GROUP 4

February 27, 2012

Family Enterobacteriaceae: Morphology and Cultural Characteristics

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_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 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/62915dat.pd f
Characteristic reactions of some Enterobacteriaceae cultured on Lysine Iron Agar: Microorganisms
Arizona Salmonella * Proteus mirabilis

Butt
violet violet yellow

Slant surface
violet violet red-brown

H2S production
+ + +

Proteus vulgaris Proteus morganii Proteus rettgeri Providencia Citrobacter Escherichia Shigella Klebsiella

yellow yellow yellow yellow yellow violet

red-brown red-brown violet violet violet violet

+ -

* 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 filtersterilized 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 Potassium phosphate, monobasic Potassium phosphate, dibasic Urea

Amount 0.1 g 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. 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 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 pinkred 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 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) Proteose peptone (Difco) or Polypeptone (BBL) Lactose NaCl Crystal Violet Neutral Red Bile Salts Agar Distilled Water 17.0 g 3.0 g 10.0 g 5.0 g 1.0 mg 30.0 mg 1.5 g 13.5 g 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 nonfastidious 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

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. Nonfermenters 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 Enzymatic Digest of Animal Tissue.....................................16.5 g Yeast Extract............................................................................3 g Bile Salts Mixture...................................................................4.5 g Lactose...................................................................................12 g Sucrose...................................................................................12 g Salicin.......................................................................................2 g Sodium Chloride.......................................................................5 g Sodium Thiosulfate...................................................................5 g Ferric Ammonium Citrate.......................................................1.5 g Bromthymol Blue...............................................................0.065 g Acid Fuchsin..........................................................................0.1 g Agar.....................................................................................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. 3. DO NOT AUTOCLAVE.

Quality Control Specifications 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. 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. Limitations of the Procedure 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 confirm the 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 this medium.
http://www.neogen.com/Acumedia/pdf/ProdInfo/7138_PI.pdf

Xylose-Lysine Deoxycholate Agar (XLD Agar) M031

Xylose-Lysine Deoxycholate Agar (XLD Agar) is a selective medium recommended for the isolation and enumeration of Salmonella Typhi and other Salmonella species. Composition** Ingredients Gms / Litre Yeast extract 3.000 L-Lysine 5.000 Lactose 7.500 Sucrose 7.500 Xylose 3.500 Sodium 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 other Salmonella 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

overgrowth of other organisms over Salmonella 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 Shigellae but practically by all enterics. This helps in the differentiation of Shigella 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 may change 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. Some Proteus strains may give red to yellow colouration with most colonies developing black centers, giving rise to false positive reactions. Non-enterics like Pseudomonas and Providencia may exhibit red colonies. S. Paratyphi A , S. Choleraesuis , S. Pullorum and S. Gallinarum may form 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 medium Red 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

CIN Agar (Yersinia Selective)

Intended Use: CIN (cefsulodin-Irgasan-novobiocin) Agar is a differential and selective medium used in qualitative procedures for the isolation of Yersinia enterocolitica from a variety of clinical and nonclinical specimens.

Product Summary: CIN Agar was first described by Schiemann as an alternative to MacConkey Agar and other commonly used media for isolation of Yersinia enterocolitica, a causative agent of gastroenteritis.1 CIN Agar has been 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 federal regulations or accreditation requirements and your laboratory? standard Quality Control procedures. It is s recommended that the user refer to pertinent CLSI (formerly NCCLS) guidance and CLIA regulations for appropriate Quality Control practices.

Reagents: CIN Agar Approximate Formula* Per Liter Purified Water Pancreatic Digest of Gelatin Peptic Digest of Animal Tissue Beef Extract Yeast Extract Mannitol Sodium Pyruvate Sodium Chloride Magnesium Sulfate Sodium Desoxycholate Agar Crystal Violet Neutral Red Cefsulodin Irgasan Novobiocin *Adjusted and/or supplemented as required to meet performance criteria. Warnings and Precautions: For in vitro Diagnostic Use. If excessive moisture is observed, invert the bottom over an off-set lid and allow to air dry in order to prevent formation of a seal between the top and bottom of the plate during incubation. 10.0 g 5.0 g 5.0 g 2.0 g 20.0 g 2.0 g 1.0 g 0.001 g 0.5 g 12.0 g 0.001 g 0.03 g 15.0 mg 4.0 mg 2.5 mg

Pathogenic microorganisms, including hepatitis viruses and Human Immunodeficiency Virus, may be present in clinical specimens. "Standard Precautions"3-6 and institutional guidelines should be followed in handling all items contaminated with blood and other body fluids. After use, prepared plates, specimen containers and other contaminated materials must be sterilized by autoclaving before discarding.
http://www.bd.com/ds/productCenter/221848.asp

YERSINIA SELECTIVE AGAR

Intended Use Yersinia Selective Agar is used with cefsulodin and novobiocin for the isolation of Yersinia enterocolitica. Product Summary and Explanation Yersinia enterocolitica is a significant food or water borne enteric pathogen.1,2 Yersinia Selective Agar is a selective and differential medium supporting good growth of Y. enterocolitica and some Yersinia spp. The formula is based on Cefsulodin-Irgasan-Novobiocin (CIN) Agar developed by Schiemann.3-6 In comparison with MacConkey Agar and SS Agar, Schiemann found CIN Agar provided increased inhibition of normal enteric organisms and provided improved direct recovery of Y. enterocolitica from feces.4 Schiemann later modified his formula to further improve growth and recovery of Y. enterocolitica.6 Principles of the Procedure Enzymatic Digest of Gelatin, Enzymatic Digest of Casein, and Enzymatic Digest of Animal Tissue provide the nitrogen and amino acids in Yersinia Selective Agar. Yeast Extract is the vitamin source in this formula. Selectivity of Yersinia Selective Agar is due to the presence of Sodium Deoxycholate, Sodium Cholate, Crystal Violet, and Irgasan that markedly inhibit growth of Gram-positive and many Gramnegative organisms. Supplementation with Cefsulodin and Novobiocin improves inhibition of normal enteric organisms. Differentiation is based on Mannitol fermentation. Organisms capable of fermenting Mannitol lower the pH around the colony, allowing absorption of Neutral Red and turning the colony a red color. Due to the localized pH decrease, a zone of precipitated bile may also be present. Organisms that do not metabolize Mannitol to acid end products will form colorless, translucent colonies. Sodium Chloride maintains the osmotic balance of the medium. Sodium Pyruvate and Magnesium Sulfate stimulate organism growth. Agar is the solidifying agent. Formula / Liter Antimicrobic / 10 mL Enzymatic Digest of Gelatin .................................................... 17 g Cefsulodin 4 mg Enzymatic Digest of Casein ................................................... 1.5 g Novobiocin 2.5 mg Enzymatic Digest of Animal Tissue........................................ 1.5 g Yeast Extract ............................................................................. 2 g Mannitol .................................................................................. 20 g Sodium Deoxycholate ............................................................ 0.5 g Sodium Cholate ..................................................................... 0.5 g Sodium Chloride ....................................................................... 1 g Sodium Pyruvate ....................................................................... 2 g Magnesium Sulfate .............................................................. 0.01 g Neutral Red .......................................................................... 0.03 g Crystal Violet ...................................................................... 0.001 g Irgasan ............................................................................ 0.004 g Agar ..................................................................................... 13.5 g Final pH: 7.4 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 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 121C for 15 minutes. 4. Cool sterilized medium to 45 - 50C and aseptically add 10 mL of a sterilized aqueous solution containing 4 mg of Cefsulodin and 2.5 mg of Novobiocin. Quality Control Specifications 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 to slightly hazy. Results Yersinia enterocolitica colonies appear translucent or translucent with dark pink centers. Colony edges are entire or irregular. After 48 hour incubation, colonies appear dark pink with a translucent border and may be surrounded by a zone of precipitated bile. Growth of non-Yersinia organisms is markedly to completely inhibited. Storage Store sealed bottle containing 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. Limitations of the Procedure 1. Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on this medium. Further tests are necessary for confirmation of Yersinia spp. 2. Some strains of normal enteric organisms may be encountered that are not inhibited or only partially inhibited on complete medium, such as Citrobacter freundii, Serratia liquefaciens, and Enterobacter agglomerans. 3. Growth of Yersinia frederiksenii, Y. kristensenii, Y. pseudotuberculosis and Y. intermedia is not inhibited on complete medium. Colonies of these organisms must be differentiated from Y. enterocolitica on the basis of additional characteristics. http://www.neogen.com/Acumedia/pdf/ProdInfo/7257_PI.pdf

MacCONKEY AGAR W/ SORBITOL (7320)

Intended Use MacConkey Agar W/ Sorbitol is used for the isolation of pathogenic Escherichia coli. Product Summary and Explanation MacConkey Agar W/ Sorbitol is based on the formula by Rappaport and Henig.1 Originally developed for isolating enteropathogenic (EPEC) serotypes, O11 and O55, this medium is recommended for the isolation and differentiation of enterohemorrhagic E. coli O157:H7. This organism causes hemorrhagic colitis, which results in bloody diarrhea and can lead to kidney failure and death.2 Serotype O157 has been implicated in serious foodborne diseases. MacConkey Agar W/ Sorbitol contains sorbitol instead of lactose for differentiating enteropathogenic E. coli serotypes; these strains are typically sorbitol negative. MacConkey Agar W/ Sorbitol is recommended for clinical and food testing.2-4 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 W/ Sorbitol. Sorbitol is the fermentable carbohydrate;

typically enteropathogenic strains produce colorless colonies. Bile Salts Mixture and Crystal Violet are the selective agents, inhibiting Gram-positive cocci. Sodium Choride maintains the osmotic environment, and Neutral Red is the pH indicator. 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 Sorbitol .................................................................................... 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. 4. Cool to 45 - 50C and dispense into sterile Petri dishes.

Quality Control Specifications Dehydrated 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. Results E. coli O157: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 serological testing should be performed on suspected colonies. Storage Store dehydrated medium at 2 - 30C. Once opened and recapped, place the container in a low humidity environment at the same storage temperature. Protect from moisture and light by keeping container tightly closed. Limitations of the Procedure 1. Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on this medium. 2. Colonies that are sorbitol positive can revert, and can be mistaken for sorbitol negative.5 3. E. coli O157:H7 can ferment sorbitol after prolonged incubation. http://www.neogen.com/Acumedia/pdf/ProdInfo/7320_PI.pdf

TRIPLE SUGAR IRON AGAR

Intended Use Triple Sugar Iron Agar is 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 clinical specimens, dairy samples, and food products.

Principles of the Procedure Enzymatic Digest of Casein, Enzymatic Digest of Animal Tissue, and Yeast Enriched Peptone provide the nitrogen, carbon, and vitamins required for organism growth. Triple Sugar Iron Agar contains three carbohydrates, Dextrose, Lactose and Sucrose. When the carbohydrates are fermented, acid production is detected by the Phenol Red pH indicator. Sodium Thiosulfate is reduced to hydrogen sulfide, and hydrogen sulfide reacts with an iron salt yielding the typical black iron sulfide. Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator. Sodium Chloride maintains the osmotic balance of the medium. Agar is the solidifying agent. Formula / Liter Enzymatic Digest of Casein ...................................................... 5 g Enzymatic Digest of Animal Tissue........................................... 5 g Yeast Enriched Peptone ......................................................... 10 g Dextrose .................................................................................... 1 g Lactose ................................................................................... 10 g Sucrose ................................................................................... 10 g Ferric Ammonium Citrate ....................................................... 0.2 g Sodium Chloride ....................................................................... 5 g Sodium Thiosulfate ................................................................ 0.3 g Phenol Red ........................................................................ 0.025 g Agar ..................................................................................... 13.5 g Final pH: 7.3 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. 3. Heat with frequent agitation and boil for one minute to completely dissolve the medium. 3. Dispense into tubes and autoclave at 121C for 15 minutes. 4. After autoclaving, allow medium to solidify in a slanted position. Quality Control Specifications Dehydrated Appearance: Powder is homogeneous, free flowing, and light pink-beige to beige. Prepared Appearance: Prepared medium is reddish-orange and trace to slightly hazy. Results An 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 in medium indicate gas production. A black precipitate in butt indicates hydrogen sulfide production. 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 by keeping container tightly closed. Limitations of the Procedure 1. 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 as Proteus and Citrobacter spp.9 3. 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.9 4. It is recommended to streak only half way up the prepared slant to avoid reversion of sugar to an alkaline reaction (pink/red). http://www.neogen.com/Acumedia/pdf/ProdInfo/7162_PI.pdf

BAM
Blood Agar is a general purpose enriched medium often used to grow fastidious organisms and to differentiate bacteria 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 g Papaic digest of soy meal USP 5.0 g NaCl 5.0 g Agar 15.0 g Distilled 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 defibrinated blood that has been warmed to room temperature. Swirl the flask to mix thoroughly, avoiding the formation of bubbles, and dispense into sterile plates, continuing to avoid bubbles and froth on the surface. (NOTE: Cooling the agar and warming the blood are essential steps in this procedure. Hot agar can damage red blood cells, and cold blood 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 of Salmonellae (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 of raw, undercooked, or improperly processed foods contaminated with Salmonella.

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

indicator and turns the medium yellow with the formation of acid when lactose and/or sucrose is fermented. Agar is the solidifying agent. Formula / Liter Enzymatic Digest of Animal Tissue........................................ 5.0 g Peptone ................................................................................ 10.0 g Yeast Extract .......................................................................... 3.0 g Lactose ................................................................................ 10.0 g Sucrose ................................................................................ 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 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 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 Specifications Dehydrated Appearance: Powder is homogeneous, free flowing, and beige with a green tint. Prepared Appearance: Prepared medium is brown-red, trace to slightly hazy, and slightly opalescent. Test Procedure (as outlined by Edel and Kampelmacher)4 1. 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 15 minutes 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 48 hours 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 moisture and light. Limitations of the Procedure 1. Lactose-fermenting Salmonella species may be present in foods. 2. Salmonella typhi and Shigella species may not growth on this medium. 3. Proteus, Citrobacter, and Pseudomonas species may mimic enteric pathogens by producing small red colonies. http://www.neogen.com/Acumedia/pdf/ProdInfo/9212_PI.pdf

Bismuth Sulfite Agar

Intended Use
Bismuth Sulfite Agar is a highly selective medium used for isolating Salmonella spp., particularly Salmonella Typhi, from food and clinical specimens.

Principles of the Procedure

In Bismuth Sulfite Agar, beef extract and peptone provide nitrogen, vitamins and minerals. Dextrose is an energy source. Disodium phosphate is a buffering agent. Bismuth sulfite indicator and brilliant green are complementary in inhibiting gram-positive bacteria and members of the coliform group, while allowing Salmonella to grow luxuriantly. Ferrous sulfate is included for detection of H2S production. When H2S is present, the iron in the formula is precipitated, giving positive cultures the characteristic brown to black color with metallic sheen. Agar is the solidifying agent.

Formula

Difco Bismuth Sulfite Agar

Approximate Formula* Per Liter Beef Extract.................................................................. 5.0 g Peptone..................................................................... 10.0 g Dextrose...................................................................... 5.0 g Disodium Phosphate.................................................... 4.0 g Ferrous Sulfate............................................................. 0.3 g Bismuth Sulfite Indicator.............................................. 8.0 g Agar.......................................................................... 20.0 g Brilliant Green............................................................ 25.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 52 g of the powder in 1 L of purified water. Mix thoroughly. 2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. DO NOT AUTOCLAVE. 3. Evenly disperse the precipitate when dispensing. Use the medium the same day it is prepared. 4. Test samples of the finished product for performance using stable, typical control cultures.

User Quality Control

Identity Specifications
Difco Bismuth Sulfite Agar
Dehydrated Appearance: Light beige to light green, free-flowing, homogeneous. Solution: 5.2% solution, soluble in purified water upon boiling. Solution is light green, opaque with a flocculent precipitate that can be dispersed by swirling contents of flask. Prepared Appearance: Light gray-green to medium green, opaque with a flocculent precipitate. Reaction of 5.2% Solution at 25C: pH 7.7 0.2

Expected Results
The typical discrete S. Typhi surface colony is black and surrounded by a black or brownishblack zone which may be several times the size of the colony. By reflected light, preferably daylight, this zone exhibits a distinctly characteristic metallic sheen. Plates heavily seeded with 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 sparsely populated with discrete S. Typhi colonies. Other strains of Salmonella produce black to green colonies with little or no darkening of the 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 with depressed centers and exhibit a crater-like appearance. Escherichia coli is partially inhibited. Occasionally a strain will be encountered that will grow as small brown or greenish glistening colonies. This color is confined entirely to the colony itself and shows no metallic sheen. A few strains of Enterobacter aerogenes may develop on this medium, forming raised, mucoid colonies. Enterobacter colonies may exhibit a silvery sheen, 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 colonies similar in appearance to S. Typhi. These coliforms may be readily differentiated because they produce gas from lactose in differential media, 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. To isolate S. Typhi for agglutination or fermentation studies, pick characteristic black colonies from Bismuth Sulfite Agar and subculture them on MacConkey Agar. The purified colonies from MacConkey Agar may then be picked to differential tube media such as Kligler Iron Agar, Triple Sugar Iron Agar or other satisfactory differential media for partial identification. All cultures that give reactions consistent with Salmonella spp. On these media should be confirmed biochemically as Salmonella spp. before any serological testing is performed. Agglutination tests may be performed from the fresh growth 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 for inoculating carbohydrate media for fermentation studies.

Limitations of the Procedure

1. It is important to streak for well-isolated colonies. In heavy growth areas, S. Typhi appears light green and may be misinterpreted as negative growth for S. Typhi.20 2. S. Typhi and S. arizonae are the only enteric organisms to exhibit typical brown zones on the medium. Brown zones are not produced by other members of the Enterobacteriaceae. However, S. arizonae is usually inhibited.20 3. Colonies on Bismuth Sulfite Agar may be contaminated with other viable organisms; therefore, isolated colonies should be subcultured to a less selective medium (e.g., MacConkey Agar).20 4. Typical S. Typhi colonies usually develop within 24 hours; however, all plates should be incubated for a total of 48 hours to allow growth of all typhoid strains.20 5. DO NOT AUTOCLAVE. Heating this medium for a period longer than necessary to just dissolve the ingredients destroys its selectivity. http://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/273300.pdf

SELENITE BROTH
Intended Use Selenite Broth is used for the selective enrichment of Salmonella spp. Product Summary and Explanation Selenite Broth was originated by Leifson,1 while observing good recovery of Salmonella spp. and reduced growth of fecal coliforms. Selenite Broth is used as a selective enrichment for the cultivation of Salmonella spp. that may be present in small numbers and competing with intestinal flora. Salmonella organisms are also injured in food-processing procedures, including exposure to low temperatures, submarginal heat, drying, radiation, preservatives or sanitizers.2 Although injured cells may not form colonies on 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 clinical applications.4,6 Many modifications of Selenite Broth exist, including Selenite Cystine Broth, from the original formula described as Selenite F Broth by Leifson. Principles of the Procedure Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue are the nitrogen and vitamin sources in Selenite Broth. Lactose is the fermentable carbohydrate. Sodium Phosphate is the buffer. A rise in pH decreases selective activity of Selenite. The acid produced by lactose fermentation helps to maintain a neutral pH. Sodium Selenite inhibits the growth of Gram-positive bacteria and many Gram-negative bacteria. Formula/Liter Enzymatic Digest of Casein ................................................... 2.5 g Enzymatic Digest of Animal Tissue....................................... 2.5 g Lactose ..................................................................................... 4 g Sodium Phosphate .................................................................. 10 g Sodium Selenite ........................................................................ 4 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. Harmful. Harmful by inhalation and if swallowed. Irritating to respiratory system. Directions 1. Dissolve 23 g of the medium in one liter of purified water. 2. Heat to boiling. Avoid overheating. 3. DO NOT AUTOCLAVE.

Quality Control Specifications Dehydrated Appearance: Powder is homogeneous, free flowing, and off-white. Prepared Appearance: Prepared medium is clear, with no to light precipitate and very pale yellow. Storage Store dehydrated medium at 2 - 30C. Once opened and recapped, place the container in a low humidity environment at the same storage temperature. Protect from moisture and light by keeping container tightly closed.

Limitation 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/7155_PI.pdf