Enterobacteriaceae culture media

JOVE, JESSICA JANE B February 27, 2013

Experiment #

TRIPLE SUGAR IRON AGARPurpose

Determine whether a gram negative rod utilizes glucose and lactose or sucrose fermentative and forms hydrogen sulfide

PrincipleThe TSI is a two reaction chamber with an aerobic slant portion and an anaerobic deep portion. The slant of the tube is exposed to atmospheric oxygen and will become alkaline due to oxidative decarboxylation of peptides and amino acids. The slant tends to become and remain alkaline (red). Amino acid degradation is minimal in the deep (anaerobic) portion, and thus a small quantity of acid produced can be detected because few amines are being formed from amino acids.

IngredientsEnzymatic Digest of Casein ...................................................... 5 gEnzymatic Digest of Animal Tissue........................................... 5 gYeast Enriched Peptone ......................................................... 10 gDextrose.................................................................................... 1 gLactose ................................................................................... 10 gSucrose................................................................................... 10 gFerric Ammonium Citrate....................................................... 0.2 gSodium Chloride ....................................................................... 5 gSodium Thiosulfate ................................................................ 0.3 gPhenol Red ........................................................................ 0.025 gAgar ..................................................................................... 13.5 g

Purpose of ingredientsEnzymatic 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.

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

Quality controlA/Ag: Escherichia coliK/A H2S+: Salmonella typhiK/NC: Pseudomonas aeruginosa

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

http://www.neogen.com/Acumedia/pdf/ProdInfo/7162_PI.pdf

INDOLE PRODUCTIONPurpose

Indole broth is used for distinguishing Enterobacteriaceae based on the ability to produce indole from tryptophan. The test is particularly useful for the identification of lactose-fermenting member of Enterobacteriaceae. Escherichia coli are indole positive, whereas Enterobacter and Klebsiella are indole negative. Indole is also useful in the inspection of Proteus. P. mirabilis is indole negative, P. vulgaris is positive.

Principle:Tryptophan present in peptone is oxidized by certain bacteria to indole, skatole, and indole-acetic acid. The intracellular enzymes that metabolize tryptophan are known as tryptophanse. Indole is detected in broth cultures of bacteria with an alcoholic pdimethlyaminobenzaldehyde reagent. Indole reacts with the acetaldehyde to form a red product. Two reagents may be used to detect indole, Kovac’s and Ehrlich’s. Ehrlich’s reagent is believed to be more sensitive than Kovac’s reagent and is recommended for indole detection in anaerobes and non-fermentative bacteria. Kovac’s reagent was initially used to classify members of the Enterobacteriaceae and should be used with these organisms.

TrytophanaseTryptophan Indole + pyruvic acid + NH3

H2OIngredients

Kovac’s reagentp-DimethylaminobenzaldehydeAmyl alcoholHCl

Indole Reagentp-DimethylaminocinamaldehydeHClDeionized water

Purpose of IngredientsKovac’s Reagent contain the aldehyde p-dimethylaminobenzaldehyde. Comparatively, Indole Reagent (PACA) is the most sensitive reagent and can detect as little as 3.0µg of indole per mL. Indole Reagent is the reagent of choice for performing the spot indole test. The spot indole test is a rapid procedure designed by Vracko and Sherris. It can be used to presumptively characterize Escherichia coli, and to differentiate swarming Proteus species. It has also been found useful for examining anaerobic organisms.

ConsiderationsCheck for signs of deterioration and the performance of the reagent weekly using known quality control organisms.

Quality controlA. Kovac’s method

Positive: Escherichia coliNegative: Klebsiella pneumonia

ResultsPositive: Pink to wine colored ring after addition of

appropriate reagentNegative: No color change after the addition of the

appropriate reagent

http://www.pmlmicro.com/assets/TDS/845.pdf


METHYL RED TEST / VOGES-PROSKAUER TEST

PurposeMR-VP broth is a dextrose broth medium buffered with peptone. Glucose is fermented to pyruvic acid by one of two pathways, which results in either a positive MR or a positive VP test. The tests are particularly useful for the lactose-fermenting Enterobacteriaceae. Escherichia coli is MR positive and VP negative, whereas most members of the Klebsiella- Enterobacter- Serratia-Hafnia group are VP positive..

PrincipleThis is to detect the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Some bacteria produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system. Methyl Red is a pH indicator, which remains red in color at a pH of 4.4 or less. VP test detects butylene glycol producers. Acetyl-methyl carbinol (acetoin) is an intermediate in the production of butylene glycol. If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl. Diacetyl then reacts with guanidine components of peptone, in the presence of alpha-naphthol to produce red color.

IngredientsMR-VP broth; glucose baseMR pH indicator5% alpha-napthol in absolute methyl alcohol40% KOH containing 0.3% creatine

Purpose of ingredientsMethyl red act as a pH indicatorAlpha-naphthol is that of a catalyst and a color intensifier.

ConsiderationsRemember Some organisms do not produce stable acid end products and instead further metabolize acids to more neutral end products like 2,3 butanediol. The reagents used however, don't test for 2,3 butanediol, but rather it's precursor acetoin.

Quality controlMethyl Red

Positive: Escherichia coliNegative: Enterobacter cloacae

Voger ProskauerPositive: Enterobacter cloacaeNegative: Escherichia coli

ResultsMethyl Red

Positive: Presence of red color (B)Negative: Yellow or orange coloration (A)

Voger ProskauerPositive: Red color (A)Negative: Yellow color (B)

http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/MRVP%20Results.html

http://homepages.wmich.edu/~rossbach/bios312

CITRATE UTILIZATION

Purpose: It is used to determine the ability of an organism to utilize sodium citrate as its only carbon source and inorganic ammonium salt as its only nitrogen source.

Principle:Bacteria can grow on this medium turn the bromthymol blue indicator from green to blue.

IngredientsMagnesium sulfate (heptahydrate) 0.2 gAmmonium dihydrogen phosphate 1.0gDipotassium phosphate 1.0gSodium citrate (dehydrate) 2.0gSodium chloride 5.0gAgar 15.0gBromothymol blue 0.08gDeionized water 1000mL

Purpose of ingredientsAmmonium Dihydrogen Phosphate is the sole source of nitrogen in Simmons Citrate Agar. Dipotassium Phosphate acts as a buffer. Sodium Chloride maintains the osmotic balance of the medium. Sodium Citrate is the sole source of carbon in this medium. Magnesium Sulfate is a cofactor for a variety of metabolic reactions. Bromthymol Blue is the pH indicator. Organisms that can utilize Ammonium Dihydrogen Phosphate and Sodium Citrate as their sole sources of nitrogen and carbon will grow on this medium and produce a color change from green (neutral) to blue (alkaline). Agar is the solidifying agent.

ConsiderationsDue to nutritional variation, some strains may be encountered that grow poorly or fail to grow on this medium.Some citrate positive organisms require a 48 hour incubation or longer for a pH change to occur.

Quality controlPositive: Klebsiella pneumoniaNegative: Escherichia coli

Result:Positive: Growth on the medium, with or without a change in the color of the indicator. The color change of the indicator is due to acid or alkali production by the test organism as it grows on the medium. Growth usually results in the bromthymol blue indicator turning from green to blue.Negative: Absence of growth



UREASE TEST

PurposeUrease is an enzyme that splits urea into alkaline end products. The reaction is useful in the identification of rapid urease producers, such as Proteus and Morganella, as well as weak urease producers, such as Klebsiella pneumoniae and some species of Enterobacter.

PrincipleUrease splits the urea molecule into ammonia (NH3), carbon dioxide (CO2), and water (H2O). Ammonia reacts in solution to form an alkaline compound, ammonium carbonate, which results in an increased pH of the medium and a color change in the indicator to pink-red.

IngredientsEnzymatic Digest of Gelatin...................................................... 1 g Dextrose.................................................................................... 1 gSodium Chloride ....................................................................... 5 gMonopotassium Phosphate ...................................................... 2 gUrea ........................................................................................ 20 gPhenol Red ........................................................................ 0.012 gFinal pH: 6.8 ± 0.2 at 25°C

Purpose of ingredientsEnzymatic Digest of Gelatin provides nitrogen, carbon, and amino acids required for organism growth in Urea Agar Base. Dextrose is an energy source. Sodium Chloride maintains the osmotic balance of the medium. Monopotassium Phosphate is the buffer. Urea provides a nitrogen source for organisms producing urease. The splitting of urea by urease causes the release of ammonia, increasing pH of the medium to the alkaline side. This is indicated by a color change of the pH indicator, Phenol Red, from yellow (pH 6.8) to red (pH 8.1). Agar is added as a supplement to solidify the medium.

Considerations1. The alkaline reaction produced in this medium after prolonged incubation may not be caused by urease activity. False positive reactions may occur due to the utilization of peptones or other proteins that raise the pH due to protein hydrolysis and the release of excessive amino acid residues. To eliminate possible protein hydrolysis, perform a control test with the same test medium without urea. Do not autoclave medium because excessive heat may alter ingredients.2. Do not heat or reheat the medium because urea decomposes very easily. 3. Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on this medium. Urea Agar Base detects rapid urease activity of only the urease-positive Proteus spp.

Quality controlPositive: Proteus vulgarisNegative: Escherihia coli

ResultsPositive: Change in color of slant from light orange to magenta (A)Negative: No color change (B)


MOTILITY TEST

PurposeThis procedure determines the motility of the bacteria through semisolid media. Shigella and Klebsiella are the only non-motile Enterobacteriaceae. Yersinia enterocolitica is non-motile at 37°C but is motile at 22°C.

PrincipleThe medium contains a small amount of agar, which allows motile bacteria to move out from the line of inoculation. Organisms grow only along the line of inoculation; 1% triphenytetrazolium chloride may be added to medium to aid visualization of the reaction. Bacteria incorporate this colorless dye and reduce it to a red pigment. Thus reddening of the medium can be used as an indication for the extent of bacterial growth.

IngredientsTryptoseSodium chlorideAgar

Purpose of ingredientsTryptose serve as a source of essential growth nutrients required for bacterial metabolism. Sodium chloride maintains the osmotic equilibrium of the medium. Small amount of agar helps to create a semisolid medium.

ConsiderationsStore below 30°C and the prepared medium at 2 - 8°C. Use before expiry date on the label.

Quality controlPositive: Escherichia coliNegative: Klebsiella pneumoniae

ResultsA. Hanging drop

Positive: In true motility, the organism change position with respect to each other, often darting across the field.

Negative: In Brownian movement, the organisms may appear quite active but they remain in the same relative position to other organisms or debris in the field.

B. Semisolid agar deepPositive: Motile organisms will spread out into the medium from the site of inoculationNegative: Non-motile organisms remain at the site of inoculation

First tube: PositiveSecond tube: Negative

https://us.vwr.com/stibo/hi_res/8041824.pdf

ONPG (o-Nitrophenyl-B-D-Galactopyranoside) TEST

PurposeThis test is used to demonstrate the presence or absence of the enzyme B-galactosidase using the substrate Ortho-nitrophenyl-D-galactopyranoside in order to differentiate Lactose-fermenting from non-lactose-fermenting organisms.

PrincipleThis test is used to determine the ability of an organism to produce B-galactosidase, an enzyme that hydrolyzes the substrate ONPG to form a visible (yellow) product, orthonitrophenol.

Ingredientso-Nitrophenyl-B-D-GalactopyranosideMonosodium phosphate

Purpose of ingredientsO-Nitrophenyl-B-D-Galactopyranoside capable of penetrating the bacterial cell without the presence of permease and it increases the presence of sodium ions.

ConsiderationsReagent should not be used if there are signs of deterioration (evaporation or discoloration) or if the expiration date has passed.

Quality controlPositive: Escherichia coliNegative: Salmonella typhimurium

ResultsPositive: Yellow (presence of B-galactosidase) (A)Negative: Colorless (absence of enzyme) (B)


PHENYLALANINE DEAMINASE (PAD) TEST

Purpose

This test determines whether the microbe produces the enzyme phenylalanine deaminase, which is needed for it to use the amino acid phenylalanine as a carbon and energy source for growth.

PrincipleThis test is used to determine the ability of an organism to oxidatively deaminate phenylalanine to phenyl-pyruvic acid which is detected by adding a few drops of 10% ferric chloride; a green-colored complex is formed between these two compounds.

IngredientsFerric chlorideHClDeionized water

Purpose of ingredientsFerric chloride is used to detect the deamination of phenylalanine in the PAD or sometimes referred to as TDA test

ConsiderationsThe PAD agar should be brought to room temperature but upon receipt, store at 2-8 degrees Celsius. Reagents should not be used after expiration date.

Quality controlPositive: Proteus vulgarisNegative: Escherichia coli

ResultsPositive: Green color develops on slant after ferric chloride is added (1st tube)Negative: Slant remains original color after the addition of ferric chloride (2nd tube)


Hektoen Enteric Agar

PURPOSE: Hektoen enteric medium selects for stool pathogens by inhibiting the normal flora of the lower GI tract.

PRINCIPLE:A high concentration of bile salts inhibits gram positive bacteria and gram negative coliforms. Lactose, sucrose, and salicin are carbohydrate sources. Bromthymol blue indicator has the following pH ranges:

> 7.6 – blue6.0-7.6 – green< 6.0 – yellow

Sodium thiosulfate is the sulfur source of H2S detection. H2S combines with ferric ammonium citrate to form ferric sulfide (FeS), which is represented by black-centered colonies. If one, two, or three of the carbohydrates are fermented, the colonies are orange in color. Non-fermenters produce green colonies.

PROCEDURE:1. Streak agar for isolation.2. Incubate at 35-37 °C for 18-24 hours and observe for growth and color.

INTERPRETATION:Pathogens = green colonies or green colonies with black centersNormal flora (except Yersinia enterocolitica, which produce yellowcolonies due to its fermentation of sucrose) = yellow colonies

Differential capabilities ofHE agar:Lactose-fermenting,gram negative bacilli (e.g E. coli, arrow A),Non-lactose fermenters (e.gShigella spp., arrow B) andH2S producers (e.g Salmonella spp., arrow C).


Lysine Iron Agar (LIA)

PurposeLIA can be used to determine the ability of the organism to deaminate lysine, decarboxylate lysine and produce H2S gas. It is useful in the identification of Salmonella, Proteus, Providencia, and Morganella. Members of the Proteus group (Proteus, Providencia and Morganella) are the only members of the Enterobacteriaceae that are deaminase positive.

PrincipleLIA contains a small amount of protein, glucose, lysine, sulfur, H2S indicator, agar, and the pH indicator bromcresol purple. As glucose fermentation occurs, the deep of the tube turns yellow. Lysine decarboxylation produces alkaline cadaverine and leads to reversion of the deep from yellow to purple. Lysine deamination occurs in the presence of oxygen (on the slant) and results in production of a red color. H2S production is noted by a black precipitate in the deep as H2S gas reacts with ferric ammonium citrate.

MediaLIA slants

Procedure1. Inoculate LIA by using straight wire to stab the deep and to streak the slant.2. Incubate at 35C for 18 to 24 hours, if necessary, incubate for 48 hours.

Interpretation· Lysine decarboxylase positive: purple/purple· Lysine decarboxylase negative: purple/yellow· Deaminase positive: red/yellow· H2S positive: blackening

Indicator: Bromcresol purple

· Acid: Yellow· Alkaline state: Purple

Results to be observed:1. Deamination on the slant portion only:

· Positive: red slant· Negative: purple slant

2. Lysine decarboxylation on the butt portion only:· Positive: purple butt· Negative: yellow slant

A. Uninoculated: · Cadaverine color

B. K/K· alkaline slant (purple): negative deamination· alkaline butt (purple): positive lysine decarboxylation

C. K/A· alkaline slant (purple): negative deamination· acid butt (yellow): negative lysine decarboxylation

D. R/Y or R/A· red slant: positive deamination· acid butt (yellow): negative lysine decarboxylation

Lysine Iron AgarA. Alkaline slant/alkaline butt (K/K).B. Alkaline slant/alkaline butt, H2S positive (K/K H2S+).C. Alkaline slant/acid butt (K/A).D. Red slant/acid butt (R/A).E. Un-inoculated tube

Documents

Enterobacteriaceae culture media