KLIGLER IRON AGARINTENDED USE Kligler Iron Agar is used for the
identification of enterobacteria by the rapid detection of lactose
and glucose fermentation (with or without gas production), as well
as the production of hydrogen sulfide. PRINCIPLES - The
fermentations of lactose and glucose, used to differentiate species
of enterobacteria, result in acidification which makes phenol red
(pH indicator) turn yellow. - Microorganisms not fermenting lactose
(Salmonella or Shigella) initially product a yellow slant due to
the acidification resulting from glucose present in small
quantities. When the glucose has been exhausted in the aerobic
portion of the slant, the reaction becomes basic by oxidation of
the acids produced, resulting in the phenomenon of a red color on
the surface of the medium. This color does not appear in depth in
the butt, where the color remains yellow. - Bacteria fermenting
lactose and glucose make the slant and the butt turn yellow because
of the production of large quantities of acid. This is sufficient
to maintain an acid pH on the surface. - Microorganisms which
ferment neither of these two sugars do not change the color of the
medium. - The production of H2S is revealed in the base of the
medium by the appearance of black iron sulfide, due to the
reduction of thiosulfate in the presence of ferric citrate. - The
production of gas (H2, CO2) resulting from sugar fermentations is
shown by the appearance of gas bubbles or by a fragmentation of the
agar. PREPARATION - Suspend 58.0 g of dehydrated medium (BK034) in
1 liter of distilled or deionized water. - Slowly bring to boiling,
stirring with constant agitation until complete dissolution. -
Dispense in tubes. - Sterilize in an autoclave at 121C for 15
minutes. - Incline the tubes so as to obtain a butt 3 cm in height
and an oblique slant. NOTE 1 : Incomplete agar melting during
preparation will invariably lead to significant inconsistency in
the gel strength of the solidified agar, after sterilization and
cooling. NOTE 2 : If the medium is not used within one week of its
preparation, it is recommended to regenerate it in a boiling water
bath and to resolidify it in a slanted position.
INSTRUCTIONS FOR USE - Using a suspected colony taken from a
selective isolation medium, inoculate the butt by stabbing in the
center and the inclined surface by closely spaced streaks. - Pure
cultures taken from the center of well isolated colonies must be
used to avoid cross reactions which will make identification
impossible. - Incubate at 37C for 24 hours with caps loosely
screwed to favor gas exchanges. RESULTS Kligler's medium supplies
four types of information: (1) Glucose fermentation Red butt :
glucose not fermented Yellow butt : glucose fermented (2) Lactose
fermentation Red slant : lactose not fermented Yellow slant :
lactose fermented (3) Gas production Production of gas bubbles in
the butt of the tube. (4) Formation of H2S Formation of a black
color between the butt and the slant or along the inoculation stab.
For 1 liter of medium : -
Tryptone.........................................................................................20.0
g - Yeast extract
....................................................................................3.0
g - Meat extract
.....................................................................................3.0
g -
Glucose............................................................................................1.0
g - Lactose
..........................................................................................10.0
g - Sodium chloride
...............................................................................5.0
g - Sodium thiosulfate
...........................................................................0.5
g - Ferric ammonium citrate
..................................................................0.5
g - Phenol
red...................................................................................25.0
mg - Bacteriological agar
.......................................................................15.0
g pH of the ready-to-use medium at 25C : 7.4 0.2.
QUALITY CONTROL - Dehydrated medium : pinkish powder,
free-flowing and homogeneous. - Prepared medium : orange-red agar.
- Typical culture response after 18-24 hours of incubation at 37C
:
Microorganisms Escherichia coli ATCC 25922 Escherichia coli RIVM
WR1 Citrobacter freundii CIP 57.32T Proteus vulgaris ATCC 13315
Salmonella Enteritidis CIP 82.97 Pseudomonas aeruginosa CIP
82.118
Growth good, score 2 good, score 2 good, score 2
Slant yellow yellow yellow
Butt yellow yellow yellow
H2S +
Gas + + +
good, score 2 good, score 2
red red
yellow yellow
(+) +
+
good, score 2
red
red
-
-
http://www.biokardiagnostics.com/solabia/produitsDiagnostic.nsf/0/397C6C2EDA342538C12574B100496CA0/$file/TDS_B
K034_v6.pdf
Lysine Iron AgarTest agar for the simultaneous detection of
lysine decarboxylase, lysine deaminase enzymes and formation of
hydrogen sulfide in the identification of Enterobacteriaceae, in
particular Salmonella and Arizona according to Edwards and Fife.
Primarily used for the examination of foods.
Composition: Ingredients Grams/Litre Meat peptone 5.0 Yeast
extract 3.0 D(+)-Glucose 1.0 L-Lysine monohydrochloride 10.0 Sodium
thiosulfate 0.04 Ammonium ferric citrate 0.5 Bromocresol purple
0.02 Agar 12.5 Final pH 6.7 +/- 0.2 at 25C Store prepared media
below 8C, protected from direct light. Store dehydrated powder, in
a dry place, in tightly-sealed containers at 2-25C. Directions:
Dissolve 32 g in 1 litre distilled water and pour into tubes.
Autoclave at 121C for 15 minutes and let set as slants.
Principle and Interpretation: Lysine Iron Agar was developed to
detect lactose fermenting Salmonellae which are known to
decarboxylate lysine rapidly and produce large amounts of hydrogen
sulfide. This medium is a sensitive medium for the detection of
Iactose fermenting and lactose non-fermenting Salmonella species.
Many strains of this group, ferment Iactose very rapidly thus
suppressing H2S production on Triple Sugar Iron Agar (Fluka 44940).
It is recommended to use Lysine Iron Agar and Triple Sugar Iron
together for better discrimination between coliform organisms e.g.
Escherichia and Shigella. Meat peptone and Yeast extract is a
source of nitrogen, sulfur, carbon, coenzym and Vitamine B
complexes. D(+)- Glucose is a source of fermentable carbohydrate.
Ferric ammonium citrate and sodium thiosulphate are indicators of
H2S formation. Cultures that produce hydrogen sulphide cause
blackening of the medium due to ferrous sulphide production.
Proteus species producing H2S do not blacken this medium.
Bromocresol purple is a pH indicator which has a yellow color below
pH 5.3 and a purple color above pH 6.7. Lysine decarboxylation
causes an alkaline reaction (purple color) to give the amine
cadaverine and the organisms which do not decarboxylate lysine,
produce acid butt (yellow colour) due to the glucose
fermentation. Species of the Proteus-Providencia group, with the
exception of a few Proteus morganii strains, deaminate the lysine
to -Ketocarboxylic acid, which reacts with iron salt near the
surface of the medium under the influence of oxygen to form
reddish-brown compounds. The medium is stabbed to the base of the
butt and streaked on slant.
http://www.sigmaaldrich.com/etc/medialib/docs/Fluka/Datasheet/62915dat.Par.0001.File.tmp/62
915dat.pdf
Characteristic reactions of some Enterobacteriaceae cultured on
Lysine Iron Agar:
MicroorganismsArizona Salmonella * Proteus mirabilis Proteus
vulgaris Proteus morganii Proteus rettgeri Providencia Citrobacter
Escherichia Shigella Klebsiella
Buttviolet violet yellow yellow yellow yellow yellow yellow
violet
Slant surfaceviolet violet red-brown red-brown red-brown violet
violet violet violet
H2S production+ + + + -
* Exception: Salm. paratyphi A (no lysine decarboxyloase
production, butt = yellow, slant surface violet)
Quality control Test strainsShigella flexneri ATCC 12022
Escherichia coli ATCC 25922 Salmonella typhimurium ATCC 14028
Salmonella enteritidis NCTC 5188 Citrobacter freundii ATCC 8090
Proteus mirabilis ATCC 29906 Morganella morganii ATCC 25830
Growthgood / very good good / very good good / very good good /
very good good / very good good / very good good / very good
Buttyellow yellow violet and black violet and black yellow and
black yellow and black yellow
Slantviolet violet violet violet violet reddish-brown
reddish-brown / violet
http://85.238.144.18/analytics/Micro_Manual/TEDISdata/prods/1_11640_0500.html
Two media types are commonly used to detect urease activity.
Christensens urea agar is used to detect urease activity in a
variety of microorganisms. Stuarts urea broth is used primarily for
the differentiation of Proteus species. Both media types are
available commercially as prepared tubes or as a powder.
Christensens Urea Agar (2, 4, 5)
Ingredient Peptone Dextrose Sodium chloride Potassium phosphate,
monobasic Urea Phenol red Agar
Amount
1g 1g 5g 2g 20 g 0.012 g 15 to 20 g
To prepare the urea base, dissolve the first six ingredients in
100 ml of distilled water and filter sterilize (0.45-mm pore size).
Suspend the agar in 900 ml of distilled water, boil to dissolve
completely, and autoclave at 121oC and 15 psi for 15 minutes. Cool
the agar to 50 to 55oC. Aseptically add 100 ml of filter-sterilized
urea base to the cooled agar solution and mix thoroughly.
Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the
tubes during cooling until solidified. It is desirable to have a
long slant and short butt. Prepared media will have a yellow-orange
color. Store the prepared media in the refrigerator at 4 to 8oC
until needed. Once prepared, do not reheat the medium as the urea
will decompose. Stuarts Urea Broth (4, 5, 7)
Ingredient Yeast extract
Amount
0.1 g
Potassium phosphate, monobasic Potassium phosphate, dibasic
Urea
9.1 g 9.5 g 20 g 0.01 g
Phenol red
Dissolve all ingredients in 1 liter of distilled water and
filter sterilize (0.45-mm pore size). Distribute 3 ml of prepared
broth per sterile tube (13 x 100 mm). Prepared media will have a
yellow-orange color. Store the prepared broth in the refrigerator
at 4 to 8oC until needed. Do not heat the medium as the urea will
decompose. PROTOCOL Christensens Urea Agar (4, 5) Use a heavy
inoculum from an 18- to 24-hour pure culture to streak the entire
slant surface. Do not stab the butt as it will serve as a color
control (Fig. 1c). Incubate tubes with loosened caps at 35oC.
Observe the slant for a color change at 6 hours, 24 hours, and
every day for up to 6 days. Urease production is indicated by a
bright pink (fuchsia) color on the slant that may extend into the
butt. Note that any degree of pink is considered a positive
reaction. Prolonged incubation may result in a false-positive test
due to hydrolysis of proteins in the medium. To eliminate protein
hydrolysis as the cause of a positive test, a control medium
lacking urea should be used. Rapidly urease-positive Proteeae
(Proteus spp., Morganella morganii, and some Providencia stuartii
strains) will produce a strong positive reaction within 1 to 6
hours of incubation. Delayedpositive organisms (e.g., Klebsiella or
Enterobacter) will typically produce a weak positive reaction on
the slant after 6 hours, but the reaction will intensify and spread
to the butt on prolonged incubation (up to 6 days). The culture
medium will remain a yellowish color if the organism is urease
negative (Fig. 1).
a
b
c
d
FIG. 1. Urea agar test results. Urea agar slants were inoculated
as follows: (a) uninoculated, (b) Proteus mirabilis (rapidly urease
positive), (c) Klebsiella pneumoniae (delayed urease positive), (d)
Escherichia coli (urease negative). All samples were incubated at
37oC for 16 hours. Stuarts Urea Broth (4, 5) Use a heavy inoculum
from an 18- to 24-hour pure culture to inoculate the broth. Shake
the tube gently to suspend the bacteria. Incubate tubes with
loosened caps at 35oC. Observe the broth for a color change at 8,
12, 24, and 48 hours. Urease production is indicated by a bright
pink (fuchsia) color throughout the broth. Rapidly urease-positive
Proteeae (Proteus spp., Morganella morganii, and some Providencia
stuartii strains) for which this medium is differential, will
produce a strong positive reaction as early as 8 hours, but always
within 48 hours of incubation. Delayed-positive organisms (e.g.,
Enterobacter) will not produce a positive reaction due to the high
buffering capacity of this medium.
a
b
FIG. 2. Urea broth test results. Urea broth test tubes were
inoculated as follows: (a) Proteus vulgaris (urease positive) and
(b) Escherichia coli (urease negative). All samples were incubated
at 37oC for 16 hours.
http://www.microbelibrary.org/index.php/library/laboratory-test/3223-urease-test-protocol
Methyl Red and Voges-Proskauer (MR-VP)Principle: MR-VP is a
buffered Peptone-Glucose broth. Organisms that ferment dextrose
will release acid into the broth. Adding Methyl Red, an indicator
dye which turns red at pH 4.4 and yellow at pH 6.2, to the
inoculated MR-VP medium indicates if the bacteria fermented
dextrose. The Voges and Proskauer test was originally developed in
1898 by two German associates of Robert Koch. (Pioneers in Medical
Laboratory Science. Retrieved 06/09/04
http://www.hoslink.com/PIONEERS.htm) Some bacteria can be
distinguished on the basis of their production of acetoin, a
neutral end product, after incubation in buffered pepton-glucose
media. The addition of alpha-napthol and KOH solutions will result
in a pink-red color within a few minutes. Test Procedure: 1.
Lightly inoculate the tube from a single colony, preferably an
18-24 hour culture. 2. Slightly loosen the cap and incubate the
tubes at 35-37C for 48 hours. 3. After incubation, use a sterile
pipette to remove two - 1mL aliquots and place into two small
tubes. One tube is for the methyl red test and the other for the
Voges-Proskauer test. You do not want to contaminate your original
broth tube in case you have to do further incubation. 4. Add 5
drops of methyl red to one tube. Read the result immediately. Do
NOT mix the tube. 5. For the Voges-Proskauer test add 15 drops of
Voges-Proskauer A reagent. Mix well to aerate the sample. Oxygen is
needed to complete the reaction. 6. Add 5 drops of Voges-Proskauer
B to the tube and mix well to aerate the sample. 7. Read the
results within 5-15 minutes. Results: Methyl Red - A red color at
the surface is considered a positive result. A negative test is
indicated by a yellow color at the surface. Voges-Proskauer - A
positive test is indicated by a pink-red color developing within 5
minutes. Limitations of Procedure: Other tests are needed to
definitively identify the Enterobacteriaceae. The VP test should be
done at 48 hours. Longer incubation times could result in false
positives. The VP reagents must be added in the order listed and
with mixing to avoid weak-positive or falsenegative results.
The broth must be incubated for a minimum of 48 hours for the MR
test. Negative MR tests should be incubated for an additional 48
hours.http://biolabs.tmcc.edu/Micro%20Web/MRVP.pdf
MacConkey AgarPurpose MacConkey agar is used for the isolation
of gram-negative enteric bacteria and the differentiation of
lactose fermenting from lactose non-fermenting gram-negative
bacteria. It has also become common to use the media to
differentiate bacteria by their abilities to ferment sugars other
than lactose. In these cases lactose is replaced in the medium by
another sugar. These modified media are used to differentiate
gram-negative bacteria or to distinguish between phenotypes with
mutations that confer varying abilities to ferment particular
sugars. RECIPE Peptone (Difco) or Gelysate (BBL) 17.0 g Proteose
peptone (Difco) or Polypeptone (BBL) 3.0 g Lactose 10.0 g NaCl 5.0
g Crystal Violet 1.0 mg Neutral Red 30.0 mg Bile Salts 1.5 g Agar
13.5 g Distilled Water Add to make 1 Liter Adjust pH to 7.1 +/-0.2.
Boil to dissolve agar. Sterilize at 121 C for 15 minutes. (Holt and
Krieg, 1994, Remel 2005) PROTOCOL Streak a plate of MacConkey's
agar with the desired pure culture or mixed culture. If using a
mixed culture use a streak plate or spread plate to achieve colony
isolation. Good colony separation will ensure the best
differentiation of lactose fermenting and non-fermenting colonies
of bacteria.
Streak plate of Escherichia coli and Serratia marcescens on
MacConkey agar. Both microorganisms grow on this selective media
because they are gram-negative non-fastidious rods. Growth of E.
coli, which ferments lactose, appears red/pink on the agar. Growth
of S. marcescsens, which does not ferment lactose, appears
colorless and translucent.
http://www.microbelibrary.org/component/resource/laboratory-test/2855-macconkey-agar-platesprotocols
Intended UseMacConkey Agar is used for the isolation and
differentiation of Gram-negative enteric bacilli. Conforms to
Harmonized USP/EP/JP Requirements.1,2,3 Product Summary and
Explanation MacConkey Agar is based on the bile salt-neutral
red-lactose agar of MacConkey.4 The original MacConkey medium was
used to differentiate strains of Salmonella typhosa from members of
the coliform group. Formula modifications improved growth of
Shigella and Salmonella strains. These modifications include the
addition of 0.5% sodium chloride, decreased agar content, altered
bile salts, and neutral red concentrations. The formula
modifications improved differential reactions between enteric
pathogens and coliforms. MacConkey Agar is recommended for the
detection and isolation of Gram-negative organisms from clinical,5
dairy,6 food,7,8 water,9 pharmaceutical,1,2,3 and industrial10
sources. MacConkey Broth conforms to Harmonized United States
Pharmacopoeia (USP), European Pharmacopoeia (EU), and Japanese
Pharmacopoeia (JP).1,2,3 Principles of the Procedure Enzymatic
Digest of Gelatin, Enzymatic Digest of Casein, and Enzymatic Digest
of Animal Tissue are the nitrogen and vitamin sources in MacConkey
Agar. Lactose is the fermentable carbohydrate. During Lactose
fermentation a local pH drop around the colony causes a color
change in the pH indicator, Neutral Red, and bile precipitation.
Bile Salts Mixture and Crystal Violet are the selective agents,
inhibiting Gram-positive cocci and allowing Gram-negative organisms
to grow. Sodium Chloride maintains the osmotic environment. Agar is
the solidifying agent. Formula / Liter Enzymatic Digest of Gelatin
.................................................... 17 g Enzymatic
Digest of Casein
................................................... 1.5 g Enzymatic
Digest of Animal Tissue........................................ 1.5
g Lactose
...................................................................................
10 g Bile Salts Mixture
...................................................................
1.5 g Sodium Chloride
.......................................................................
5 g Neutral Red
..........................................................................
0.03 g Crystal Violet
......................................................................
0.001 g Agar
.....................................................................................
13.5 g Final pH: 7.1 0.2 at 25C Formula may be adjusted and/or
supplemented as required to meet performance specifications.
Precautions 1. For Laboratory Use. 2. IRRITANT. Irritating to
eyes, respiratory system, and skin. Directions 1. Suspend 50 g of
the medium in one liter of purified water. 2. Heat with frequent
agitation and boil for one minute to completely dissolve the
medium. 3. Autoclave at 121C for 15 minutes. Quality Control
Specifications Dehydrated Appearance: Powder is homogeneous, free
flowing, and light pink-beige. Prepared Appearance: Prepared
MacConkey Agar is medium to dark pink-purple and trace to slightly
hazy. Results Lactose-fermenting organisms grow as pink colonies
with or without a zone of precipitated bile. Nonlactose fermenting
organisms grow as colorless or clear colonies. Storage Store
dehydrated medium at 2 - 30C. Once opened and recapped, place
container in a low humidity environment at the same storage
temperature. Protect from moisture and light by keeping container
tightly closed. Expiration Refer to expiration date stamped on
container. The dehydrated medium should be discarded if not free
flowing, or if appearance has changed from the original color.
Expiry applies to medium in its intact container when stored as
directed. Limitations of the Procedure 1. Some strains may be
encountered that grow poorly or fail to grow on this medium. 2.
Although MacConkey Agar is a selective medium primarily for
Gram-negative enteric bacilli, biochemical and serological testing
using pure cultures are recommended for complete identification. 3.
Incubation of MacConkey Agar plates under increased CO2 has been
reported to reduce growth and recovery of a number of strains of
Gram-negative
bacilli.http://www.neogen.com/Acumedia/pdf/ProdInfo/7102_PI.pdf
EOSIN METHYLENE BLUE AGAR, LEVINE (7103)Intended Use Eosin
Methylene Blue Agar, Levine is used for the isolation and
differentiation of Gram-negative enteric bacilli. Product Summary
and Explanation Eosin Methylene Blue Agar, abbreviated EMB, was
developed by Holt-Harris and Teague.1 This formula contains lactose
and sucrose with two indicator dyes, Eosin Y and Methylene Blue.
Levine modified the formula by removing sucrose and doubling the
concentration of lactose.2,3 Eosin Methylene Blue Agar, Levine is
used for testing clinical materials, food, and dairy products.4-8
This medium is primarily used for the detection and confirmation of
coliforms. Principles of the Procedure Enzymatic Digest of Gelatin
is the nitrogen source in EMB Agar, Levine. Lactose is the
carbohydrate and Dipotassium Phosphate is the buffer. Eosin Y and
Methylene Blue are the indicators. Methylene Blue is also a
selective agent. During strong acidic conditions, the dyes impart a
metallic sheen to certain lactose fermenters, such as E. coli.
Formula / Liter Enzymatic Digest of Gelatin
....................................... 10 g
Lactose
.......................................................................
10 g Dipotassium Phosphate
............................................. 2 g Eosin Y
......................................................................
0.4 g Methylene Blue
...................................................... 0.065 g Agar
............................................................................
15 g Final pH: 7.1 0.2 at 25CFormula may be adjusted and/or
supplemented to meet performance specifications.
Precautions 1. For Laboratory Use. 2. IRRITANT. Irritating to
eyes, skin, and respiratory system. Directions 1. Suspend 37.5 g of
the medium in one liter of purified water. 3. Heat with frequent
agitation and boil for one minute to completely dissolve the
medium. 4. Autoclave at 121C for 15 minutes. Quality Control
Specifications Dehydrated Appearance: Powder is homogeneous, free
flowing, and light red-purple. Prepared Appearance: Prepared medium
is trace to slightly hazy, with or without a fine precipitate, and
medium to dark red to blue-purple Results Colonies of lactose
fermenters are blue-black with or without a green metallic sheen.
E. coli colonies typically are dark centered and usually have a
green metallic sheen. Colonies of non-lactose fermenting bacteria
are colorless and translucent. Refer to appropriate references for
specific results and biochemical reactions.4-8 Storage Store the
sealed bottle containing the dehydrated medium at 2 - 30C. Once
opened and recapped, place container in a low humidity environment
at the same storage temperature. Protect from moisture and light by
keeping container tightly closed. Expiration Refer to expiration
date stamped on the container. The dehydrated medium should be
discarded if not free flowing, or if appearance has changed from
the original color. Expiry applies to medium in its intact
container when stored as directed. Limitations of the Procedure Due
to nutritional variation, some strains may be encountered that grow
poorly or fail to grow on this
medium.http://www.neogen.com/Acumedia/pdf/ProdInfo/7103_PI.pdf
SALMONELLA SHIGELLA AGAR (7152)Intended Use Salmonella Shigella
Agar is used for the isolation of Salmonella spp. and some strains
of Shigella spp. Product Summary and Explanation Salmonellosis
continues to be an important public health problem worldwide.
Infection with non-typhi Salmonella often causes a mild,
self-limiting illness. Typhoid fever, caused by Salmonella typhi,
is
characterized by fever, headache, diarrhea, abdominal pain, and
can result in fatal respiratory, hepatic, and or neurological
damage.1 This infection can result from the consumption of raw,
undercooked, or improperly processed foods contaminated with
Salmonella spp. Shigellosis, caused by Shigella spp., is an
intestinal illness characterized by abdominal pain, fever, and
watery diarrhea. When associated with outbreaks, shigellosis is
usually transmitted through contaminated food and/or water.
Salmonella Shigella Agar is a modification of the Desoxycholate
Citrate Agar described by Leifson.2 Salmonella Shigella Agar is
superior to a number of other media for the isolation of Salmonella
spp. and Shigella spp.3 Salmonella Shigella Agar is recommended for
testing clinical specimens and food testing for the presence of
Salmonella spp. and some Shigella spp.1,4,5 Principles of the
Procedure Beef Extract, Enzymatic Digest of Casein, and Enzymatic
Digest of Animal Tissue provide sources of nitrogen, carbon, and
vitamins required for organism growth. Lactose is the carbohydrate
present in Salmonella Shigella Agar. Bile Salts, Sodium Citrate and
Brilliant Green inhibit Gram-positive bacteria, most coliform
bacteria, and inhibit swarming Proteus spp., while allowing
Salmonella spp. to grow. Sodium Thiosulfate and Ferric Citrate
permit detection of hydrogen sulfide by the production of colonies
with black centers. Neutral Red is the pH indicator. Formula /
Liter Beef Extract
..............................................................................
5 g Enzymatic Digest of Casein
................................................... 2.5 g Enzymatic
Digest of Animal Tissue........................................ 2.5
g Lactose
...................................................................................
10 g Bile Salts
................................................................................
8.5 g Sodium Citrate
.......................................................................
8.5 g Sodium Thiosulfate
................................................................
8.5 g Ferric Citrate
.............................................................................
1 g Brilliant Green
................................................................
0.00033 g Neutral Red
........................................................................
0.025 g Agar
.....................................................................................
13.5 g Final pH: 7.0 0.2 at 25CFormula may be adjusted and/or
supplemented as required to meet performance specifications.
Precautions 1. For Laboratory Use. 2. IRRITANT. Irritating to
eyes, respiratory system, and skin. Directions 1. Suspend 60 g of
the medium in one liter of purified water. 2. Heat with frequent
agitation and boil for one minute to completely dissolve the
medium. 3. DO NOT AUTOCLAVE. Quality Control Specifications
Dehydrated Appearance: Powder is homogeneous, free-flowing, and
light to medium pinkish-beige. Prepared Appearance: Prepared medium
is reddish-orange to peach and trace to slightly hazy. Test
Procedure For isolation of Salmonella spp. and Shigella spp. from
clinical specimens, inoculate fecal samples and rectal swabs onto
one quadrant of Salmonella Shigella Agar, streak for isolation.
Incubate plates at 35C, and examine after 24 and 48 hours for
colonies resembling Salmonella spp. or Shigella spp. Consult
appropriate references for food testing. Results
Enteric organisms are differentiated by their ability to ferment
lactose. Salmonella spp. and Shigella spp. are non-lactose
fermenters and form colorless colonies on Salmonella Shigella Agar.
H2S positive Salmonella spp. produce black-center colonies. Some
Shigella spp. are inhibited on Salmonella Shigella Agar. E. coli
produces pink to red colonies and may have some bile precipitation.
Storage Store sealed bottle containing the dehydrated medium at 2 -
30C. Once opened and recapped, place container in a low humidity
environment at the same storage temperature. Protect from moisture
and light. Expiration Refer to expiration date stamped on the
container. The dehydrated medium should be discarded if not free
flowing, or if appearance has changed from the original color.
Limitations of the Procedure 1. Salmonella Shigella Agar is highly
selective and not recommended as the primary isolation of Shigella.
1,2,6 Some Shigella spp. may be inhibited. 2. A few nonpathogenic
organisms may grow on Salmonella Shigella Agar. These organisms can
be differentiated by their ability to ferment lactose and other
confirmatory tests.
http://www.neogen.com/Acumedia/pdf/ProdInfo/7152_PI.pdf
Hektoen Enteric Agar Hektoen Enteric Agar (HE) is a selective
and differential medium designed to isolate and differentiate
members of the species Salmonella and Shigella from other
Enterobacteriaceae. Bile salts and the dyes bromthymol blue and
acid fuchsin inihibit the growth of most Gram positive organisms.
Lactose, sucrose, and salicin provide fermentable carbohydrates to
encourage the growth and differentiation of enterics. Sodium
thiosulfate provides a source of sulfur. Ferric ammonium citrate
provides a source of iron to allow production of hydrogen sulfide
from sodium thiosulfate, which provides a source of sulfur. Ferric
ammonium citrate also allows the visualiztion of hydrogen sulfide
production by reacting with hydrogen sulfide gas to form a black
precipitate. Enterics that ferment one or more of the carbohydrates
will produce yellow to salmon-colored colonies. Non-fermenters will
produce blue-green colonies. Organisms that reduce sulfur to
hydrogen sulfide will produce black colonies or blue-green colonies
with a black
center.http://www.austincc.edu/microbugz/hektoen_enteric_agar.php
Principles of the Procedure Enzymatic Digest of Animal Tissue
provides nitrogen, carbon, and amino acids required for organism
growth. Yeast Extract is a vitamin source. Bile Salts Mixture and
Acid Fuchsin inhibit Gram-positive organisms. Lactose, Sucrose, and
Salicin are fermentable carbohydrates. Sodium Chloride maintains
the osmotic balance of the medium. Ferric Ammonium Citrate, a
source of iron, allows production of hydrogen sulfide (H2S) present
from Sodium Thiosulfate. H2S-positive colonies have black centers.
Bromothymol Blue is added as the pH indicator. Agar is the
solidifying agent.
Formula / Liter
...............16.5 g ....................3 g
..................4.5 g ..................12 g
...................12 g ....................2 g
....................5 g .....................5 g
..................1.5 g .............0.065 g .................0.1 g
...............13.5 g Final pH: 7.6 0.2 at 25CFormula may be
adjusted and/or supplemented as required to meet performance
specifications.
Precautions 1. For Laboratory Use. 2. IRRITANT. Irritating to
eyes, respiratory system, and skin. Directions 1. Suspend 75 g of
the medium in one liter of purified water. 2. Heat with frequent
agitation and boil for one minute to completely dissolve the
medium.