exercise no. 28

7/29/2019 exercise no. 28

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GROUP 4 February 27, 2012

Family Enterobacteriaceae: Morphology and Cultural Characteristics

KLIGLER IRON AGAR

INTENDED USEKligler Iron Agar is used for the identification of enterobacteria by the rapid detection of lactose andglucose 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 inacidification which makes phenol red (pH indicator) turn yellow.

- Microorganisms not fermenting lactose (Salmonella orShigella) initially product a yellow slant due to theacidification resulting from glucose present in small quantities. When the glucose has been exhausted inthe 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 inthe butt, where the color remains yellow.

- Bacteria fermenting lactose and glucose make the slant and the butt turn yellow because of theproduction 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 gasbubbles 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 gelstrength 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 inthe 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 reactionswhich will make identification impossible.- Incubate at 37C for 24 hours with caps loosely screwed to favor gas exchanges.


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RESULTSKligler's medium supplies four types of information:

(1) Glucose fermentationRed butt : glucose not fermentedYellow butt : glucose fermented

(2) Lactose fermentationRed slant : lactose not fermentedYellow slant : lactose fermented

(3) Gas productionProduction of gas bubbles in the butt of the tube.

(4) Formation of H2SFormation 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 gpH 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 :


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Microorganisms Growth Slant Butt H2S Gas

Escherichia coliATCC 25922

Escherichia coliRIVM WR1

CitrobacterfreundiiCIP57.32T

Proteus vulgarisATCC 13315

SalmonellaEnteritidis CIP82.97

Pseudomonasaeruginosa CIP

82.118

good, score 2 yellow yellow - +

good, score 2 yellow yellow - +

good, score 2 yellow yellow + +

good, score 2 red yellow (+) -

good, score 2 red yellow + +

good, score 2 red red - -

http://www.biokar-

diagnostics.com/solabia/produitsDiagnostic.nsf/0/397C6C2EDA342538C12574B100496CA0/$file/TDS_BK034_v6.pd

f

Lysine Iron Agar

Test agar for the simultaneous detection of lysine decarboxylase, lysine deaminase enzymes and

formation of

hydrogen sulfide in the identification of Enterobacteriaceae, in particular Salmonella andArizona

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
http://www.biokar-diagnostics.com/solabia/produitsDiagnostic.nsf/0/397C6C2EDA342538C12574B100496CA0/$file/TDS_BK034_v6.pdfhttp://www.biokar-diagnostics.com/solabia/produitsDiagnostic.nsf/0/397C6C2EDA342538C12574B100496CA0/$file/TDS_BK034_v6.pdfhttp://www.biokar-diagnostics.com/solabia/produitsDiagnostic.nsf/0/397C6C2EDA342538C12574B100496CA0/$file/TDS_BK034_v6.pdfhttp://www.biokar-diagnostics.com/solabia/produitsDiagnostic.nsf/0/397C6C2EDA342538C12574B100496CA0/$file/TDS_BK034_v6.pdfhttp://www.biokar-diagnostics.com/solabia/produitsDiagnostic.nsf/0/397C6C2EDA342538C12574B100496CA0/$file/TDS_BK034_v6.pdfhttp://www.biokar-diagnostics.com/solabia/produitsDiagnostic.nsf/0/397C6C2EDA342538C12574B100496CA0/$file/TDS_BK034_v6.pdf


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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 sodiumthiosulphate 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/62915dat.pd

f

Characteristic reactions of some Enterobacteriaceae cultured on Lysine Iron Agar:

Microorganisms Butt Slant surface H 2S productionArizona violet violet +

Salmonella * violet violet +

Proteus mirabilis yellow red-brown +
http://www.sigmaaldrich.com/etc/medialib/docs/Fluka/Datasheet/62915dat.Par.0001.File.tmp/62915dat.pdfhttp://www.sigmaaldrich.com/etc/medialib/docs/Fluka/Datasheet/62915dat.Par.0001.File.tmp/62915dat.pdfhttp://www.sigmaaldrich.com/etc/medialib/docs/Fluka/Datasheet/62915dat.Par.0001.File.tmp/62915dat.pdfhttp://www.sigmaaldrich.com/etc/medialib/docs/Fluka/Datasheet/62915dat.Par.0001.File.tmp/62915dat.pdf


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Proteus vulgaris

Proteus morganiiProteus rettgeri

yellow red-brown -

Providencia yellow red-brown -

Citrobacter yellow violet +

Escherichia yellow violet -

Shigella yellow violet -

Klebsiella violet violet -

* Exception: Salm. paratyphi A (no lysinedecarboxyloase production, butt = yellow, slant surface violet)

Quality control

Test strains Growth Butt Slant Shigella flexneri ATCC 12022 good / very good yellow violet

Escherichia coli ATCC 25922 good / very good yellow violet

Salmonella typhimurium ATCC 14028 good / very good violet and black violet

Salmonella enteritidis NCTC 5188 good / very good violet and black violet

Citrobacter freundii ATCC 8090 good / very good yellow and black violet

Proteus mirabilis ATCC 29906 good / very good yellow and black reddish-brown

Morganella morganii ATCC 25830 good / very good yellow 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 detecturease activity in a variety of microorganisms. Stuarts urea broth is used primarily for the differentiation ofProteus species. Both media types are available commercially as prepared tubes or as a powder.

Christensens Urea Agar(2, 4, 5)

Ingredient

Amount

Peptone 1 g

Dextrose 1 g

Sodium chloride 5 g

Potassium phosphate,

monobasic2 g

Urea 20 g

Phenol red 0.012 g

Agar 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
http://85.238.144.18/analytics/Micro_Manual/TEDISdata/prods/1_11640_0500.htmlhttp://85.238.144.18/analytics/Micro_Manual/TEDISdata/prods/1_11640_0500.html


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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 andshort butt. Prepared media will have a yellow-orange color. Store the prepared media in the refrigerator at4 to 8oC until needed. Once prepared, do not reheat the medium as the urea will decompose.

Stuarts Urea Broth (4, 5, 7)

Ingredient

Amount

Yeast extract 0.1 g

Potassium phosphate,

monobasic9.1 g

Potassium phosphate, dibasic 9.5 g

Urea 20 g

Phenol red

0.01 g

Dissolve all ingredients in 1 liter of distilled water and filter sterilize (0.45-mm pore size). Distribute 3 ml ofprepared broth per sterile tube (13 x 100 mm). Prepared media will have a yellow-orange color. Store theprepared broth in the refrigerator at 4 to 8oC until needed. Do not heat the medium as the urea willdecompose.

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 itwill serve as a color control (Fig. 1c). Incubate tubes with loosened caps at 35oC. Observe the slant for a colorchange 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 eliminateprotein 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 stuartiistrains) willproduce a strong positive reaction within 1 to 6 hours of incubation. Delayed-positive organisms (e.g., Klebsiella or

Enterobacter) will typically produce a weak positive reaction on the slant after 6 hours, but the reaction will intensifyand spread to the butt on prolonged incubation (up to 6 days). The culture medium will remain a yellowish color if theorganism is urease negative (Fig. 1).


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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 tosuspend 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 thebroth.

Rapidly urease-positive Proteeae (Proteus spp., Morganella morganii, and some Providencia stuartiistrains) 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 producea 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 16hours.

http://www.microbelibrary.org/index.php/library/laboratory-test/3223-urease-test-protocol
http://www.microbelibrary.org/index.php/library/laboratory-test/3223-urease-test-protocolhttp://www.microbelibrary.org/index.php/library/laboratory-test/3223-urease-test-protocol


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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 pH4.4 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 false-negative 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 Agar

Purpose

MacConkey agar is used for the isolation of gram-negative enteric bacteria and thedifferentiation 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
http://biolabs.tmcc.edu/Micro%20Web/MRVP.pdfhttp://biolabs.tmcc.edu/Micro%20Web/MRVP.pdf


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another sugar. These modified media are used to differentiate gram-negative bacteria or todistinguish between phenotypes with mutations that confer varying abilities to ferment

particular sugars.

RECIPE

Peptone (Difco) or Gelysate (BBL) 17.0 gProteose peptone (Difco) or

Polypeptone (BBL)3.0 g

Lactose 10.0 g

NaCl 5.0 gCrystal Violet 1.0 mg

Neutral Red 30.0 mgBile Salts 1.5 g

Agar 13.5 g

Distilled WaterAdd to make 1

Liter

Adjust pH to 7.1 +/-0.2. Boil to dissolve agar. Sterilize at 121 C for 15 minutes. (Holt andKrieg, 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 colonyseparation 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-plates-protocols

Intended UseMacConkey Agaris used for the isolation and differentiation of Gram-negative enteric bacilli. Conforms toHarmonized USP/EP/JP Requirements.1,2,3
http://www.microbelibrary.org/component/resource/laboratory-test/2855-macconkey-agar-plates-protocolshttp://www.microbelibrary.org/component/resource/laboratory-test/2855-macconkey-agar-plates-protocolshttp://www.microbelibrary.org/component/resource/laboratory-test/2855-macconkey-agar-plates-protocolshttp://www.microbelibrary.org/component/resource/laboratory-test/2855-macconkey-agar-plates-protocols


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Product Summary and ExplanationMacConkey Agar is based on the bile salt-neutral red-lactose agar of MacConkey.4 The originalMacConkey medium was used to differentiate strains ofSalmonella typhosa from members of thecoliform group. Formula modifications improved growth ofShigella and Salmonella strains. Thesemodifications include the addition of 0.5% sodium chloride, decreased agar content, altered bile salts, andneutral red concentrations. The formula modifications improved differential reactions between entericpathogens and coliforms.MacConkey Agar is recommended for the detection and isolation of Gram-negative organisms fromclinical,5 dairy,6 food,7,8 water,9 pharmaceutical,1,2,3 and industrial10 sources. MacConkey Brothconforms to Harmonized United States Pharmacopoeia (USP), European Pharmacopoeia (EU), andJapanese Pharmacopoeia (JP).1,2,3

Principles of the ProcedureEnzymatic Digest of Gelatin, Enzymatic Digest of Casein, and Enzymatic Digest of Animal Tissue are thenitrogen and vitamin sources in MacConkey Agar. Lactose is the fermentable carbohydrate. DuringLactose 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, inhibitingGram-positive cocci and allowing Gram-negative organisms to grow. Sodium Chloride maintains the

osmotic environment. Agar is the solidifying agent.Formula / LiterEnzymatic Digest of Gelatin .................................................... 17 gEnzymatic Digest of Casein ................................................... 1.5 gEnzymatic Digest of Animal Tissue........................................ 1.5 gLactose ................................................................................... 10 gBile Salts Mixture ................................................................... 1.5 gSodium Chloride ....................................................................... 5 gNeutral Red .......................................................................... 0.03 gCrystal Violet ...................................................................... 0.001 g

Agar ..................................................................................... 13.5 gFinal pH: 7.1 0.2 at 25 CFormula may be adjusted and/or supplemented as required to meet performance specifications.

Precautions1. For Laboratory Use.2. IRRITANT. Irritating to eyes, respiratory system, and skin.Directions1. 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 121 C for 15 minutes.Quality Control SpecificationsDehydrated Appearance: Powder is homogeneous, free flowing, and light pink-beige.Prepared Appearance: Prepared MacConkey Agar is medium to dark pink-purple and trace to slightlyhazy.

ResultsLactose-fermenting organisms grow as pink colonies with or without a zone of precipitated bile. Non-lactose fermenting organisms grow as colorless or clear colonies.StorageStore dehydrated medium at 2 - 30 C. Once opened and recapped, place container in a low humidity environment at the same storage temperature. Protect from moisture and light by keeping containertightly closed.Expiration


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Refer to expiration date stamped on container. The dehydrated medium should be discarded if not freeflowing, or if appearance has changed from the original color. Expiry applies to medium in its intactcontainer when stored as directed.Limitations of the Procedure1. 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 andrecovery of a number of strains of Gram-negative bacilli.

http://www.neogen.com/Acumedia/pdf/ProdInfo/7102_PI.pdf

EOSIN METHYLENE BLUE AGAR, LEVINE

Intended UseEosin Methylene Blue Agar, Levine is used for the isolation and differentiation of Gram-negative entericbacilli.

Product Summary and ExplanationEosin Methylene Blue Agar, abbreviated EMB, was developed by Holt-Harris and Teague.1 This formulacontains lactose and sucrose with two indicator dyes, Eosin Y and Methylene Blue. Levine modified theformula 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 forthe detection and confirmation of coliforms.

Principles of the ProcedureEnzymatic Digest of Gelatin is the nitrogen source in EMB Agar, Levine. Lactose is the carbohydrate andDipotassium Phosphate is the buffer. Eosin Y and Methylene Blue are the indicators. Methylene Blue isalso a selective agent. During strong acidic conditions, the dyes impart a metallic sheen to certain lactosefermenters, such as E. coli.

Formula / LiterEnzymatic Digest of Gelatin ....................................... 10 gLactose ....................................................................... 10 gDipotassium Phosphate ............................................. 2 gEosin Y ...................................................................... 0.4 gMethylene Blue ...................................................... 0.065 g

Agar ............................................................................ 15 gFinal pH: 7.1 0.2 at 25CFormula may be adjusted and/or supplemented to meet performance specifications.

Precautions1. For Laboratory Use.2. IRRITANT. Irritating to eyes, skin, and respiratory system.

Directions1. 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.
http://www.neogen.com/Acumedia/pdf/ProdInfo/7102_PI.pdfhttp://www.neogen.com/Acumedia/pdf/ProdInfo/7102_PI.pdf


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Prepared Appearance: Prepared medium is trace to slightly hazy, with or without a fine precipitate, andmedium to dark red to blue-purple

ResultsColonies of lactose fermenters are blue-black with or without a green metallic sheen. E. colicoloniestypically are dark centered and usually have a green metallic sheen. Colonies of non-lactose fermentingbacteria are colorless and translucent. Refer to appropriate references for specific results andbiochemical reactions.4-8

StorageStore the sealed bottle containing the dehydrated medium at 2 - 30 C. Once opened and recapped, place container in a low humidity environment at the same storage temperature. Protect from moistureand light by keeping container tightly closed.

ExpirationRefer to expiration date stamped on the container. The dehydrated medium should be discarded if notfree flowing, or if appearance has changed from the original color. Expiry applies to medium in its intactcontainer 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 thismedium.


SALMONELLA SHIGELLA AGAR (7152)

Intended UseSalmonella Shigella Agaris used for the isolation ofSalmonella spp. and some strains ofShigella spp.

Product Summary and ExplanationSalmonellosis continues to be an important public health problem worldwide. Infection with non-typhiSalmonella often causes a mild, self-limiting illness. Typhoid fever, caused by Salmonella typhi, ischaracterized 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, orimproperly processed foods contaminated with Salmonella spp.Shigellosis, caused by Shigella spp., is an intestinal illness characterized by abdominal pain, fever, andwatery diarrhea. When associated with outbreaks, shigellosis is usually transmitted through contaminatedfood and/or water.Salmonella Shigella Agar is a modification of the Desoxycholate Citrate Agar described by Leifson.2Salmonella Shigella Agar is superior to a number of other media for the isolation ofSalmonella spp. andShigella spp.3 Salmonella Shigella Agar is recommended for testing clinical specimens and food testingfor the presence ofSalmonella spp. and some Shigella spp.1,4,5

Principles of the ProcedureBeef Extract, Enzymatic Digest of Casein, and Enzymatic Digest of Animal Tissue provide sources ofnitrogen, carbon, and vitamins required for organism growth. Lactose is the carbohydrate present inSalmonella 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 colonieswith black centers. Neutral Red is the pH indicator.

Formula / Liter


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Beef Extract .............................................................................. 5 gEnzymatic Digest of Casein ................................................... 2.5 gEnzymatic Digest of Animal Tissue........................................ 2.5 gLactose ................................................................................... 10 gBile Salts ................................................................................ 8.5 gSodium Citrate ....................................................................... 8.5 gSodium Thiosulfate ................................................................ 8.5 gFerric Citrate ............................................................................. 1 gBrilliant Green ................................................................ 0.00033 gNeutral Red ........................................................................ 0.025 g


Precautions1. For Laboratory Use.2. IRRITANT. Irritating to eyes, respiratory system, and skin.

Directions1. 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.


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 ProcedureFor isolation ofSalmonella spp. and Shigella spp. from clinical specimens, inoculate fecal samples andrectal swabs onto one quadrant of Salmonella Shigella Agar, streak for isolation. Incubate plates at35 C, and examine after 24 and 48 hours for colonies resembling Salmonella spp. orShigella spp.

Consult appropriate references for food testing.

ResultsEnteric 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 positiveSalmonella spp. produce black-center colonies. Some Shigella spp. are inhibited on Salmonella Shigella

Agar. E. coliproduces pink to red colonies and may have some bile precipitation.

StorageStore sealed bottle containing the dehydrated medium at 2 - 30C. Once opened and recapped, placecontainer in a low humidity environment at the same storage temperature. Protect from moisture and light.

ExpirationRefer to expiration date stamped on the container. The dehydrated medium should be discarded if notfree flowing, or if appearance has changed from the original color.

Limitations of the Procedure1. Salmonella Shigella Agar is highly selective and not recommended as the primary isolation ofShigella.1,2,6 Some Shigella spp. may be inhibited.2. A few nonpathogenic organisms may grow on Salmonella Shigella Agar. These organisms can bedifferentiated by their ability to ferment lactose and other confirmatory tests.



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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 otherEnterobacteriaceae. 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 organismgrowth. Yeast Extract is a vitamin source. Bile Salts Mixture and Acid Fuchsin inhibit Gram-positiveorganisms. Lactose, Sucrose, and Salicin are fermentable carbohydrates. Sodium Chloride maintains theosmotic balance of the medium. Ferric Ammonium Citrate, a source of iron, allows production of hydrogensulfide (H2S) present from Sodium Thiosulfate. H2S-positive colonies have black centers. BromothymolBlue is added as the pH indicator. Agar is the solidifying agent.

Formula / LiterEnzymatic Digest of Animal Tissue.....................................16.5 gYeast Extract............................................................................3 gBile Salts Mixture...................................................................4.5 gLactose...................................................................................12 gSucrose...................................................................................12 gSalicin.......................................................................................2 gSodium Chloride.......................................................................5 gSodium Thiosulfate...................................................................5 gFerric Ammonium Citrate.......................................................1.5 gBromthymol Blue...............................................................0.065 g

Acid Fuchsin..........................................................................0.1 gAgar.....................................................................................13.5 g

Final pH: 7.6 0.2 at 25CFormula may be adjusted and/or supplemented as required to meet performance specifications.


Directions1. 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.3. DO NOT AUTOCLAVE.
http://www.austincc.edu/microbugz/hektoen_enteric_agar.phphttp://www.austincc.edu/microbugz/hektoen_enteric_agar.php


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Dehydrated Appearance: Powder is homogeneous, free flowing, and light green-beige.Prepared Appearance: Prepared medium is trace to slightly hazy and light to dark green.

StorageStore sealed bottle containing the dehydrated medium at 2 - 30C. Once opened and recapped, placecontainer in a low humidity environment at the same storage temperature. Protect from moisture and light.


1. Do not autoclave medium because excessive heat may alter ingredients.

2. Proteus spp. may resemble salmonellae or shigellae. Further testing should be conducted to confirmthe presumptive identification or organisms isolated on this medium.

3. Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on thismedium.


Xylose-Lysine Deoxycholate Agar (XLD Agar) M031

Xylose-Lysine Deoxycholate Agar (XLD Agar) is a selective medium recommended for the isolation and

enumeration ofSalmonella Typhi and otherSalmonella species.

Composition**

Ingredients Gms / Litre

Yeast extract 3.000

L-Lysine 5.000

Lactose 7.500

Sucrose 7.500

Xylose 3.500Sodium chloride 5.000

Sodium deoxycholate 2.500

Sodium thiosulphate 6.800

Ferric ammonium citrate 0.800

Phenol red 0.080

Agar 15.000

Final pH ( at 25C) 7.40.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 56.68 grams in 1000 ml distilled water. Heat with frequent agitation until the medium boils. DO NOT

AUTOCLAVE OR OVERHEAT. Transfer immediately to a water bath at 50C. After cooling, pour into sterile

Petri plates. It is advisable not to prepare large volumes that will require prolonged heating.

Principle And Interpretation

XLD Agar was formulated by Taylor (1-5) for the isolation and differentiation of enteric pathogens including

Salmonella

Typhi from otherSalmonella species. XLD Agar has been recommended for the identification of

Enterobacteriaceae (7) and for the microbiological testing of foods, water and dairy products (8-12). XLD Agar

exhibits increased selectivity and sensitivity as compared to other plating media e.g. SS Agar (M108), EMB Agar

(M022) and Bismuth Sulphite Agar (M027) (2, 4, 6, and 13-16). The media formulation does not allow the


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overgrowth of other organisms overSalmonella and Shigella (17). Samples suspected of containing enteric

pathogens, along with other mixed flora, are initially enriched in Modified Semisolid RV Medium Base (M1482)

(18). The medium contains yeast extract, which provides nitrogen and vitamins required for growth. Though the

sugars xylose, lactose and sucrose provide sources of fermentable carbohydrates, xylose is mainly incorporated into

the medium since it is not fermented by Shigellaebut practically by all enterics. This helps in the differentiation ofShigella species. Sodium chloride maintains the osmotic balance of the medium. Lysine is included to differentiate

the Salmonella group from the non-pathogens. Salmonellae rapidly ferment xylose and exhaust the supply.Subsequently lysine is decarboxylated by the enzyme lysine decarboxylase to form amines with reversion to an

alkaline pH that mimics the Shigella reaction. However, to prevent this reaction by lysine-positive coliforms, lactose

and sucrose are added to produce acid in excess. Degradation of xylose, lactose and sucrose to acid causes phenol

red indicator to change its colour to yellow. Bacteria that decarboxylate lysine to cadaverine can be recognized by

the appearance of a red colouration around the colonies due to an increase in pH. These reactions can proceed

simultaneously or successively, and this may cause the pH indicator to exhibit various shades of colour or it maychange its colour from yellow to red on prolonged incubation.

To add to the differentiating ability of the formulation, an H2S indicator system, consisting of sodium thiosulphate

and ferric ammonium citrate, is included for the visualization of hydrogen sulphide produced, resulting in the

formation of colonies with black centers. The non-pathogenic H2S producers do not decarboxylase lysine; therefore,

the acid reaction produced by them prevents the blackening of the colonies (1).

XLD Agar is both selective and differential medium. It utilizes sodium deoxycholate as the selective agent and

therefore it is inhibitory to gram-positive microorganisms. SomeProteus strains may give red to yellow colourationwith most colonies developing black centers, giving rise to false positive reactions. Non-enterics likePseudomonas

andProvidencia may exhibit red colonies. S. Paratyphi A , S. Choleraesuis , S. Pullorum and S. Gallinarum mayform red colonies without H2S, thus resembling Shigella species (19).

Quality Control

Appearance

Light yellow to pink homogeneous free flowing powder

Gelling

Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared mediumRed coloured clear to slightly opalescent gel forms in Petri plates

Reaction

Reaction of 5.67% w/v aqueous solution at 25C . pH : 7.40.2

pH

7.20-7.60

Cultural Response

Cultural response was observed after an incubation at 35-37C for specified time. Recovery rate is considered as

100% for

bacteria growth on Soyabean Casein Digest Agar.

Storage and Shelf Life

Store below 30C in a tightly closed container and use freshly prepared medium. Use before expiry date on the

label.

http://himedialabs.com/TD/M031.pdf
http://himedialabs.com/TD/M031.pdfhttp://himedialabs.com/TD/M031.pdf


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CIN Agar (Yersinia Selective)

Intended Use: CIN (cefsulodin-Irgasan-novobiocin) Agar is a differential and selective medium used inqualitative procedures for the isolation ofYersinia enterocolitica from a variety of clinical and nonclinicalspecimens.

Product Summary:

CIN Agar was first described by Schiemann as an alternative to MacConkey Agar and other commonlyused media for isolation ofYersinia enterocolitica, a causative agent of gastroenteritis.1 CIN Agar hasbeen found to be far superior to MacConkey, SS, CAL or Y agars.2

User Quality Control:

See "Quality Control Procedures."

Quality control requirements must be performed in accordance with applicable local, state and/or federalregulations or accreditation requirements and your laboratory?s standard Quality Control procedures. It isrecommended that the user refer to pertinent CLSI (formerly NCCLS) guidance and CLIA regulations forappropriate Quality Control practices.

Reagents:

CIN Agar

Approximate Formula* Per Liter Purified WaterPancreatic Digest of Gelatin 10.0 gPeptic Digest of Animal Tissue 5.0 gBeef Extract 5.0 gYeast Extract 2.0 gMannitol 20.0 gSodium Pyruvate 2.0 gSodium Chloride 1.0 gMagnesium Sulfate 0.001 gSodium Desoxycholate 0.5 gAgar 12.0 gCrystal Violet 0.001 gNeutral Red 0.03 gCefsulodin 15.0 mgIrgasan 4.0 mg

Novobiocin 2.5 mg*Adjusted and/or supplemented as required to meet performance criteria.

Warnings and Precautions:

Forin vitro Diagnostic Use.

If excessive moisture is observed, invert the bottom over an off-set lid and allow to air dry in order toprevent formation of a seal between the top and bottom of the plate during incubation.


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Directions1. Suspend 59.5 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 121 C for 15 minutes.4. Cool sterilized medium to 45 - 50 C and aseptically add 10 mL of a sterilized aqueous solution containing

4 mg of Cefsulodin and 2.5 mg of Novobiocin.


Dehydrated Appearance: Powder is homogeneous, free-flowing, and pink-beige to beige.Prepared Appearance: Prepared medium is medium to dark red-orange to red-purple, and trace toslightly hazy.

ResultsYersinia enterocolitica colonies appear translucent or translucent with dark pink centers. Colony edgesare entire or irregular. After 48 hour incubation, colonies appear dark pink with a translucent border andmay be surrounded by a zone of precipitated bile. Growth of non-Yersinia organisms is markedly tocompletely inhibited.

Storage

Store sealed bottle containing dehydrated medium at 2 - 30C. Once opened and recapped, placecontainer in a low humidity environment at the same storage temperature. Protect from moisture and lightby keeping container tightly closed.


1. Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on thismedium. Further tests are necessary for confirmation ofYersinia spp.2. Some strains of normal enteric organisms may be encountered that are not inhibited or only partiallyinhibited on complete medium, such as Citrobacter freundii, Serratia liquefaciens, and Enterobacteragglomerans.3. Growth of Yersinia frederiksenii, Y. kristensenii, Y. pseudotuberculosis and Y. intermedia is notinhibited on complete medium. Colonies of these organisms must be differentiated from Y. enterocoliticaon the basis of additional characteristics.


MacCONKEY AGAR W/ SORBITOL (7320)

Intended UseMacConkey Agar W/ Sorbitol is used for the isolation of pathogenic Escherichia coli.

Product Summary and ExplanationMacConkey Agar W/ Sorbitol is based on the formula by Rappaport and Henig.1 Originally developed forisolating enteropathogenic (EPEC) serotypes, O11 and O55, this medium is recommended for theisolation and differentiation of enterohemorrhagic E. coliO157:H7. This organism causes hemorrhagic

colitis, which results in bloody diarrhea and can lead to kidney failure and death.2 Serotype O157 hasbeen implicated in serious foodborne diseases.MacConkey Agar W/ Sorbitol contains sorbitol instead of lactose for differentiating enteropathogenic E.coliserotypes; these strains are typically sorbitol negative. MacConkey Agar W/ Sorbitol is recommendedfor clinical and food testing.2-4

Principles of the ProcedureEnzymatic Digest of Gelatin, Enzymatic Digest of Casein, and Enzymatic Digest of Animal Tissue are thenitrogen and vitamin sources in MacConkey Agar W/ Sorbitol. Sorbitol is the fermentable carbohydrate;


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typically enteropathogenic strains produce colorless colonies. Bile Salts Mixture and Crystal Violet are theselective agents, inhibiting Gram-positive cocci. Sodium Choride maintains the osmotic environment, andNeutral Red is the pH indicator. Agar is the solidifying agent.

Formula / Liter

Enzymatic Digest of Gelatin .................................................... 17 gEnzymatic Digest of Casein ................................................... 1.5 gEnzymatic Digest of Animal Tissue........................................ 1.5 gSorbitol .................................................................................... 10 gBile Salts Mixture ................................................................... 1.5 gSodium Chloride ....................................................................... 5 gNeutral Red .......................................................................... 0.03 gCrystal Violet ...................................................................... 0.001 g

Agar ..................................................................................... 13.5 gFinal pH: 7.1 0.2 at 25CFormula may be adjusted and/or supplemented as required to meet performance specifications.Precautions1. For Laboratory Use.2. IRRITANT. Irritating to eyes, respiratory system, and skin.

Directions1. 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.4. Cool to 45 - 50C and dispense into sterile Petri dishes.

Quality Control SpecificationsDehydrated Appearance: Powder is homogeneous, free flowing, and light pink-beige.Prepared Appearance: Prepared medium is trace to slightly hazy, and medium to dark pink-purple.

ResultsE. coliO157:H7, and other organisms that do not ferment sorbitol, are colorless on MacConkey Agar W/Sorbitol. Sorbitol-fermenting organisms produce pink colonies. Confirmatory biochemical and serologicaltesting should be performed on suspected colonies.

StorageStore dehydrated medium at 2 - 30C. Once opened and recapped, place the container in a low humidityenvironment at the same storage temperature. Protect from moisture and light by keeping containertightly closed.

Limitations of the Procedure1. Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on thismedium.2. Colonies that are sorbitol positive can revert, and can be mistaken for sorbitol negative.5

3. E. coliO157:H7 can ferment sorbitol after prolonged incubation.


TRIPLE SUGAR IRON AGAR


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Intended UseTriple Sugar Iron Agaris used for the differentiation of microorganisms on the basis of dextrose, lactose,and sucrose fermentation and hydrogen sulfide production.

Triple Sugar Iron Agar is recommended for differentiation of enteric, Gram-negative bacilli from clinicalspecimens, dairy samples, and food products.

Principles of the ProcedureEnzymatic Digest of Casein, Enzymatic Digest of Animal Tissue, and Yeast Enriched Peptone provide thenitrogen, carbon, and vitamins required for organism growth. Triple Sugar Iron Agar contains threecarbohydrates, Dextrose, Lactose and Sucrose. When the carbohydrates are fermented, acid productionis detected by the Phenol Red pH indicator. Sodium Thiosulfate is reduced to hydrogen sulfide, andhydrogen sulfide reacts with an iron salt yielding the typical black iron sulfide. Ferric Ammonium Citrate isthe hydrogen sulfide (H2S) indicator. Sodium Chloride maintains the osmotic balance of the medium. Agaris the solidifying agent.

Formula / LiterEnzymatic Digest of Casein ...................................................... 5 g

Enzymatic Digest of Animal Tissue........................................... 5 gYeast Enriched Peptone ......................................................... 10 gDextrose .................................................................................... 1 gLactose ................................................................................... 10 gSucrose ................................................................................... 10 gFerric Ammonium Citrate ....................................................... 0.2 gSodium Chloride ....................................................................... 5 gSodium Thiosulfate ................................................................ 0.3 gPhenol Red ........................................................................ 0.025 g



Directions1. Suspend 60 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.3. Dispense into tubes and autoclave at 121 C for 15 minutes.4. After autoclaving, allow medium to solidify in a slanted position.

Quality Control SpecificationsDehydrated Appearance: Powder is homogeneous, free flowing, and light pink-beige to beige.Prepared Appearance: Prepared medium is reddish-orange and trace to slightly hazy.

ResultsAn alkaline slant-acid butt (red/yellow) indicates fermentation of dextrose only. An acid slant-acid butt(yellow/yellow) indicates fermentation of dextrose, lactose and/or sucrose. An alkaline slant-alkaline butt(red/red) indicates dextrose or lactose were not fermented (non-fermenter). Cracks, splits, or bubbles inmedium indicate gas production. A black precipitate in butt indicates hydrogen sulfide production.

Storage


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Store sealed bottle containing the dehydrated medium at 2 - 30C. Once opened and recapped, placecontainer in a low humidity environment at the same storage temperature. Protect from moisture and lightby keeping container tightly closed.

Limitations of the Procedure1. Padron and Dockstader8 found not all H2S positive Salmonella are positive on TSI.2. Sucrose is added to TSI to eliminate some sucrose-fermenting non-lactose fermenters, such asProteus and Citrobacterspp.93. Do not use inoculating loop to inoculate a tube of Triple Sugar Iron Agar. While stabbing butt,mechanical splitting of medium occurs, causing a false positive result for gas production.94. It is recommended to streak only half way up the prepared slant to avoid reversion of sugar to analkaline reaction (pink/red).


BAMBlood Agar is a general purpose enriched medium often used to grow fastidious organisms and to differentiatebacteria based on their hemolytic properties.

RECIPE

One commonly used formula:

Soybean-Casein Digest Agar

(Also referred to as "Trypticase Soy Agar" or "Tryptic Soy Agar" or "TSA" or "Blood Agar Base")

Pancreatic digest of casein USP 15.0 gPapaic digest of soy meal USP 5.0 gNaCl 5.0 gAgar 15.0 gDistilled water 1,000 ml

Combine the ingredients and adjust the pH to 7.3. Boil to dissolve the agar, and sterilize by autoclaving.

Blood Agar

To sterile Blood Agar Base which has been melted and cooled to 45 to 50C, add 5% (vol/vol) sterile defibrinatedblood that has been warmed to room temperature. Swirl the flask to mix thoroughly, avoiding the formation ofbubbles, and dispense into sterile plates, continuing to avoid bubbles and froth on the surface. (NOTE: Cooling theagar and warming the blood are essential steps in this procedure. Hot agar can damage red blood cells, and coldblood can cause the agar to gel before pouring.)

BRILLIANT GREEN AGAR (ISO) (9212)

Intended Use Brilliant Green Agar (ISO) is used for the selection and differentiation ofSalmonellae(other than S. typhi) from foods or feedstuffs. Product Summary and Explanation Salmonellosis

continues to be an important public health problem worldwide. Infection can result from consumption ofraw, undercooked, or improperly processed foods contaminated with Salmonella.

Principles of the ProcedureEnzymatic Digest of Casein and Enzymatic Digest of Animal Tissue provide sources of nitrogen, aminoacids, and carbon. Yeast Extract supplies vitamins required for organism growth. Sodium Phosphate isthe buffering agent. Lactose and Sucrose are the carbohydrates in the medium. Brilliant Green inhibitsGram-positive bacteria and most Gram-negative bacilli other than Salmonella spp. Phenol Red is the pH


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indicator and turns the medium yellow with the formation of acid when lactose and/or sucrose isfermented. Agar is the solidifying agent.

Formula / LiterEnzymatic Digest of Animal Tissue........................................ 5.0 gPeptone ................................................................................ 10.0 g YeastExtract .......................................................................... 3.0 gLactose ................................................................................ 10.0 gSucrose ................................................................................ 10.0 g Sodium Phosphate,dibasic ................................................... 1.0 g Sodium Phosphate,monobasic ............................................. 0.6 g Phenol Red ..........................................................................0.09 g Brilliant Green .................................................................. 0.0047 g

Agar ..................................................................................... 12.0 g Final pH: 6.9 0.2 at 25C Formula maybe adjusted and/or supplemented as required to meet performance specifications.


Directions1. Suspend 52 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 SpecificationsDehydrated Appearance: Powder is homogeneous, free flowing, and beige with a green tint.Prepared Appearance: Prepared medium is brown-red, trace to slightly hazy, and slightlyopalescent.

Test Procedure (as outlined by Edel and Kampelmacher)41. Add one part of the sample to 20 parts of Muller Kauffmann Tetrathionate Broth Base (9221).

2. After agitation, incubate Muller Kauffmann Tetrathionate Broth Base into a 45C waterbath for 15minutes only.3. Transfer the inoculated flask to a 43C incubator.4. Subculture the Muller Kauffmann Tetrathionate Broth Base to Brilliant Green Agar (ISO) after 18 48hours using the streak method.5. Incubate Brilliant Green Agar (ISO) at 35C for 18 24 hours.

Results Typical Salmonella spp. colonies are red. Serological confirmation is required.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 moistureand light.


1. Lactose-fermenting Salmonella species may be present in foods.2. Salmonella typhiand Shigella species may not growth on this medium.3. Proteus, Citrobacter, and Pseudomonas species may mimic enteric pathogens by producing small redcolonies.



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Bismuth Sulfite AgarIntended UseBismuth Sulfite Agar is a highly selective medium used for isolating Salmonella spp.,particularly SalmonellaTyphi, from food and clinical specimens.

Principles of the ProcedureIn Bismuth Sulfite Agar, beef extract and peptone providenitrogen, vitamins and minerals. Dextrose is an energy source.Disodium phosphate is a buffering agent. Bismuth sulfiteindicator and brilliant green are complementary in inhibitinggram-positive bacteria and members of the coliform group,while allowing Salmonella to grow luxuriantly. Ferroussulfate is included for detection of H2S production. WhenH2S is present, the iron in the formula is precipitated, givingpositive cultures the characteristic brown to black color withmetallic sheen. Agar is the solidifying agent.

FormulaDifco Bismuth Sulfite Agar

Approximate Formula* Per LiterBeef Extract.................................................................. 5.0 gPeptone..................................................................... 10.0 gDextrose...................................................................... 5.0 gDisodium Phosphate.................................................... 4.0 gFerrous Sulfate............................................................. 0.3 gBismuth Sulfite Indicator.............................................. 8.0 gAgar.......................................................................... 20.0 gBrilliant Green............................................................ 25.0 mg*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation fromDehydrated Product1. Suspend 52 g of the powder in 1 L of purified water. Mix

thoroughly.2. Heat with frequent agitation and boil for 1 minute tocompletely dissolve the powder. DO NOT AUTOCLAVE.3. Evenly disperse the precipitate when dispensing. Use themedium the same day it is prepared.4. Test samples of the finished product for performance usingstable, typical control cultures.

User Quality ControlIdentity Specifications

Difco Bismuth Sulfite AgarDehydrated Appearance: Light beige to light green, free-flowing, homogeneous.Solution: 5.2% solution, soluble in purified water uponboiling. Solution is light green, opaque with aflocculent precipitate that can be dispersed byswirling contents of flask.Prepared Appearance: Light gray-green to medium green, opaque witha flocculent precipitate.Reaction of 5.2%Solution at 25C: pH 7.7 0.2


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Expected ResultsThe typical discrete S.Typhi surface colony is black and surrounded by a black or brownish-black zone which may be several times the size of the colony. By reflected light, preferablydaylight, this zone exhibits a distinctly characteristic metallic sheen. Plates heavily seededwith S.Typhi may not show this reaction except near the margin of the mass inoculation. In

these heavy growth areas, this organism frequently appears as small light green colonies.This fact emphasizes the importance of inoculating plates so that some areas are sparselypopulated with discrete S.Typhi colonies. Other strains ofSalmonella produce black to greencolonies with little or no darkening ofthe surrounding medium.Generally, Shigella spp. other than S. flexneri and S. sonnei are inhibited. S. flexneri and S.sonnei strains that do grow on this medium produce brown to green, raised colonies withdepressed centers and exhibit a crater-like appearance.Escherichia coli is partially inhibited. Occasionally a strain will be encountered that will growas small brown or greenish glistening colonies. This color is confined entirely to the colonyitself and shows no metallic sheen. A few strains ofEnterobacter aerogenes may develop onthis medium, forming raised, mucoid colonies. Enterobactercolonies may exhibit a silverysheen, appreciably lighter in color than that produced by S.Typhi. Some members of the

coliform group that produce hydrogen sulfide may grow on the medium, giving coloniessimilar in appearance to S.Typhi. These coliforms may be readily differentiated becausethey produce gas from lactose in differentialmedia, for example, Kligler Iron Agar or Triple Sugar Iron Agar. The hydrolysis of urea,demonstrated in Urea Broth or on Urea Agar Base, may be used to identify Proteus sp. Toisolate S.Typhi for agglutination or fermentation studies, pick characteristic black coloniesfrom Bismuth Sulfite Agar and subculture them on MacConkey Agar. The purified coloniesfrom MacConkey Agar may then be picked to differential tubemedia such as Kligler Iron Agar, Triple Sugar Iron Agar or other satisfactory differentialmedia for partial identification. All cultures that give reactions consistent with Salmonellaspp. On these media should be confirmed biochemically as Salmonella spp. before anyserological testing is performed. Agglutination tests may be performed from the freshgrowth on the differential tube media or from the growth on nutrient agar slants inoculated

from the differential media. The growth on the differential tube media may also be used forinoculating carbohydrate media for fermentation studies.

Limitations of the Procedure1. It is important to streak for well-isolated colonies. In heavygrowth areas, S.Typhi appears light green and may bemisinterpreted as negative growth for S.Typhi.202. S.Typhi and S. arizonae are the only enteric organisms toexhibit typical brown zones on the medium. Brown zones arenot produced by other members of the Enterobacteriaceae .However, S. arizonae is usually inhibited.203. Colonies on Bismuth Sulfite Agar may be contaminated withother viable organisms; therefore, isolated colonies should

be subcultured to a less selective medium (e.g., MacConkeyAgar).204. Typical S.Typhi colonies usually develop within 24 hours;however, all plates should be incubated for a total of48 hours to allow growth of all typhoid strains.205. DO NOT AUTOCLAVE. Heating this medium for a periodlonger than necessary to just dissolve the ingredients destroysits selectivity.

http://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/273300.pdf
http://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/273300.pdfhttp://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/273300.pdf


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SELENITE BROTH

Intended Use

Selenite Broth is used for the selective enrichment ofSalmonella spp.

Product Summary and ExplanationSelenite Broth was originated by Leifson,1 while observing good recovery ofSalmonella spp. and reducedgrowth of fecal coliforms. Selenite Broth is used as a selective enrichment for the cultivation ofSalmonella spp. that may be present in small numbers and competing with intestinal flora. Salmonellaorganisms are also injured in food-processing procedures, including exposure to low temperatures, sub-marginal heat, drying, radiation, preservatives or sanitizers.2Although injured cells may not form colonieson selective media, they cause infection if ingested.3 Salmonella spp. cause many types of infections,from mild self-limiting gastroenteritis to life-threatening typhoid fever.Selenite Broth conforms with the American Public Health Association (APHA),and is specified for clinicalapplications.4,6 Many modifications of Selenite Broth exist, including Selenite Cystine Broth, from theoriginal formula described as Selenite F Broth by Leifson.

Principles of the ProcedureEnzymatic Digest of Casein and Enzymatic Digest of Animal Tissue are the nitrogen and vitamin sourcesin Selenite Broth. Lactose is the fermentable carbohydrate. Sodium Phosphate is the buffer. A rise in pHdecreases selective activity of Selenite. The acid produced by lactose fermentation helps to maintain aneutral pH. Sodium Selenite inhibits the growth of Gram-positive bacteria and many Gram-negativebacteria.

Formula/LiterEnzymatic Digest of Casein ................................................... 2.5 gEnzymatic Digest of Animal Tissue....................................... 2.5 gLactose ..................................................................................... 4 gSodium Phosphate .................................................................. 10 g

Sodium Selenite ........................................................................ 4 gFinal pH: 7.0 0.2 at 25CFormula may be adjusted and/or supplemented as required to meet performance specifications.

Precautions1. For Laboratory Use.2. Harmful. Harmful by inhalation and if swallowed. Irritating to respiratory system.

Directions1. Dissolve 23 g of the medium in one liter of purified water.2. Heat to boiling. Avoid overheating.3. DO NOT AUTOCLAVE.

Quality Control SpecificationsDehydrated Appearance: Powder is homogeneous, free flowing, and off-white.Prepared Appearance: Prepared medium is clear, with no to light precipitate and very pale yellow.

StorageStore dehydrated medium at 2 - 30C. Once opened and recapped, place the container in a low humidityenvironment at the same storage temperature. Protect from moisture and light by keeping containertightly closed.


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Limitation of the ProcedureDue to nutritional variation, some strains may be encountered that grow poorly or fail to grow on thismedium.


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