Overview of Bacterial Identification Methods and Strategies

(Bailey and Scott’s Diagnostic Microbiology)

Test Incubation Expected Results (+) Control (-) Control Comments
Acetamide utilization 35’C for up (+) Blue Pseudomonas Stenotrophomonas
to 7 days (-) Green aeruginosa maltophilia
Acetate utilization 35’C for up (+) Blue Escherichia coli Shigella flexneri
to 7 days (-) Green
Bacitracin test 35’C for (+) Any zone of Streptococcus Streptococcus Amount of Bacitracin:
18-24 hrs inhibition pyogenes agalactiae 0.04 U
(-) No zone
Bile Esculin agar 35’C for (+) Blackening of agar Enterococcus Streptococcus mitis Conc.: 40% Bile
48 hrs (-) No blackening faecalis Indicator:
Ferric ammonium citrate
Bile Solubility test 35’C for (+) Colony disintegrates Streptococcus Enterococcus Rgt: 10% Sodium desoxycholate
30 mins (-) Intact colonies pneumoniae faecalis
Butyrate disk Room temp (+) Blue color Moraxella Neisseria Rgt: Bromo-chloro-indolyl
for 5 mins (-) No color change catarrhalis gonorrhoeae butyrate
CAMP test 35’C (+) Arrowhead zone of Streptococcus Streptococcus CAMP:
overnight beta-hemolysis agalactiae pyogenes Christie
(-) No enhancement of Atkins
hemolysis Munch
Peterson
Catalase test (+) Bubble formation Staphylococcus Streptococcus Rgt: 30% H2O2
(-) No or few bubbles aureus pyogenes BAP contains blood
(Hgb  false positive)
Cetrimide 35’C for up (+) Growth Pseudomonas Escherichia coli
to 7 days (-) No growth aeruginosa
Citrate utilization 35-37’C for (+) Growth w/ or w/o Klebsiella Escherichia coli Indicator: Bromthymol blue
up to 7days change in color from pneumoniae Medium: Simmon’s citrate agar
green to blue
(-) No growth
Coagulase test b. tube test: a. Slide test Staphylococcus Staphylococcus Slide test: detects clumping
a. Slide test 35’C for (+) Clumping aureus epidermidis factor (bound coagulase)
b. Tube test 4 hours (-) No clumping Tube test: detects free coagulase
b. Tube test Rgt: Rabbit EDTA plasma
(+) Clot formation
(-) No clot

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 Test Incubation Expected Results (+) Control (-) Control Comments
Decarboxylase test Glucose non (+) Alkaline (purple Lysine Lysine
(Moeller’s method) fermenters: color) Klebsiella Enterobacter
35’C for 7d (-) Acid (yellow color) pneumoniae cloacae
Glucose Ornithine Ornithine
fermenters: Enterobacter Klebsiella
35’C for 4d cloacae pneumoniae
Arginine Arginine
Enterobacter Klebsiella
cloacae pneumoniae
DNA Hydrolysis 35’C for (+) Colorless Staphylococcus Staphylococcus Medium: DNase agar (DNA-
(DNase) 13-24 hours (-) Green aureus epidermidis methyl green complex)
Serratia
marcescens
Esculin Hydrolysis 35’C for (+) Blackened medium Klebsiella Shigella flexneri
7 days and loss of fluorescence pneumoniae
under Wood’s lamp
(-) No blackening and no
loss of fluorescence
under Wood’s lamp
Fermentation Media a. Peptone: a. Peptone medium a. Peptone a. Peptone medium a. Peptone medium:
a. Peptone medium 35’C for up (+) Pink w/ or w/o gas medium Pseudomonas Indicator: Andrade’s indicator
b. Heart Infusion to 7 days formation in Durham w/ gas: aeruginosa b. Heart infusion broth:
b. Heart tube Escherichia coli b. Heart infusion Indicator: Bromcresol purple
infusion: (-) Growth but no change w/o gas: broth
35’C for in color Shigella flexneri Sorbitol:
4 days b. Heart infusion broth b. Heart infusion Streptococcus mitis
(+) Yellow broth
(-) Growth but no change Sorbitol:
in color (purple) Streptococcus
mutans

Flagella Stain (+) Flagella Peritrichous: Klebsiella Rgt: RYU flagella stain
(Wet-Mount technique) a. Peritrichous Escherichia coli pneumoniae
b. Lophotrichous Polar:
c. Polar Pseudomonas
d. Tufted aeruginosa

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 Test Incubation Expected Results (+) Control (-) Control Comments
Gelatin Hydrolysis 35’C (+) Partial or total Proteus vulgaris Enterobacter
(Gelatinase) (or 25’C) liquefaction at 4’C aerogenes
for up to 14 (-) Complete
days solidification at 4’C
Growth at 42’C 35’C and (+) Good growth at both Pseudomonas Pseudomonas
42’C for 35’C and 24’C aeruginosa fluorescens
18-24 hrs (-) No growth at 42’C but
good growth at 35’C
Hippurate test 35’C for (+) Deep purple color Streptococcus Streptococcus
2 hrs (-) Colorless or slightly agalactiae pyogenes
yellow pink color
Indole production 35’C for (+) Pink- to wine-colored a. Kovac’s method a. Kovac’s method
a. Kovac’s method 48 hours ring Escherichia coli Klebsiella
b. Ehrlich’s method (-) No color change b. Ehrlich’s pneumoniae
method b. Ehrlich’s method
Elizabethkingia CDC group EO-2
meningoseptica
LAP test Room temp (+) Red color Enterococcus Leuconostoc sp Rgt: cinnamaldehyde
for 5 mins (-) No color change or faecalis
dev’t of slight yellow
color
Litmus milk 35-37’C Color of Indicator: Alkaline: Indicator: Litmus paper
Acid: Pink, mauve Alcaligenes
Alkaline: Blue faecalis
No change: Purple Acid:
White: Reduction of Enterococcus
indicator faecium
Consistency of Milk Peptonization:
Clot/Coagulation: Acid or Burkholderia
Alkaline pH cepacia
Digestion: Dissolution of
clot w/ shrunken,
insoluble pink clot (acid)
Peptonization:
Dissolution of clot w/
shrunken, insoluble blue
clot (alk)

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 Test Incubation Expected Results (+) Control (-) Control Comments
Lysine Iron Agar 35’C for K/K: K/K w/ H2S: K/A: Decarboxylation: Butt (Purple)
18-24 hrs (+) Decarboxylation Salmonella Shigella flexneri Deamination: Slant (Red)
(-) Glucose fermentation typhimurium H2S indicator: Ferric ammonium
K/A: R/A: citrate
(+) Glucose fermentation Proteus vulgaris
(-) Decarboxylation
K/A or K/K w/ H2S:
(+) Black precipitate
(FeS)
R/A:
(+)Deamination
(+) Glucose fermentation
Methyl Red test 35-37’C for (+) Bright red color Escherichia coli Enterobacter MR detects mixed acid
48 hrs (W+) Red-orange color cloacae fermentation
(-) Yellow Indicator: Methyl red
Voges-Proskauer b. Coblentz (+) Red color a. Barritt’s method a. Barritt’s method VP detects acetoin
a. Barritt’s method (For method: (-) Yellow color Enterobacter Escherichia coli (acetylmethylcarbinol) formation
Gram-negative rods) Room temp cloacae b. Coblentz method Rgts:
b. Coblentz method (For for 30 mins b. Coblentz Streptococcus mitis a. Barritt’s mtd: α-naphthol +
Streptococci) method KOH
Streptococcus b. Coblentz mtd: α-naphthol +
mutans 40% KOH w/ creatine
Microdase test Room temp (+) Blue to purple-blue Micrococcus luteus Staphylococcus Modified oxidase rgt:
for 2 mins color aureus Tetramethyl-p-
(-) No color change phenylenediamine
dihydrochloride in
dimethylsulfoxide
Motility testing b. Semisolid a. Hanging drop Escherichia coli Klebsiella Coverslip size: 22 x 22 mm
a. Hanging drop agar deep: (+) True motility pneumoniae In Brownian movement, the
b. Semisolid agar deep 35-37’C for (-) Brownian movement organisms may appear quite
up to 7days b. Semisolid agar deep active, but they remain in the
(+) Motile: spread out same relative position to other
into the medium from organisms or debris in the field
the site of inoculation
(-) Nonmotile: remain at
the site of inoculation

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 Test Incubation Expected Results (+) Control (-) Control Comments
MRS Broth 35’C for (+) Gas production Leuconostoc Pediococcus Durham tube detects gas
24-48 hours (-) No gas production formation (bubbles)
MUG test 35’C for (+) Electric blue Escherichia coli Pseudomonas MUG: 4-methylumbelliferyl-beta-
up to 2 hrs fluorescence aeruginosa D-glucuronide
(-) Lack of fluorescence Uses 366nm UV light
Nitrate reduction 35’C for 48 After addition of the rgts: NO3+, no gas: Acinetobacter spp. Reagents:
(NO3NO2) hours or (+) Red color Escherichia coli Sulfanilic acid
max. of 7d (-) Colorless NO3+, w/ gas: Alpha-naphthylamine
If colorless, add Zinc Pseudomonas Zinc dust: to make sure the
dust: aeruginosa nitrite has not been oxidized to
(+) Colorless nitrate
(-) Red color
Nitrite reduction 35’C for (+) Colorless w/ gas Alcaligenes Alcaligenes Durham tube detects nitrogen
(NO2N2) 48 hours (-) Red w/o gas faecalis piechaudii gas formation
Zinc dust: to make sure the
nitrite has not been oxidized to
nitrate
ONPG 37’C for (+) Yellow Escherichia coli Salmonella ONPG: O-Nitrophenyl-beta-D-
4 hours (-) Colorless typhimurium galactopyranoside
Optochin test 35’C for (+) Zone of inhibition Streptococcus Streptococcus mitis Optochin (P disk):
18-24 hrs >14mm pneumoniae Ethylhydrocupreine
w/ 5% CO2 (-) No zone of inhibition hydrochloride
Equivocal: <14 mm zone Medium: 5% sheep BAP
of inhibition (perform
bile solubility test)
Oxidase test (+) Dark purple color Neisseria Escherichia coli Oxidase rgt:
(Kovac’s method) (-) Absence of color gonorrhoeae Tetramethyl-p-
phenylenediamine
dihydrochloride
Oxidation/Fermentation 35’C for up (+) Yellow (Acid deep) Fermenter: Nonutilizer: Stab agar deeps using a needle 4-
(OF) medium to 7 days (W+) Deeper red (Alk) Escherichia coli Alcaligenes faecalis 5x to a depth of 1 cm
(CDC method) (-) Red (alkaline deep) Oxidizer:
(NC or N): No change or Pseudomonas
Neutral, there is growth aeruginosa
in the media
(NG): No growth

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 Test Incubation Expected Results (+) Control (-) Control Comments
Phenylalanine 35’C for (+) Green color Proteus vulgaris Escherichia coli Rgt: 10% Ferric chloride
Deaminase 18-24 hrs (-) No color change
PYR test Room temp (+) Bright red color Enterococcus Streptococcus mitis PYR: L-pyrrolidonyl-beta-
(PYRase) for 2 mins (-) No color or an orange faecalis naphthylamide
color Rgt: N,N-
Methylaminocinnamaldehyde
Pyruvate broth 35’C for (+) Yellow Enterococcus Enterococcus
24-48 hours (-) No color change or faecalis faecium
yellow-Green
Salt Tolerance test 35’C for (+) Visible turbidity w/ Enterococcus Streptococcus mitis Conc.: 6.5% NaCl
48 hours or w/o color change faecalis Indicator: Bromcresol purple
from purple to yellow
(-) No turbidity and no
color change
Spot Indole test (+) Blue color Escherichia coli Enterobacter Rgt: 1% paradimethylamino-
(-) No color or slightly cloacae cinnamaldehyde
pink color Inoculum should not be selected
from MAC because the color of
lactose-fermenting colonies can
interfere w/ test interpretation
Triple Sugar Iron agar 35’C for K/K: A/A w/ gas: K/K: TSI contains:
(TSI) 18-24 hrs Glucose, lactose and Escherichia coli Pseudomonas 1 part glucose
sucrose nonutilizer K/A w/ H2S: aeruginosa 10 parts lactose
K/A: Salmonella typhi 10 parts sucrose
Glucose fermentation Peptone
only Indicators:
A/A: pH: phenol red
Glucose, sucrose, and/or H2S: ferrous sulfate
lactose fermenter
Black ppt (butt):
H2S production
Bubbles/cracks:
CO2 or H2 (gas)
production
Urea Hydrolysis 35’C for (+) Magenta color Proteus vulgaris Escherichia coli Indicator: phenol red
(Christensen’s method) 48 hrs up to (-) Light orange Ammonia alkalinizes the medium
7 days pH 6.8: light orange
pH 8.1: magenta

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 Test Incubation Expected Results (+) Control (-) Control Comments
X and V factor test Overnight (+) Growth: Around XV disk: Haemophilus X factor: hemin/hematin
at 35’C Growth around XV disk: Haemophilus aphrophilus (does V factor: NAD
Shows a requirement for influenzae not need X, V, or XV Medium: TSB
both factors Around XV and V factors for growth) Avoid carrying over of any X-
Growth around V disk, disks: factor contained in the medium
no growth around X disk, Haemophilus the organism is taken from
and light growth around parainfluenzae A loop, not a swab, should be
XV disk: used to make a suspension
Shows a V factor
requirement
(-) Growth over entire
surface of the agar

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