B A C T 2 1 1 / LABORATORY / B S M L S
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FINALS | BACT 211 | BIOCHEMICAL IDENTIFICATION OF GRAM-NEGATIVE BACTERIA
Family Enterobacteriaceae

Broth cultures of Y. pestis exhibit a characteristic
“stalactite pattern”, Y. enterocolitica produces bull’sGram negative bacilli and coccobacilli, non-spore
eye colonies
forming, facultative anaerobic
Does not produce cytochrome C oxidase except for
Plesiomonas which is oxidase positive
All ferment glucose
Motile at body temperatures except for Klebsiella,
Shigella, and Yersinia
Often referred to as enterics
Rod-shaped or elongated in shape
LABORATORY DIAGNOSIS
Specimen Collection
To ensure isolation of both opportunistic and fastidious
pathogens, laboratories must provide appropriate transport
media, such as Cary-Blair, Amies, or Stuart media.
Typically isolated from variety of sources in combination with other
more fastidious organisms. We have to collect fecal specimen or
stool sample usually within the first four days of illness and prior
to administering antibiotics.
These specimens should be processed within 1-2 hours of
collection, otherwise, it must be placed on transport media. It should
be kept in 4°C.
Gram Stain
Direct smear examination of stool samples is not
particularly helpful in identifying enteric pathogens [because it is
normal to have these in stool samples] but may reveal the presence
of inflammatory cells.
Culture

Grow at an optimal temperature of 35°C to 37°C
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Low temperatures (1°C to 5°C such as Serratia and
Yersinia)
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Or tolerate high temperatures (45°C to 50°C, such as E.
coli)
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Visible after 18 to 24 hours of incubation
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BAP, CAP, MacConkey Agar and other selective medium
MacConkey Agar
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Best used to characterize gram negative rods
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Lactose fermenters can be differentiated with non-lactose
fermenters
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Lactose fermenter: dark pink colonies
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Non-lactose fermenter: clear colonies
Lactose fermenter

Rapid Lactose Fermenter [can ferment lactose within 18 to 24
hours of incubation]: Escherichia, Enterobacter, Klebsiella

Late Lactose Fermenter [can ferment lactose within 48 to 72
hours of incubation]: Hafnia, Serratia, Citrobacter, Salmonella
arizonae, Shigella sonnei, and Yersinia enterocolitica.
Non-lactose fermenter

All Salmonella except S. arizonae

All Shigella except S. sonnei

All Yersinia except Y. enterocolitica

Proteus

Providencia

Morganella

Edwardsiella
In performing culture, we can use:
1. General media

BAP

CAP
2. Selective and Differential

MacConkey Agar

Eosin Methylene Blue Agar

Hektoen Enteric Agar
—depending on the suspected organism.
Colonial Appearance

All Enterobacteriaceae produce similar growth on blood
and chocolate agars; colonies are large, gray, and
smooth
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Klebsiella or Enterobacter may be mucoid [because of the
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
presence of their polysaccharide capsule]
P. mirabilis, P. penneri, and P.vulgaris “swarm” on blood
and chocolate agars [swaming motility]
Y. pestis on 5% sheep blood agar are pinpoint at 24
hours but exhibit a rough, cauliflower appearance at
48 hours
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TRANS: BACT – LEC - 211
Nonfermenters:
Translucent, either
amber or colorless.
Gram-negative
broth (GN)
BIOCHEMICAL MEDIA USED IN THE DIFFERENTIATION
AND ISOLATION OF ENTEROBACTERIACEA
Media
Selective
Blood agar
(sheep) (BA)
Bismuth
sulfite agar
Brilliant
green agar
Cefiximetelluritecontaining
MacConkeys
(CT-SMAC)
Cefsulodinirgasan
novobiocin
(CIN)
Media
Nutritional
Hemolysis of RBCs:
Beta: complete lysis
Alpha: partial,
greening
Gamma: nonhemolytic
Routinely used to
cultivate moderately
fastidious
organisms;
trypticase soy agar
with 5%-10%
defibrinated blood
Selective inhibition
of most grampositive and gramnegative bacteria
(bismuth sulfite
and brilliant green)
Production of
hydrogen sulfide
(H2S): ferrous sulfate.
Positive reactions
appear as brown to
black precipitate
Selective; brilliant
green is inhibitory
to most grampositive and gramnegative bacteria
Lactose and sucrose
fermentation; positive
(phenol red)
fermenters appear as
yellow to green
colonies with bright
yellow to green halos.
Salmonella spp.
Appear as white to
pink or red colonies
surrounded by a bright
halo.
Selective inhibition
of most nonvercytotoxigenic E.
coli and nonsorbitol fermenter
(sorbitol, cefixime,
tellurite)
Fermentation of
sorbitol in the
presence of neutral
red. Positive colonies
appear pink and nonfermenters will appear
colorless.
Selective inhibition
of gram-negative
and gram-positive
organisms
Fermentation of
mannitol in the
presence of neutral
red. Macroscopic
colonial appearance:
colorless or pink
colonies with red
center.
Selective
Differential
Citrate as the sole
carbon source,
ammonium salt as
nitrate. Ammonium
salt alteration
changes pH to
alkaline,
bromothymol blue
shifts from green to
blue
Incorporate amino
acid as differential
media (e.g., lysine,
arginine, or
ornithine).
Decarboxylation
yields alkaline, pHsensitive
bromocresol purple
dye. Basal medium
serves as a control.
Citrate agar,
Simmons
(CIT)
Decarboxylase
(ornithine,
arginine,
lysine)
Eosin /
methylene
blue agar
(EMB)
Differential
Eosin Y and
methylene blue
dyes inhibit the
growth of the
gram-positive
bacteria
Incubate for up to 4
days. Fermentative
organisms turn
media yellow,
using glucose. (H+)
increases, making
optimal conditions
for
decarboxylation.
Conversion of the
amino acids to free
amine groups
raising the pH,
reversing the
yellow to purple.
Nonfermenters turn
the purple a deeper
color.
Lactose and
sucrose for
differentiation
based on
fermentation.
Sucrose is an
alternate energy
source for slow
lactose fermenters,
allowing quick
differentiation from
pathogens.
Beef extract,
peptones and
dextrose
Bile salts inhibit
gram-positive
and many gram
negative normal
intestinal flora.
MacConkey
agar (MAC)
Bile salts and
crystal violet
inhibit most
gram-positive
organisms and
permit growth of
gram-negative
rods
Screening
colonies for
the oxidase
enzyme
Isolation of
Salmonella
spp.
Enzymatic digests of
animal tissue,
casein, lactose and
sucrose
Deoxycholate
and citrate salts
inhibit grampositive
bacteria.
Hektoen
enteric agar
(HE)
Purpose
Isolation of
Salmonella
spp.
Increasing
mannitol, which
temporarily
favors the
growth of
mannitolfermenting,
gram-negative
rods (e.g.,
Salmonella and
Shigella spp.)
Differential lactose,
salicin, and
sucrose with a pH
indicator
bromothymol blue
and ferric salts to
detect hydrogen
sulfide (H2S). Most
pathogens ferment
one or both sugars
and appear bright
orange to salmon
pink because of the
pH interaction with
the dye.
Lactose serves as
the sole
carbohydrate.
Lactose fermenters
produce pink or red
colonies;
precipitated bile
salts may surround
colonies. Nonlactose fermenters
appear colorless or
transparent.
Detection of enteric
pathogens from
feces or from
selective enrichment
broth
Selection for gramnegatice organisms
and differentiating
Enterobacteriaceae
Same as regular
MacConkey except
D-sorbitol is
substituted for
lactose. Sorbitolnegative organisms
are clear and may
indicate E. coli
O157:H7
MacConkeysorbitol (MACSOR)
Enhances the
recovery of enteric
pathogens from
fecal specimens
Used to isolate
Escherichia coli
O157:H7
Determine motility
for an organism.
Pancreatic digests
of gelatin, peptone,
and sorbitol
Isolation and
identification
of E. coli
O157-H7
Motility test
medium
Isolation of
Yersinia
enterocolitica
Nutritional
Purpose
Media
Detect organisms
capable of citrate
utilization
Differentiate
fermentative and
nonfermentative
gram-negative
bacteria
Identification of
gram-negative
bacteria.
Escherichia coli:
Lactose fermenter,
forms blue-back with
a metallic green
sheen
Selective
SalmonelaShigella agar
(SS)
Bile salts,
sodium citrate,
and brilliant
green, which
inhibit grampositive
organisms and
some lactosefermenting,
gram-negative
rods normally
found in the
stool.
Selenite
broth
Selective
inhibition of
gram-positive
and many gramnegative
organisms
Urea agar
Other coliform
fermenters: from
pink colonies
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Identification and
differentiation of
Enterobacteriaceae
Shigella and
Klebsiella spp.:
nonmotile;
Nonmotile
organisms grow
clearly only on stab
line, and the
surrounding
medium remains
clear. Motile
organisms move
out of the stab line
and make the
medium appear
diffusely cloudy.
Differential
Yersinia sp.: motile
at room temperature
Listeria
monocytogenes (not
an
Enterobacteriaceae):
umbrella-shaped
motility
Nutritional
Lactose is the sole
carbohydrate, and
the neutral red is the
pH indicator.
Fermenters produce
acid and change the
indicator to pink-red.
Sodium thiosufate is
added as a source
of sulfur for the
production of
hydrogen sulfide.
Also includes ferric
ammonium citrate to
react with H2S and
produce a black
precipitate in the
center of the colony.
Shigella spp. appear
colorless.
Salmonella spp. are
colorless with a
black center
Select for
Salmonella spp.
and strains of
Shigella from stool
specimens
Enzymatic
digestion of
casein and animal
tissue and lactose
Urea is hydrolyzed
to form carbon
dioxide, water, and
ammonia. Ammonia
reacts with the
components of the
medium to form
ammonium
carbonate, raising
the pH, which
changes the pH
indicator, phenol
red, to pink. Limited
protein in the
medium prevents
protein metabolism
from causing a
false-positive
reaction.
Purpose
Selective
enrichment for the
growth of
Salmonella spp.
Identification of
Enterobacteriaceae
species capable of
producing urease.
(Citrobacter,
Klebsiella, Proteus,
Providencia, and
Yersinia spp.)
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TRANS: BACT – LEC - 211

Sucrose and lactose
in excess
concentrations and
xylose in lower
amounts. Phenol
red is the pH
indicator.

Lysine is included to
detect
decarboxylation.
A. CARBOHYDRATE UTILIZATION
Two enzymes are necessary for a bacterium to take up lactose
and to cleave it into monosaccharides:
A. ß-galactoside permease (lactose permease) [transport
Sodium
thiosulfate/ferric
ammonium citrate
allows the
production of H2S.
Xyloselysinedeoxycholate
agar (XLD)
Sodium
deoxycholate
inhibits grampositive cocci
and some gramnegative rods.
Contains less
bile salts than
other
formulations of
enteric media
(e.g., SS, HE)
and therefore
permits better
recovery.
The following types
of colonies may be
seen:
Yellow:
Fermentation of
excess
carbohydrates to
produce acid;
because of the
carbohydrate use,
the organisms do
not decarboxylate
lysine, even though
they may have the
enzyme
Lysine iron agar (LIA) to determine lysine decarboxylase
activity
Sulfide-indole-motility (SIM) or motility-indole-ornithine
(MIO) media
enzymes which facilitates the entry of lactose into the
plasma membrane of the bacteria]
ß-galactosidase [hydrolyzes lactose into glucose and
galactose]
B.
Selective media
used to isolate
Salmonella and
Shigella spp. from
stool and other
specimens
containing mixed
flora
Colorless or red:
Produced by
organisms that do
not ferment any of
the sugars.
LFs: possess both ß-galactoside permease and ßgalactosidase
NLFs: do not possess either enzyme
LLFs: lack ß-galactoside permease but possess ßgalactosidase
A1. Oxidation-Fermentation Tests
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Yellow to red:
Fermentation of
xylose (yellow), but
because it is in
small amounts, it is
used up quickly, and
the organisms
switch to
decarboxylation of
lysine, turning the
medium back to red.
Oxidation: to utilize carbohydrate aerobically
Fermentation: to utilize carbohydrate aerobically
OF Basal Medium [contains 1% carbohydrate that is also present in
TSI]
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Black precipitate is
formed from the
production of H2S.
pH indicator: bromthymol blue
Uninoculated medium: green
Acid Environment: yellow
Alkaline Environment: blue
When O/F tests are performed, two tubes of Hugh-Leifson
OFBM are inoculated; one is overlaid with sterile mineral oil
to create an anaerobic environment (closed), and the other
tube is left aerobic (open), without mineral oil overlay.
Reactions:
Acid produced on both tubes: oxidizer
and fermenter
Acid only in closed tube: fermenter and
possible obligate anaerobe
Acid in open tube: oxidizer
A2. Triple Sugar Iron (TSI)
Used to determine glucose and lactose or sucrose utilization
and H2S production, gas production
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Serratia marcescens produce red pigment due to the production
of prodigiosin.
E. coli has greenish metallic sheen on Eosin Methylene Blue
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Composition: 10 parts lactose, 10 parts sucrose, 1 part
glucose, and peptone
pH indicator: phenol red
H2S indicator: Ferrous sulfate and sodium thiosulfate
The picture on the left shows an uninoculated TSI
agar.
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Slant portion is aerobic; used to determine
if
the
organism
can
ferment
lactose/sucrose

Butt portion is anaerobic; used to
determine if the organism can ferment
glucose
BIOCHEMICAL TESTS
TRADITIONAL BIOCHEMICAL TESTS FOR IDENTIFICATION
FOR GRAM-NEGATIVE BACTERIA
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Triple sugar iron (TSI) agar or Kligler iron agar (KIA) to
determine glucose and lactose, or sucrose, utilization
(sucrose in TSI only) and hydrogen sulfide production
Methyl red and Voges-Proskauer tests to determine end
products of glucose fermentation
Indole test to determine whether indole is formed from
tryptophan by tryptophanase
Urease test to determine hydrolysis of urea
Simmon’s citrate to determine whether citrate can be used
as the sole carbon source
Carbohydrate fermentation
Slant / butt turns yellow at 6.8 pH / acidic
environment which implies that fermentation has
occurred. If slant turns yellow, the organism can
ferment lactose/sucrose. If the butt turns yellow, the
organism can ferment glucose.
Reactions should be read within 18 to 24-hour incubation
period; otherwise, erroneous results are possible
Reactions
[A stands for acid; K stands for alkaline]
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1. A/A (Acid(yellow) slant/acid (yellow) butt): Lactose /
sucrose, glucose fermenter
2. K/A (Alkaline (red) slant/acid (yellow) butt): Glucose
fermenter
3. K/K (Alkaline (red) slant/alkaline (red) butt): No
fermentation; not a member of Enterobacteriaceae [because all
of them ferment glucose]
4. H2S production: (K/A, H2S) or (A/A, H2S) black precipitate;
requires acid environment
This shows the 2-step process through which a black precipitate is
formed.
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H2S is formed from the sodium thiosulfate;

Because H2S gas is colorless, we need an indicators (ferrous
sulfate) which is why a black precipitate is formed.
Since H2S can only be produced in acid environment, it is safe to
assume that if the butt / slant portion has turned black, it is yellow
underneath (fermenter).
Enterobacteriaceae membersthat are H2S producers

Salmonella

Proteus

Arizona

Citrobacter

Edwardsiella
5. Gas production: formation of bubbles or splitting of the
medium in butt
B. GLUCOSE METABOLISM AND ITS METABOLIC
PRODUCTS
B.1 Methyl Red Test

If glucose is metabolized by the mixed acid
fermentation pathway, stable acid end products are
produced, which results in a low pH
[if glucose is metabolized with the use of pyruvic acid, the pH will
decrease; upon the addition of methyl red indicator, it will turn red]


Positive result: red color
Negative result: yellow
In an MR-VP (Methyl Red and Vogues-Proskauer) broth, we inoculate
an organism then incubate it for 48 hours at 35°C – 37°C. After that,
we are going to divide the content of the test tube; one for MR test and
one for VP test. For MR test, we add methyl red as the reagent.
MR and VP results are always opposite. This is because organisms
that undergo butylene glycol pathway (VP) produce small acid during
glucose fermentation thus they will not test positive in MR.
B.2 Voges-Proskauer Test

detects the organism's ability to convert the acid products
to acetoin and 2,3-butanediol (Butylene glycol
pathway)

After incubation, a-naphthol is added first as a catalyst
or color intensifier. Next, 40% potassium hydroxide
(KOH) or sodium hydroxide (NaOH) is added
Positive Result: red
Negative Result: yellow
C. AMINO ACID UTILIZATION
C.1 Lysine Iron Agar
Purpose: To differentiate gram-negative bacilli based on
decarboxylation or deamination of lysine and the formation of
hydrogen sulfide (H2S).
pH indicator: bromcresol purple
Lysine Deaminase (Slant)
Positive: Burgundy/ Red
Negative: Purple
Lysine Decarboxylase (Butt)
Positive: Purple (CADAVERINE)
Negative: Yellow (acidic - glucose is fermented)
Lysine Iron agar is composed of anaerobic slant and
anaerobic butt. When glucose is fermented, the butt of
the medium will become acidic (yellow).
A.3 Ortho-Nitrophenyl-ß-D-Galactopyranoside Test (ONPG
Test)

Used to determine the ability of organism to produce ßGalactosidase

ß-Galactosidase hydrolyzes ONPG, a colorless
compound, into galactose and o-nitrophenol, a yellow
compound [this also means that the organism is a lactose
Lysine Decarboxylase:

If the organism is able to produce lysine
decarboxylase, cadaverine will be formed.
Cadaverine will neutralize the organic acids
formed during glucose fermentation. It will revert
the acidic butt (result of glucose fermentation)
back to alkaline. That’s why positive result is
purple.
fermenter]

ONPG remains colorless if the organism is an NLF.

The test can be performed by
making a heavy suspension of bacteria in
sterile saline [0.85%] and adding
commercially prepared ONPG disks or
tablets.

The suspension is incubated [for 4
hours] at 35°C [37°C], and positive results
can generally be seen within 6 hours.
METHOD:
1. With a straight inoculating needle, inoculate LIA by twice
stabbing through the center of the medium to the bottom of the
tube and then streaking the slant.
2. Cap the tube tightly and incubate ate 35° - 37°C in ambient
air for 18-24 hrs.
A. Alkaline slant (purple)/alkaline butt (purple) (K/K): lysine
decarboxylation and no fermentation of glucose
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TRANS: BACT – LEC - 211
B. Alkaline slant (purple)/acid butt (yellow) (K/A):glucose
fermentation
C. Red slant/acid butt (yellow) (R/A): lysine deamination and
glucose fermentation
Deaminaseproducing:

Proteus

Providencia

Morganella
H2S-producing:

Salmonella

Arizona

Citrobacter

Edwardsiella
C.2 Phenylalanine Deaminase (PAD) Test

determines whether an organism possesses the enzyme
that deaminates phenylalanine to phenylpyruvic acid
Deamination of Phenylalanine
Phenylalanine → Phenylalanine deaminase → Phenyl pyruvic acid + (FeCl 3) green
 contains 0.2% concentration of phenylalanine
 The surface of the slant is inoculated with a
bacterial colony. After incubation, addition of a
10% ferric chloride reagent results in a green
color if the phenylpyruvic acid is present.
D.3 Urease Test
 determines whether a microorganism can
hydrolyze urea
 Urease hydrolyzes urea to form ammonia,
water, and CO2
 Christensen's Urea Agar
Positive: Change in color from light orange (pH
6.1) to Magenta (pink) (pH 8.1)
Negative: No change in color
Rapid urease producers:

Proteus

Providencia

Morganella
Slow urease producers:

Citrobacter

Klebsiella

Enterobacter except E. gergoviae

Yersinia

Serratia
D.4 DNA Hydrolysis (DNAse Test Agar)
Purpose: This test is used to differentiate organisms based on
the production of deoxyribonuclease.

to distinguish Serratia sp. (positive) from Enterobacter sp.,
Staphylococcus aureus (positive) from other species, and
Moraxella catarrhalis (positive) from Neisseria sp.
Positive: Medium will turn colorless around the test organism.
Negative: If no degradation of DNA occurs, the medium remains
green.
D. MISCELLANEOUS TESTS
D.1 Sulfide Indole Motility Agar [semi-solid]
a. Sulfide (H2S): black precipitate
b. Indole

Organisms that possess the enzyme tryptophanase
are capable of deaminating tryptophan with the
formation of the intermediate degradation products of
indole, pyruvic acid, and ammonia.

Reagent: 0.5mL Erlich’s
reagent

pink to red color [if
positive for indole, it will turn
pink or red upon addition of
Erlich’s reagent]
c. Motility: cloudiness spreading from inoculation line
.
D.2 Citrate Utilization

Purpose: It determines whether an organism can use
sodium citrate as a sole carbon source

part of a series referred to as IMVIC (indole, methyl red,
Vogues-Proskauer, and citrate)
Simmon's Citrate Agar
 contains ammonium salts as the sole nitrogen
source
 pH indicator: bromthymol blue
 Positive: blue / growth in the medium
 Negative: green
 use light inoculum.
Inoculate organism on the agar and then incubate it at 35 to 37°C for up to 7
days. After this, observe the growth in the medium and development of blue
color (positive result).
We use DNAse Test Agar where we inoculate the organism by
streaking one streak and then incubate it at 35 – 37°C for 18 - 24
hours.
D.5 Gelatin Liquefaction

Numerous bacteria produce
gelatinase,
proteolytic
enzymes that break down
gelatin into amino acids.

Gelatinase
activity
is
detected by loss of gelling
(liquefaction) of gelatin.
[positive result, liquefaction; negative result, no liquefaction]
D.6 Malonate Utilization
 pH indicator: bromthymol blue
 Bacteria able to use malonate as a
sole carbon source also use
ammonium sulfate as a nitrogen
source.
 A positive test results in increased
alkalinity from utilization of the
ammonium sulfate, changing the
indicator from green to blue
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TRANS: BACT – LEC - 211
D.7 Nitrate and Nitrite Reduction

The nitrate reduction test determines whether an organism
has the ability to reduce nitrate to nitrite and reduce nitrite
further to nitrogen gas (N2).

After 24 hours of incubation, N,N-dimethyl-αnaphthylamine and sulfanilic acid is added.

Α red color indicates the presence of nitrite.
D.8 Oxidase Test

The oxidase test determines the presence of the
cytochrome oxidase system that oxidizes reduced
cytochrome with molecular oxygen.

Kovac's oxidase test uses a 0.5% or 1% aqueous solution
of tetramethyl-p-phenylenediamine dihydrochloride.

Positive: development of a lavender color within 10 to 15
seconds.
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