth (not shown).
and Leuconostoc and ammonium citrate
spp. produce gas. to prevent overgrowth
by contaminating
organisms. Growth is
considered a positive
result. A Durham tube
may be added to
differentiate
Lactobacillus spp. from
Leuconostoc spp.

4- This test is used to E. coli and other Enterobacteriaceae produce the enzyme 1. Wet the disk with one drop Positive: Escherichia coli
Methylumbellif presumptively b-d-glucuronidase, which hydrolyzes b-d-glucopyranosid- of water. Negative: Klebsiella pneumoniae
eryl-b-d- identify various uronic derivatives to aglycons and d-glucuronic acid. The 2. Using a wooden applicator
genera of substrate 4-methylumbelliferyl-b-d-glucuronide is stick, rub a portion of a colony Limitations: Do not test colonies
Glucuronide
Enterobacteriacea impregnated into the disk and is hydrolyzed by the enzyme from an 18- to 24-hour-old isolated from media containing
(MUG) Test e and verotoxin- to yield the 4-methylumbelliferyl moiety, which fluoresces pure culture onto the disk. dyes (eosin methylene blue [EMB],
producing blue under long wavelength ultraviolet light. However, 3. Incubate at 35°C to 37°C in a MacConkey [MAC]), because it may
Escherichia coli. verotoxin-producing strains of E. coli do not produce MUG, closed container for up to make the interpretation difficult.
and a negative test result may indicate the presence of a 2 hours. Only test on oxidase-positive
clinically important strain. 4. Observe the disk using a organisms, because some oxidase-
366-nm ultraviolet light. negative organisms naturally
fluoresce.
Positive: Electric blue
fluorescence (A)
Negative: Lack of fluorescence
(B)

Nitrate This test is used Anaerobic metabolism 1. Inoculate nitrate broth with The nitrate reduction test is read for the presence Positive: NO 31,
Reduction to determine requires an electron one to two drops from a young or absence of three metabolic products: gas, no gas:
the ability of an acceptor other than broth culture of the test nitrate (NO3), and nitrite (NO2). The expected Escherichia coli
organism to atmospheric oxygen (O2). organism. results can be summarized as follows: Positive: NO 31,
reduce nitrate Many gram-negative 2. Incubate for 48 hours at 35°C gas:
bacteria use nitrate as the to 37°C in ambient air (some Pseudomonas
to nitrite. All
final electron acceptor. organisms may require longer aeruginosa
members of the
The organisms produce incubation for adequate Negative:
Enterobacteriac
nitrate reductase, which growth). Test these cultures 24 Acinetobacter
eae family converts the nitrate (NO3) hours after obvious growth is baumannii
reduce nitrate, to nitrite (NO2). The detected or after a maximum of
but some reduction of nitrate to 7 days. Limitations:
members nitrite is determined by 3. After a suitable incubation Nitrate reduction
further adding sulfanilic acid and period, test the nitrate broth is a supportive
metabolize alpha-naphthylamine. The culture for the presence of gas, test for
nitrite to other sulfanilic acid and nitrite reduction of nitrate, and identification of
compounds. react to form a diazonium reduction of nitrite according to Enterobacteriace
salt. The diazonium salt the following steps: ae to the genus
then couples with the level; however,
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)
Test Purpose Principle
alpha-naphthylamine to
produce a red, water-
soluble azo dye. If no color
change occurs, the
organism did not reduce
nitrate or reduced it
further to NH 3, NO, or
N2O2. Zinc is added at this
point; if nitrate remains,
the zinc will reduce the
compound to nitrite and
the reaction will turn
positive, indicating a
negative test result for
nitrate reduction by the
organism. If no color
change occurs after the
addition of zinc, this
indicates that the
organism reduced nitrate
to one of the other
nitrogen compounds
previously described. A
Durham tube is placed in
the broth for two reasons:
(1) to detect deterioration
of the broth before
inoculation, as evidenced
by gas formation in the
tube, and (2) to identify
denitrification by
organisms that produce
gas by alternate pathways;
if gas is formed in the tube
before the addition of the
color indicator, the test
result is negative for
nitrate reduction by this
method.
Nitrite This test is used to Microorganisms
Reduction determine capable of reducing
whether an nitrite to nitrogen do
organism can not turn color and do
reduce nitrites to produce gas in the
gaseous nitrogen nitrate reduction test
or to other (Procedure 12-30). The
compounds test does not require
containing the addition of zinc
nitrogen. dust.
Method Expected Result Quality Control
a. Observe the inverted additional
Durham tube for the presence follow-up
of gas, indicated by bubbles confirmatory
inside the tube. testing is
b. Add five drops each of required for final
nitrate reagent solution A identification.
(sulfanilic acid) and B (alpha-
naphthylamine). Observe
for at least 3 minutes for a red
color to develop.
c. If no color develops, test
further with zinc powder. Dip a
wooden applicator stick into
zinc powder and transfer
only the amount that adheres
to the stick to the nitrate
broth culture to which solutions
A and B have been
added. Observe for at least 3
minutes for a red color to
develop. Breaking the stick into
the tube after the
addition of the zinc provides a
useful marker for the
stage of testing.
1. Inoculate nitrite broth with Positive: No color change to red 2 minutes after Positive: Proteus
one drop from a 24-hour broth the addition of the reagents; gas production mirabilis—
culture. observed in the Durham tube. colorless; gas
2. Incubate for 48 hours at 35°C Negative: The broth becomes red after the production
to 37°C. addition of the reagents. No gas production is Negative:
3. Examine 48-hour nitrite observed Acinetobacter
broth cultures for nitrogen gas baumannii—red;
in the no gas productio
inverted Durham tube and add
five drops each of the nitrate Limitations: If
reagents A and B to determine the broth does
whether nitrite is still present in not become red
the medium (reagents A and B and no gas
are described under the nitrate production is
reduction test in Procedure 12- observed, zinc
30). dust is added to
determine
whether the
nitrite has
not been
oxidized to
nitrate (thus
invalidating the
test). If oxidation
has occurred,
the mixture
turns red after
the addition of
zinc.
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality

Control
o-Nitrophenyl-b-D- This test is used to Lactose fermenters 1. Aseptically suspend a loop full Positive: Yellow (presence of b-galactosidase) Positive:
Galactopyranoside determine the ability must be able to of organism in 0.85% saline. Negative: Colorless (absence of enzyme) Shigella sonnei
(ONPG) Test of an organism to transport the 2. Place an ONPG disk in the Negative:
produce b- carbohydrate (b- tube. Salmonella
galactosidase, an galactoside 3. Incubate for 4 hours at 37°C in typhimurium
enzyme that permease) and ambient air.
hydrolyzes the hydrolyze (b- 4. Examine tubes for a color
substrate o- galactosidase) the change.
nitrophenyl-b-D- lactose to glucose
galactopyranoside and galactose.
(ONPG) to form a Organisms unable
visible (yellow) to produce b-
product, ortho- galactosidase may
nitrophenol. The test become genetically
distinguishes late altered through a
lactose fermenters variety of
from non–lactose mechanisms and
fermenters of be identified as
Enterobacteriaceae. late-lactose
fermenters. ONPG
enters the cells of
organisms that do
not produce the
permease but are
capable of
hydrolyzing the
ONPG to galactose
and a yellow
compound, o-
nitrophenol,
indicating the
presence of b-
galactosidase.

Optochin (P disk) This test is used to Optochin is an 1. Using an Positive: Zone of inhibition at least 14 mm in diameter, with 6- Positive:
Susceptibility Test determine the effect antibiotic that inoculating loop, mm disk Streptococcus
of optochin interferes with the streak two or Negative: No zone of inhibition pneumoniae
(ethylhydrocupreine ATPase and three suspect
hydrochloride) on an production of colonies of a pure Negative:
organism. Optochin adenosine culture onto half Streptococcus
lyses pneumococci triphosphate (ATP) of a 5% sheep pyogenes
(positive test), but in microorganisms. blood agar plate.
alpha-streptococci are The optochin- 2. Using heated Limitations:
resistant (negative impregnated disk forceps, place an Equivocal: Any
test). (TaxoP) is placed optochin disk in zone of
on a lawn of the upper third of inhibition less
organism on a the streaked area. than 14 mm is
sheep blood agar Gently tap the disk questionable
plate, allowing the to ensure for
antibiotic to adequate contact pneumococci;
diffuse into the with the agar the strain is
medium. The surface. identified as a
antibiotic inhibits 3. Incubate the pneumococcus
the growth of a plate for 18 to 24 with
susceptible hours at 35°C in confirmation
organism, creating 5% CO2. by a positive
a clearing, or zone Note: Cultures do bile-solubility
of inhibition, not grow as well in test.
around the disk. A ambient air, and
zone of 14 to 16 larger zones of
mm is considered inhibition occur.
susceptible and 4. Measure the
presumptive zone of inhibition
identification for in millimeters,
Streptococcus including the
pneumoniae. diameter of the
disk.
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality

Control
Oxidase Test This test determines To determine the 1. Moisten filter paper with the Positive: Development of a dark purple Positive:
(Kovac’s Method) the presence of presence of bacterial substrate (1% tetramethyl- color within 10 seconds Pseudomonas
cytochrome oxidase cytochrome oxidase pphenylenediamine dihydrochloride) Negative: Absence of color aeruginosa
activity in using the oxidation of or select a commercially Negative:
microorganisms for the substrate available paper disk that has been Escherichia coli
the identification of tetramethyl-p- impregnated with the substrate.
oxidase-negative phenylenediamine 2. Use a platinum wire or wooden Limitations
Enterobacteriaceae, dihydrochloride to stick to remove a small portion of a :Using nickel-
base alloy wires
differentiating them indophenol, a dark bacterial colony (preferably not more
containing
from other gram- purple–colored end than 24 hours old) from the agar chromium and
negative bacilli. product. A positive surface and rub the sample on the iron (nichrome)
test (presence of filter paper or commercial disk. to rub the colony
oxidase) is indicated 3. Observe the inoculated area of paste onto the
by the development paper or disk for a color change to filter paper may
of a dark purple color. deep blue or purple (Figure 12-34) cause false-
No color development within 10 seconds (timing is critical). positive results
indicates a negative
test and the absence
of the enzyme.

Oxidation and This test is used This test is used to determine 1. To determine whether Positive: Acid production (A) is indicated by the color Note:
Fermentation to differentiate whether an organism uses acid is produced from indicator changing to yellow in the carbohydrate-containing Appropriate
of Medium microorganisms carbohydrate substrates to carbohydrates, deep. organisms
(CDC Method) based on the produce acid byproducts. inoculateagar deeps, Weak-positive (Aw): Weak acid formation can be detected depend on
ability to oxidize Nonfermentative bacteria are each containing a single by comparing the tube containing the medium with which
or ferment routinely tested for their carbohydrate, with carbohydrate with the inoculated tube containing medium carbohydrate
specific ability to produce acid from bacterial growth from an with no carbohydrate. Most bacteria that can grow in the has been
carbohydrates six carbohydrates (glucose, 18- to 24-hour culture by OF base produce an alkaline reaction in the control tube. If added to the
xylose, mannitol, lactose, stabbing a needle four to the color of the medium in a tube containing carbohydrate basal medium.
sucrose, and maltose). In five times into the remains about the same as it was before the medium was Glucose is
addition to the six tubes medium to a depth of 1 inoculated and if the inoculated medium in the control used as an
containing carbohydrates, a cm. Note: Two tubes of tube becomes a deeper red (i.e., becomes alkaline), the example.
control tube containing the OF dextrose are usually culture being tested is considered weakly positive,
OF base without inoculated; one is assuming the amount of growth is about the same in both Fermenter:
carbohydrate is also overlaid with either tubes. Escherichia coli
inoculated. Triple sugar iron sterile melted Negative: Red or alkaline (K) color in the deep with Oxidizer:
(TSI) agar is also used to petrolatum or sterile carbohydrate equal to the color of the inoculated control Pseudomonas
determine whether an paraffin oil to detect tube. aeruginosa
organism can ferment fermentation. No change (NC) or neutral (N): There is growth in the
glucose. OF glucose is used to 2. Incubate the tubes at media, but neither the carbohydrate-containing medium Limitation:
determine whether an 35°C to 37°C in ambient nor the control base turns alkaline (red). Slow-growing
organism ferments or air for up to 7 days. organisms may
oxidizes glucose. If no Note: If screwcap tubes Note: If the organism does not grow at all in the OF not produce
reaction occurs in either the are used, loosen the medium, mark the reaction as no growth (NG). results for
TSI or OF glucose, the caps during incubation several days.
organism is considered a to allow for air
non–glucose utilizer). Hugh exchange. Otherwise,
and Leifson’s formula uses a the control tube and
low peptone-to- tubes containing
carbohydrate ratio and a carbohydrates that are
limiting amount of not oxidized might not
carbohydrate. The reduced become alkaline.
peptone limits the formation
of alkaline amines that may
mask acid production
resulting from oxidative
metabolism. Two tubes are
required for interpretation of
the OF test. Both are
inoculated, and one tube is
overlaid with mineral oil,
producing an anaerobic
environment. Production of
acid in the overlaid tube
results in a color change and
is an indication of
fermentation. Acid
production in the open tube
and color change is the result
of oxidation.
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality

Control
Phenylalanine This test is used to Microorganisms that 1. Inoculate phenylalanine slant with one Positive: Green color develops on Positive:
Deaminase Agar determine the ability of produce phenylalanine drop of a 24-hour slant after ferric chloride is added Proteus
(PDA) an organism to deaminase remove the brain-heart infusion broth. Negative: Slant remains original color mirabilis
oxidatively deaminate amine (NH2) from 2. Incubate 18 to 24 hours (or until good after the addition of ferric chloride Negative:
phenylalanine to phenylalanine. The growth is apparent) at Escherichia coli
phenylpyruvic acid. reaction results in the 35°C to 37°C in ambient air with the cap
The genera production of ammonia loose.
Morganella, Proteus, (NH3) and 3. After incubation, add four to five
and Providencia can be phenylpyruvic acid. The drops of 10% aqueous ferric
differentiated from phenylpyruvic acid is chloride to the slant.
other members of the detected by adding a
Enterobacteriaceae few drops of 10% ferric
family. chloride; a green-
colored complex is
formed between these
two compounds.
l-Pyrrolidonyl This test is used for the The enzyme l- 1. Before inoculation, moisten Positive: Bright red color within 5 minutes(A) Positive:
ArylAmidAse (Pyr) presumptive pyrrolidonyl the disk slightly with Negative: No color change or an orange color (B) -Enterococcus
Test identification of group arylamidase reagentgrade water. Do not faecalis
A streptococci hydrolyzes the flood the disk. -Streptococcus
(Streptococcus l-pyrrolidonyl-b- 2. Using a wooden applicator pyogenes
pyogenes) and naphthylamide stick, rub a small amount of
enterococci by the substrate to several colonies of an 18- to 24- Negative:
presence of the produce a hour pure culture onto a small Streptococcus
enzyme l pyrrolidonyl b-naphthylamine. area of the PYR disk. agalactiae
arylamidase. The b- 3. Incubate at room temperature
naphthylamine can for 2 minutes.
be detected in the 4. Add a drop of detector
presence of N,N- reagent, N,N-
methylamino- dimethylaminocinnamaldehyde,
cinnamaldehyde and observe for a red color
reagent by the within 1 min
production of a
bright red
precipitate.

Pyruvate Broth This test is used to Pyruvate broth is a 1. Lightly inoculate Positive: Indicator changes from green to yellow Positive:
determine the ability carbohydrate-free, nutrient- the pyruvate broth (A) Enterococcus
of an organism to limited medium. Pyruvic acid with an 18- to 24- Negative: No color change; yellow-green indicates faecalis
utilize pyruvate. This is added to the broth to hour a weak reaction and should be regarded as
aids in the determine whether the culture of the negative (B) Negative:
differentiation microorganism is able to use organism from 5% Streptococcus
between Enterococcus pyruvate, resulting in the sheep blood agar. bovis
faecalis (positive) and formation of metabolic acids. 2. Incubate at 35°C to
Enterococcus faecium Bromothymol blue indicator 37°C in ambient air
(negative). changes from blue to yellow in for 24 to 48 hours.
the presence of acid as a
result of the decrease in pH.

Salt Tolerance Test This test is used to The salt tolerance 1. Inoculate one or two colonies Positive: Visible turbidity in the broth, with or Positive:
determine the ability test is a selective from an 18- to 24-hour culture without a color change from purple to yellow Enterococcus
of an organism to grow and differential into 6.5% NaCl broth. Negative: No turbidity and no color change faecalis—
in high concentrations medium. 2. Incubate the tube at 35°C to growth; color
of salt. It is used to Enterococci are 37°C in ambient air for 48 hours. change to
differentiate resistant to high 3. Check daily for growth. yellow
enterococci (positive) salt concentration. Negative:
from nonenterococci A heart infusion Streptococcus
(negative). broth containing bovis-
6.5% NaCl is used inhibition, as
as the test demonstrated
medium. This broth by little to no
also contains a growth; no
small amount of color change
glucose and
bromocresol
purple as the
indicator for acid
production.
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality

Control
Spot Indole Test This test is used to Tryptophanase 1. Saturate a piece of filter paper Positive: Development of a blue color within 20 Positive:
determine the breaks down with the 1% seconds Escherichia coli
presence of the tryptophan to paradimethylamino- Negative: No color development or slightly pink
enzyme release indole, cinnamaldehyde reagent. color Negative:
tryptophanase. It is a which 2. Use a wooden stick or Klebsiella
rapid method that can is detected by its bacteriologic loop to remove a pneumoniae
be used in lieu of ability to combine small
the tube test. with certain portion of a bacterial colony Limitation: The
aldehydes to from the agar surface and rub bacterial
form a colored the inoculum
compound. For sample on the filter paper. Rapid should not be
indole-positive development of a blue co selected from
bacteria, the indicates a positive test result. MacConk
blue-green Most indole-positive organis agar, because
compound formed turn blue within 30 seconds the color of
by the reaction of lactose-
indole with fermenting
cinnamaldehyde is colonies on
easily visualized. this medium
The absence of can interfere
enzyme with test
results in no color interpretation.
production (indole
negative).

Triple Sugar Triple sugar iron The composition of TSI is 10 parts 1. With a straight Alkaline slant/no change in the butt (K/NC): Ⓐ, gas
Iron (TSI) Agar (TSI) agar is used lactose:10 parts sucrose:1 part glucose inoculation glucose, lactose, and sucrose nonutilizer; this may production:
to determine and peptone. Phenol red and ferrous needle, touch the also be recorded as K/K (alkaline slant/alkaline Escherichia coli
whether a gram- sulfate serve as indicators of acidification top of a butt) (A)
negative rod and H 2S formation, respectively. A wellisolated K/A, 1/2 gas
ferments glucose glucose-fermenting organism turns the colony. Alkaline slant/acid butt (K/A): glucose production,
and lactose or entire medium acidic (yellow) in 8 to 12 2. Inoculate TSI fermentation only. (B) H2S1:
sucrose and forms hours. The butt remains acidic after the (Figure 12-41, D) Salmonella
hydrogen sulfide recommended 18- to 24-hour incubation by first stabbing Acid slant/acid butt (A/A): glucose, sucrose, typhimurium
(H2S). The test is period because of the presence of organic through the and/or lactose fermenter (C)
used primarily to acids resulting from the fermentation of center of the K/K:
differentiate glucose under anaerobic conditions in the medium to the Note: A black precipitate in the butt indicates Pseudomonas
members of the butt of the tube. The slant, however, bottom of the production of ferrous sulfide and H 2S gas (H2S1) aeruginosa)
Enterobacteriaceae reverts to the alkaline (red) state because tube and then .Bubbles or cracks in the tube indicate the
family from other of oxidation of the fermentation products streaking the production of CO2 or H2. Drawing a circle around K/A, H2S1:
gram-negative under aerobic conditions on the slant. surface of the the A for the acid butt; that is,Ⓐ, usually indicates Proteus
rods. This change is a result of the formation of agar slant. this means the organism ferments glucose and mirabilis
CO2 and H 2O and the oxidation of 3. Leave the cap sucrose; glucose and lactose; or glucose, sucrose,
peptones in the medium to alkaline on loosely and and lactose, with the production of gas. K/A: Shigella
amines. When, in addition to glucose, incubate the tube flexneri
lactose and/or sucrose are fermented, the at 35°C to
large amount of fermentation products 37°C in ambient Note: gas
formed on the slant neutralizes the air for 18 to 24 production
alkaline amines and renders the slant hours. may also be
acidic (yellow), provided the reaction is indicated with
read in 18 to 24 hours. Reactions in TSI a lower case
should not be read beyond 24 hours of (g) or upper
incubation, because aerobic oxidation of case (G) as
the fermentation products from lactose indicated:
and/or sucrose proceeds, and the slant
eventually reverts to the alkaline state. Small amount
The formation of CO2 and hydrogen gas of gas: g
(H2) is indicated by the presence of
bubbles or cracks in the agar or by Large amount
separation of the agar from the sides or of gas: G
bottom of the tube. The production of
H2S (sodium thiosulfate reduced to H 2S)
requires an acidic environment, and
reaction with the ferric ammonium citrate
produces a blackening of the agar butt in
the tube.
 Overview of Bacterial Identification Methods and Strategies
(Bailey and Scott’s Diagnostic Microbiology)

Test Purpose Principle Method Expected Result Quality

Urease Test This test is used to Urea is the product of
(Christensen’s determine an decarboxylation of amino
Method) organism’s ability to acids. Hydrolysis of urea
produce the enzyme produces ammonia and CO2.
urease, which The formation of ammonia
hydrolyzes urea. alkalinizes the medium, and
Proteus spp. may be the pH shift is detected by the
presumptively color change of phenol red
identified by the from light orange at pH 6.8 to
ability to rapidly magenta (pink) at pH 8.1.
hydrolyze urea. Rapid urease-positive
organisms turn the entire
medium pink within 24 hours.
Weakly positive organisms
may take several days, and
negative organisms produce
no color change or yellow as a
result of acid production.
Control
1. Streak the surface of Positive: Change in color of slant from Positive: Proteus vulgaris
a urea agar slant with a light orange to magenta (A) Weak positive: Klebsiella
portion of a Negative: No color change (agar slant pneumoniae
well-isolated colony or and butt remain light orange) (B) Negative: Escherichia coli
inoculate slant with
one to two drops Limitations: Alkaline
from an overnight reactions may appear
brain-heart infusion after prolonged
broth culture. incubation and may be
2. Leave the cap on the result of peptone or
loosely and incubate other protein utilization
the tube at 35°C to raising the pH. To
37°C in ambient air for eliminate false-positive
48 hours to 7 days. reactions, perform a
control test with the base
medium without urea.

X and V Factor Test The X and V factor test A lawn of the test 1. Make a very light suspension Positive: Growth around the XV disk only shows a Positive:
is used to differentiate organism is (MacFarland 0.5) of the requirement for both factors(A). Growth around Haemophilus
Haemophilus spp. streaked onto organism the V disk, no growth around the X disk, and light influenza, halo
Members of the genus heart infusion in sterile saline. Note: It is growth around the XV disk shows a V factor of growth
Haemophilus require agar, tryptic soy important not to carry over any requirement.(B) around the
accessory growth agar, Haemophilus X factor in the medium from Negative: Growth over the entire surface of the XV disk, no
factors in vitro. Some agar, or nutrient which the organism is taken. agar indicates no requirement for either X or V growth on the
Haemophilus spp. agar. The Therefore a loop, not a swab, factor (C) rest of the
require X factor impregnated disks should be used to make the agar surface
(hemin) or strips (X, V, or suspension. Haemophilus
alone, V factor XV) are placed 2. Dip a sterile swab into the parainfluenza,
(nicotinamide adenine directly on organism suspension. Roll the halo of growth
dinucleotide [NAD]) the confluent swab over the entire surface of a around
alone, inoculation, trypticase soy agar plate. the XV and V
or a combination of allowing diffusion 3. Place the X, V, and XV factor disks
the two. of the accessory disks on the agar surface. If Haemophilus
growth factor into using separate disks, place them ducreyi, halo
the medium. The at least 4 to 5 cm apart. of growth
organisms will 4. Incubate overnight at 35°C to around the XV
grow only around 37°C in ambient air. and X disks
the disk that
provides the
appropriate factor
for growth of the
organism.