KLIGLER IRON AGAR

INTENDED 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 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 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:

Microorganisms
Arizona Salmonella * Proteus mirabilis Proteus vulgaris Proteus morganii Proteus rettgeri Providencia Citrobacter Escherichia Shigella Klebsiella

Butt
violet violet yellow yellow yellow yellow yellow yellow violet

Slant surface
violet 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 strains

Shigella 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

Growth
good / very good good / very good good / very good good / very good good / very good good / very good good / very good

Butt
yellow yellow violet and black violet and black yellow and black yellow and black yellow

Slant
violet 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).

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.

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 Agar
Purpose 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 Use
MacConkey 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 25C
Formula 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 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 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 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 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.