Principles of biochemical tests commonly used in the...

In the name of God

By: Dr. S. S. Khoramrooz, Ph.D.

Department of Microbiology, Faculty of Medicine,

Yasuj University of Medical Sciences, Yasuj, Iran

Principles of biochemical tests

commonly used in the identification

of gram-negative bacteria

Yasouj University of Medical Science Department

Of

Microbiology

1 Dr. Khoramrooz

Introduction

Historically, the identification of bacteria based on:

Colony morphology,

Gram staining,

Biochemical testing.

Based on the phenotype of microorganisms

This method required a lot of time and lots of space in incubators.

Over time, biochemical tests were miniaturized and multitest systems were developed

Resulting in time and space savings.

More recently, automated systems have been developed

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Clinical microbiology entered the next generation of test systems for the identification of bacteria:

Molecular biology assays.

Based on nucleic acid sequences, are highly sensitive, specific, and rapid, thereby providing accurate results in a few hours or less.

More accurate than examining the phenotype.

In this lecture discuss the principles of biochemical tests commonly used in the identification of gram-negative bacteria.

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Characters of Enterobacteriaceae

All Enterobacteriaceae

Gram-negative rods

Ferment glucose with acid production

Reduce nitrates into nitrites

Oxidase negative

Facultative anaerobic

Motile except Shigella and Klebsiella

Non-capsulated except Klebsiella

Non-fastidious

Grow on bile containing media (MacConkey agar)

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Classification of Enterobacteriaceae

Enterobacteriaceae

Lactose fermenters

E. coli, Citrobacter, Klebsiella, Enterobacter

Non-lactose fermenter

Salmonell, Shigella Proteus, Yersinia

There are several selective and differential media used to

isolate distinguishes between LF & LNF

The most important media are:

MacConkey agar

Eosin Methylene Blue (EMB) agar

Salmonella Shigella (SS) agar

In addition to Kiligler Iron agar (KIA) 5 Dr. Khoramrooz

Tests To Know Case Study Tests

Indole

Methyl Red/Voges Proskauer

Citrate

H2S production in SIM

Urea hydrolysis

Motility

Lactose fermentation

Glucose fermentation & gas production

Decarboxylation of amino acis

Fermentation of sugars

Reaction on selective media 6 Dr. Khoramrooz

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Selection of Primary Isolation Media

Used to recover the significant species of bacteria from specimens that may harbor a mixture of microorganisms.

Microbiologists must know the composition of each formula and the purpose and relative concentration of each chemical or compound that is included.

Not sufficient to know that bile salts are included in selective media

In SS agar contains about 5 times the concentration of bile salts compared with MacConkey agar and is more inhibitory to E.coli and more selective for the recovery of Salmonella species from stool cultures.

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For the recovery of the Enterobacteriaceae from clinical, three general types of media are available:

1) nonselective media for primary isolation (e.g., blood agar);

2) Selective or differential agars (e.g., MacConkey and Hektoen enteric agars);

3) enrichment broths.

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Selective Isolation Media

MacConkey and EMB agars are only moderately inhibitory and are designed primarily to prevent growth of gram positive bacteria from mixed cultures.

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Growth of Enterobacteriaceae on MacConkey agar

Uninoculated plate Lactose non feremters Salmonella, Shigella,

Proteus

Lactose feremters E. coli, Citrobacter

Klebsiella, Enterobacter

Colorless colonies Pink colonies

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MacConkey Agars

Intended Use

MacConkey agars are slightly selective and differential plating media mainly used for the detection and isolation of gram-negative organisms from clinical, dairy, food, water, pharmaceutical, cosmetic, and other industrial sources.

MacConkey Agar is used for isolating and differentiating lactose-fermenting from lactose-nonfermenting gram-negative enteric bacilli.

MacConkey Agar Base is used with added carbohydrate in differentiating coliforms based on fermentation reactions.

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MacConkey Agar without Crystal Violet is used for isolating and differentiating enteric microorganisms while permitting growth of staphylococci and enterococci.

The medium can be used also to separate Mycobacterium fortuitum and M. chelonae from other rapidly growing mycobacteria.

MacConkey Agar without Crystal Violet or Salt and MacConkey Agar without Salt are used for isolating and differentiating gram-negative bacilli while suppressing the swarming of most Proteus species.


Principles of the Procedure

Lactose is a fermentable carbohydrate.

When lactose is fermented, a local pH drop around the colony cause a color change in the pH indicator (neutral red) and bile precipitation.


Bile salts, bile salts no. 3, oxgall and crystal violet are selective agents that inhibit growth of gram-positive organisms.

Magnesium sulfate is a source of divalent cations. Agar is the solidifying agent.


Expected Results

Lactose-fermenting organisms grow as pink to brick-red colonies with or without a zone of precipitated bile.

Lactose-nonfermenting organisms grow as colorless or clear colonies.

Swarming by Proteus spp. is reduced on MacConkey agars without salt.


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Limitations of the Procedure

1. Although MacConkey media are selective primarily for gram-negative enteric bacilli, biochemical and, if indicated, serological testing using pure cultures are recommended for complete identification.

2. Incubation of MacConkey Agar plates under increased CO2 has been reported to reduce the growth and recovery of a number of strains of gram-negative bacilli.


Intended Use

Eosin Methylene Blue Agar, Levine is a slightly selective and differential plating medium for the isolation of gram-negative enteric bacteria.


The eosin Y and methylene blue dyes in Levine EMB Agar render the medium slightly selective in that they inhibit gram- positive bacteria to a limited degree.


These dyes also play a role in differentiating between lactose fermenters and lactose nonfermenters due to the presence or absence of dye uptake in the bacterial colonies.

Coliforms, as lactose-fermenting organisms, are visualized as blue-black colonies, whereas colonies of Salmonella and Shigella, as lactose nonfermenters, appear colorless, transparent or amber.

Some gram-positive bacteria, such as fecal streptococci, staphylococci and yeasts, will grow on this medium and usually form pinpoint colonies.


Expected Results

Typical colonial morphology on Eosin Methylene

Blue Agar, Levine is as follows:


Highly Selective Isolation Media Used Primarily for Gastrointestinal Specimens

Media are made highly selective by the addition of a variety of inhibitors to their formulas, generally in higher concentrations than in MacConkey and EMB agars.

These media are used primarily to inhibit the growth of E. coli and other "coli forms," but they allow Salmonella and Shigella species to grow out from stool specimens

The most commonly used are Salmonella-Shigella (SS) agar, xylose lysine deoxycholate (XLD) agar, and Hektoen enteric (HE) agar.

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Intended Use

XL (Xylose Lysine) Agar Base is used for the

isolation and differentiation of enteric pathogens

and, when supplemented with appropriate

additives, as a base for selective enteric media.

XLD Agar is the complete Xylose Lysine

Desoxycholate Agar, a moderately selective

medium recommended for isolation and

differentiation of enteric pathogens, especially

Shigella species.



Xylose is incorporated into the medium because it is fermented by practically all enterics except for the shigellae.

This property enables the differentiation of Shigella species.

Lysine is included to enable the Salmonella group to be differentiated from the nonpathogens.

Without lysine, salmonellae rapidly would ferment the xylose and be indistinguishable from nonpathogenic species.


After the salmonellae exhaust the supply of xylose, the lysine is attacked via the enzyme lysine decarboxylase, with reversion to an alkaline pH, which mimics the Shigella reaction.

To prevent similar reversion by lysine-positive coliforms, lactose and sucrose (saccharose) are added to produce acid in excess.

Degradation of xylose, lactose and sucrose generates acid products, which in the presence of the pH indicator phenol red, causes a color change in the medium from red to yellow.


To add to the differentiating ability of the

formulation, an H2S indicator system, consisting

of sodium thiosulfate and ferric ammonium citrate,

is included for the visualization of the hydrogen

sulfide produced, resulting in the formation of

colonies with black centers.

The nonpathogenic H2S producers do not

decarboxylate lysine; therefore, the acid reaction

produced by them prevents the blackening of the

colonies.


XLD Agar is both a selective and differential

medium.

It utilizes sodium desoxycholate as the selective

agent and, therefore, it is inhibitory to gram-

positive microorganisms.


Expected Results

Degradation of xylose, lactose and sucrose generates acid products, causing a color change in the medium from red to yellow.

Hydrogen sulfide production under alkaline conditions causes colonies to develop black centers.

This reaction is inhibited by the acid conditions that accompany carbohydrate fermentation.

Lysine decarboxylation in the absence of lactose and sucrose fermentation causes reversion to an alkaline condition and the color of the medium changes back to red.

Typical colonial morphology and reactions on XLD Agar are as follows:



1. Red, false-positive colonies may occur with some Proteus and Pseudomonas species.

2. Incubation in excess of 48 hours may lead to false-positive results.

3. S. Paratyphi A, S. Choleraesuis, S. pullorum and S. gallinarum may form red colonies without black centers, thus resembling Shigella species.

4. Some Proteus strains will give black-centered colonies on XLD Agar.


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Intended Use

Hektoen Enteric (HE) Agar is a moderately selective

medium used in qualitative procedures for the

isolation and cultivation of gram-negative enteric

microorganisms, especially Shigella, from a variety

of clinical and nonclinical specimens.



The selective nature of Hektoen Enteric Agar is

due to the incorporation of bile salts in the

formulation.

These substances inhibit gram-positive organisms

but also can be toxic for some gram-negative

strains.


This medium contains three carbohydrates, lactose, sucrose (saccharose) and salicin, for optimal differentiation of enteric pathogens

The lactose concentration is higher than in many other media used for enterics in order to aid in the visualization of enteric pathogens and minimize the problem of delayed lactose fermentation.

Ferric ammonium citrate and sodium thiosulfate in the medium enable the detection of hydrogen sulfide production.

The indicator system, consisting of acid fuchsin and bromthymol blue, has a lower toxicity than that of many other enteric media, resulting in improved recovery of enteric pathogens.


Procedure

A nonselective medium should also be streaked to

increase the chance of recovery when the

population of gram-negative organisms is low and

to provide an indication of other organisms present

in the specimen.

Incubate plates, protected from light, at 35 ± 2°C

for 18-24 hours.



DO NOT AUTOCLAVE.


mixed with normal fecal flora. Salmonella

Hektoen Enteric (HE) Agar:

Composition • Peptone 1.2%

• Yeast extract 0.3%

• Bile salts 0.9%

• Lactose 1.2%

• Sucrose 1.2%

• Salicin 0.2%

• Sodium chloride 0.5%

• Ferric ammonium citrate

• Acid fuchsin

• Thymol blue

• Agar 1.4%

• pH = 7.6

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HE Agar: Growth of Enteric

Pathogens and Commensals

• High bile salt concentration inhibits growth of gram-positive and gram-negative intestinal commensals, and thereby selects for pathogenic Salmonella (bile-resistant growth) present in fecal specimens.

• Salmonella species as non-lactose and non-sucrose fermenters that produce H2S form colorless colonies with black centers.

• Shigella species (non-lactose and non-sucrose fermenters, no H2S production) form colorless colonies.

• Lactose and sucrose fermenters (E. coli, K. pneumoniae) form orange to yellow colonies due to acid production.

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Intended Use

Bismuth Sulfite Agar is a highly selective medium

used for isolating Salmonella spp., particularly

Salmonella Typhi, from food and clinical

specimens.



Dextrose is an energy source.

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.


For isolation of Salmonella spp. from clinical

specimens, inoculate fecal specimens and rectal

swabs onto a small area of one quadrant of the

Bismuth Sulfite Agar plate and streak for isolation.

This will permit the development of discrete

colonies.

Incubate plates at 35°C.

Examine at 24 hours and again at 48 hours for

colonies resembling Salmonella spp.


Expected results

The typical discrete S. Typhi surface colony is black and surrounded by a black or brownish-black zone which may be several times the size of the colony.

By reflected light, 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.


Heat with frequent agitation and boil for 1 minute

to completely dissolve the powder.

DO NOT AUTOCLAVE.

Use the medium the same day it is prepared.


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Intended Use

Brilliant Green Agar is a highly selective medium for

the isolation of Salmonella other than S. Typhi from

feces and other materials.


Brilliant green dye inhibits gram-positive bacteria and a

majority of gram-negative bacilli.

Phenol red serves as a pH indicator and yields a yellow

color as a result of acid production in the fermentation of

the lactose and/or sucrose in the medium.


Procedure

A less selective medium and a nonselective medium should also be streaked to increase the chance of recovery when the population of gram-negative organisms is low and to provide an indication of other organisms present in the specimen.

Incubate plates, protected from light, at 35 ± 2°C for 18-24 hours.

If negative after 24 hours, reincubate an additional 24 hours.



Salmonella on BPLS Agar. The colonies

are red because the bacterium does not

ferment lactose or sucrose.

Escherichia coli on BPLS Agar.

The colonies are yellow due to the

low pH which is caused by the

production of acid during

fermentation of lactose and/or

sucrose.

Differential Identification Characteristics

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Carbohydrate utilization

Lactose degradation; Differentiate those (lactose fermenter LF]) and those that are nonlactose fermenters (NLFs).

Examples of sugars used to differentiate bacteria include lactose, maltose, rhamnose, sucrose, raffinose, and arabinose

Bacteria can utilize carbohydrates: oxidation (aerobically), fermentatively (anaerobically),or both.

Bacteriat hat can grow either aerobically or anaerobically are called facultative anaerobes.

Some bacteria are asacchrolytic.

They do not use any carbohydrate; instead they use other organic molecules for energy and carbon sources.

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Oxidation/Fermentation (O/F) Test

Principle :

To determine the ability of bacteria to breakdown glucose oxidative or fermentative

O/F medium ( Hugh and Leifson Medium) is formulated to detect weak acids produced from saccharolytic M.O.

O/F medium contains

Sugar (glucose 1%)

Low percentage of Agar and Peptone

pH indicator (Bromothymol blue)

• Alkaline Blue

• Neutral Green

• Acidic Yellow

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Intended Use

OF (Oxidation Fermentation) media are used for the determination of oxidative and fermentative metabolism of carbohydrates by gram-negative rods on the basis of acid reaction in either the open or closed system.

Summary and Explanation

OF Medium was developed by Hugh and Leifson who described the taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by gram-negative bacteria.


They showed that when an organism is inoculated into two tubes of OF Basal Medium containing a carbohydrate and the medium in one of the tubes is covered with melted petrolatum prior to incubation, the patterns of metabolism are of differential significance.

Oxidative organisms only produce an acid reaction in the open tube with little or no growth and no acid formation in the covered tube.

Fermentative organisms will produce an acid reaction in both types of tubes.

Changes in the covered agar are considered to be due to true fermentation, while changes in the open tubes are due to oxidative utilization of the carbohydrate present.

If the carbohydrate is not utilized by either method, there is no acid production in either tube.



Dextrose is the most important carbohydrate for

use in OF Basal Medium; however, certain

organisms may metabolize other carbohydrates

even if they are unable to utilize dextrose.

Prepared tubed media containing arabinose,

dextrose, dulcitol, fructose, galactose, lactose,

maltose, mannose, raffinose, rhamnose, salicin,

sorbitol, sucrose and xylose are provided.


Procedure

Inoculate a pair of OF tubes of each carbohydrate used with each organism being tested.

The tubes should be stabbed to approximately 1/4 inch from the bottom using an inoculating needle and a light inoculum.

Overlay one tube of each pair with sterile mineral oil.

Incubate tubes at 35 ± 2°C in an aerobic atmosphere for 48 hours.

Do not discard as negative until after 4 days of incubation.


Expected Results

Record results as acid (A) or alkaline/no change (–).

Also record whether or not the organism is motile

as evidenced by the appearance of growth away

from the line of inoculation.

Typical reaction patterns are as follows.



1. The acid reaction produced by oxidative organisms is apparent at the surface and gradually spreads throughout the medium.

If the oxidation is weak or slow, however, an initial alkaline reaction at the surface of the open tube may persist for several days and eventually convert to an acid reaction.

2. If an organism is unable to grow on OF Basal Medium, Cowan recommends adding either 2% serum or 0.1% yeast extract to each carbohydrate tube.


Triple Sugar lron Agar Kligler Iron Agar

Lactose Fermentation

Glucose fermentation

Gas Production (H2 & CO2 )

H2S Production

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glucose

lactose

Ferrous sulfate

pH indicator: phenol red

TSI and KIA

Proteins

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Intended Use

Triple Sugar Iron Agar (TSI Agar) is used for the differentiation of gram-negative enteric bacilli based on carbohydrate fermentation and the production of hydrogen sulfide.


TSI Agar contains three sugars (dextrose, lactose and sucrose),

Phenol red for detecting carbohydrate fermentation


Ferrous ammonium sulfate for detection of hydrogen sulfide production (indicated by blackening in the butt of the tube).

Carbohydrate fermentation is indicated by the production of gas and a change in the color of the pH indicator from red to yellow.

To facilitate the detection of organisms that only ferment dextrose, the dextrose concentration is one-tenth the concentration of lactose or sucrose.

The small amount of acid produced in the slant of the tube during dextrose fermentation oxidizes rapidly, causing the medium to remain red or revert to an alkaline pH.

In contrast, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower oxygen tension.


After depletion of the limited dextrose, organisms

able to do so will begin to utilize the lactose or

sucrose.

To enhance the alkaline condition of the slant, free

exchange of air must be permitted by closing the

tube cap loosely.

If the tube is tightly closed, an acid reaction

(caused solely by dextrose fermentation) will also

involve the slant.


Procedure

To inoculate, carefully touch only the center of an

isolated colony on an enteric plated medium with a

cool, sterile needle, stab into the medium in the butt

of the tube, and then streak back and forth along the

surface of the slant.

Several colonies from each primary plate should be

studied separately, since mixed infections may

occur.


Incubate with caps loosened at 35°C and examine

after 18-24 hours for carbohydrate fermentation,

gas production and hydrogen sulfide production.

Any combination of these reactions may be

observed.

Do not incubate longer than 24 hours because

the acid reaction in the slant of lactose and sucrose

fermenters may revert to an alkaline reaction.


Expected Results

Carbohydrate fermentation is indicated by a yellow coloration of the medium.

If the medium in the butt of the tube becomes yellow (acidic), but the medium in the slant becomes red (alkaline), the organism being tested only ferments dextrose (glucose).

A yellow (acidic) color in the slant and butt indicates that the organism being tested ferments dextrose, lactose and/or sucrose.

A red (alkaline) color in the slant and butt indicates that the organism being tested is a nonfermenter.


Hydrogen sulfide production results in a black

precipitate in the butt of the tube.

Gas production is indicated by splitting and

cracking of the medium.


1. Hydrogen sulfide production may be evident on Kligler Iron Agar but negative on Triple Sugar Iron Agar.

Studies by Bulmash and Fulton showed that the utilization of sucrose could suppress the enzymatic mechanisms responsible for H2S production.

Padron and Dockstader8 found that not all H2S-positive Salmonella are positive on TSI.

2. Sucrose is added to TSI to eliminate some sucrose-fermenting lactose-nonfermenters such as Proteus and Citrobacter spp.

3. Further biochemical tests and serological typing must be performed for definite identification and confirmation of organisms.


4. Do not use an inoculating loop to inoculate a tube of Triple Sugar Iron Agar.

While stabbing the butt, mechanical splitting of the medium occurs, causing a false positive result for gas production.

5. A pure culture is essential when inoculating Triple Sugar Iron Agar.

If inoculated with a mixed culture, irregular observations may occur.

6. Tubes should be incubated with caps loosened.

This allows a free exchange of air, which is necessary to enhance the alkaline condition on the slant.


Red/Red Alkaline /Alkaline K/K Lactose -/Glucose –

Yellow/Yellow Acid/Acid A/A Lactose +/Glucose +

Red/Yellow Alkaline/Acid K/A Lactose -/Glucose +

Gas - H2S -


Gas + H2S -


Gas - H2S +

Red/Black Alkaline/Acid K/A Lactose -/Glucose +

Gas + H2S +

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Example

Result

Reaction on KIA

H2S Slant

color

Butt

color

Non fermenter

e.g.

Pseudomonas

Alk/Alk/-

(No action on sugars)

Negative Red Red

LNF

e.g. Shigella A/Alk/-

(Glucose fermented

without H2S)

Negative

Red Yellow

LNF

e.g. Salmonella &

Proteus

A/Alk/+

(Glucose fermented

with H2S)

Positive

black in

butt Red Yellow

LF

e.g. E. coli,

Klebsiella,

Enterobacter

A/A/-

(three sugars are

fermented)

Negative Yellow Yellow

Result

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Ortho- Nitrophenyl - β –D- galactopyranosideTest

Organisms that are delayed lactose-fermenters appear as nonfermenting colonies.

The o-nitrophenyl β–D- galactopyranoside (ONPG) and the p-nitrophenyl-B-D-galactopyranoside (PNPG) tests determine whether the organism is a delayed lactose fermenter (one that lacks the enzyme β-galactoside permease but possesses β-galactosidase) or a true nonlactose fermenter (NLF).

The test can be performed by making a heavy suspension of bacteria in sterile saline and adding commercially prepared ONPG disks or tablets.

The suspension is incubated at 37' C, and positive results can generally be seen within 6 hours.

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IMViC Test

Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests:

The following four tests comprise a series of important determinations that are collectively called the IMViC series of reactions

The IMViC series of reactions allows for the differentiation of the various members of Enterobacteriaceae.

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IMViC: Indole test Principle

Certain microorganisms can metabolize tryptophan by tryptophanase

The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia

The presence of indole is detected by addition of Kovac's reagent.

Tryptophane amino acids

Tryptophanase Indole + Pyurvic acid + NH3

Kovac’s Reagent

Red color in upper organic layer` 102 Dr. Khoramrooz

IMViC: Indole test

Result:

A bright pink color in the top

layer indicates the presence of

indole

The absence of color means that

indole was not produced i.e.

indole is negative

Special Features:

Used in the differentiation of genera

and species. e.g. E. coli (+) from

Klebsiella (-).

Positive test e.g. E. coli

Negative test e.g. Klebsiella

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Intended Use

SIM Medium is used to differentiate enteric bacilli

on the basis of sulfide production,indole

formation and motility.



Hydrogen sulfide production, indole formation

and motility are distinguishing characteristics

which aid in the identification of the

Enterobacteriaceae, especially Salmonella and

Shigella.

SIM Medium, therefore ,is useful in the process of

identification of enteric pathogens.



Sodium thiosulfate and ferrous ammonium sulfate are indicators of hydrogen sulfide production.

The ferrous ammonium sulfate reacts with H2S gas to produce ferrous sulfide,a black precipitate.

The casein peptone is rich in tryptophan,which is attacked by certain microorganisms resulting in the production of indole.

The indole is detected by the addition of chemical reagents following the incubation period.


Motility detection is possible due to the semisolid

nature of the medium.

Growth radiating out from the central stab line

indicates that the test organism is motile.


Procedure

Loosen caps, boil and cool before use.

Using growth from a pure culture, stab an

inoculating needle two-thirds of the distance to

the bottom in the center of the tube.

Incubate tubes with loosened caps for18-24 hours

at 35±2°C in anaerobic atmosphere.


IMViC test Methyl Red-Voges Proskauer (MR-VP) Tests

Glucose

Acidic pathway

Mixed acids pH less than 4.4

Methyl Red

indicator

Red color

Principle

MR positive E. coli

Or Neutral pathway

Acety methyl carbinol (ACETOIN)

solution A

solution B

Pink color VP positive Klebsiella

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Butylene Glycol Pathway of Glucose Fermentation

In the butylene glycol pathway

pyruvic acid to acetoin and butylene glycol.

Acetoin and butylene glycol are detected by oxidation to diacteyl at an alkaline pH.

Addition of -naphthol which forms a red-colored complex with diacetyl.

Important biochemical property used for the identification of Klebsiella, Enterobacter, and Serratia.

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IMViC test: MRVP test

Inoculate the tested organism into MRVP broth

Incubate the tubes at 37°C for 24 hours

• For methyl red: Add 6-8 drops of methyl

red reagent.

• For Voges-Proskauer: Add 12 drops of

solution A (-naphthol), mix, 4 drops of

Solution B (40% KOH), mix

Method

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http://whitewolf.newcastle.edu.au/techinfo/med_bacto.html

IMViC test: MR/VP test

Results

Methyl Red test Voges-Proskauer test

Red: Positive MR (E. coli)

Yellow or orange: Negative MR (Klebsiella)

Pink: Positive VP (Klebsiella)

No pink: Negative VP (E. coli) 114 Dr. Khoramrooz

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Intended Use

MR-VP Medium and MR-VP Broth (Methyl Red-

Voges Proskauer Medium/Broth, also known as

Buffered Peptone- Glucose Broth) are used for the

differentiation of bacteria by means of the methyl

red and Voges-Proskauer reactions.



Methyl red-positive organisms produce high levels of acid during fermentation of dextrose, overcome the phosphate buffer system and produce a red color upon the addition of the methyl red pH indicator.

In the Voges-Proskauer test, the red color produced by the addition of potassium hydroxide to cultures of certain microbial species is due to the ability of the organisms to produce a neutral end product, acetoin (acetylmethylcarbinol), from the fermentation of dextrose.

The acetoin is oxidized in the presence of oxygen and alkali to produce a red color.

This is a positive Voges-Proskauer reaction.


Procedure

Using a light inoculum, inoculate tubes of MR-VP media with 18- to 24-hour pure cultures.

Incubate tubes aerobically at 35 ― 2�‹C for a minimum of 48 hours but preferably for 5 days.

Prepare the methyl red indicator: 0.1 g of methyl red in 300 mL of 95% ethyl alcohol.

Add sufficient purified water to make 500 mL.

After the appropriate incubation period, aseptically remove aliquots (1 mL for the VP test) of the medium and conduct the following tests:


1. Methyl Red Test:

Add 5 drops of methyl red indicator to an aliquot of the broth.

Interpret the color result immediately.

2. Voges-Proskauer Test:

Empty the contents (15 drops) from the reagent A dropper

5 drops from the reagent B dropper into 1 mL of broth culture.

Shake well after the addition of each reagent to aerate the sample.


Expected Results

1. Methyl Red Test

a. Positive – red color at surface of the medium.

b. Negative – yellow color at surface of the medium.

2. Voges-Proskauer Test

A positive reaction is indicated by the development of a distinct red color which occurs within 5 minutes.

Certain species within Enterobacteriaceae genera may react differently or give variable results.

Consult appropriate texts for reactions of specific species.



1. Results of the MR and VP tests need to be used in conjunction with other biochemical tests to differentiate genus and species within the Enterobacteriaceae.

2. A precipitate may form in the potassium hydroxide reagent solution.

This precipitate has not been shown to reduce the effectiveness of the reagent.


3. Most members of the family Enterobacteriaceae give either a positive MR test or a positive VP test.

However, certain organisms such as Hafnia alvei and Proteus mirabilis may give a positive result for both tests.

4. Incubation time for the Methyl Red test cannot be shortened by increasing the dextrose concentration in the medium or by heavily inoculating the broth.

5. Incubate MR-negative tests for more than 48 hours and test again.


6. Read the VP test at 48 hours. Increased incubation may produce acid conditions in the broth that will interfere with reading the results.

7. VP reagents must be added in the order and the amounts specified or a weak-positive or false-negative reaction may occur.

A weak-positive reaction may be masked by a copper-like color

which may form due to the reaction of KOH and α-naphthol.

8. Read the VP test within 1 hour of adding the reagents. The KOH and α-naphthol may react to form a copper-like color, causing a potential false-positive interpretation.

9. Due to the possible presence of acetoin, diacetyl or related substances in certain raw materials, the use of media low in these substances (such as MR-VP media) is recommended for this test.


Citrate Utilization Test Principle:

Citrate Na2CO3

Alkaline,↑pH

Blue colour Bromothymol blue

Simmone’s Citrate media

Positive test: Klebsiella, Enterobacter, Citrobacter

CO2 + Na + H2O Pyruvate

Positive test

Negative test: E. coli

Contains Citrate as a sole of C source

125 Dr. Khoramrooz

Citrate Utilization Test

Incubate at 37°C for 24 hours.

Method

Streak a Simmon's Citrate agar slant with

the organism

126 Dr. Khoramrooz




Citrate Utilization Test

Examine for growth (+)

Growth on the medium is accompanied by a rise in pH to change the medium from its initial green color to deep blue

Result

Positive Klebsiella, Enterobacter Negative

E. coli 127 Dr. Khoramrooz

Intended Use

Simmons Citrate Agar is used for the differentiation of

gram negative bacteria on the basis of citrate utilization.


Organisms able to utilize ammonium dihydrogen

phosphate and sodium citrate as the sole sources of

nitrogen and carbon, respectively, will grow on this

medium and produce an alkaline reaction as evidenced

by a change in the color of the bromthymol blue

indicator from green (neutral) to blue (alkaline).


Procedure

Inoculate slants with growth from a pure culture using a light inoculum.

Incubate all tubes for 4 days at 35 ± 2°C in an aerobic atmosphere.

Expected Results

A positive reaction is indicated by growth with an intense blue color in the slant.

A negative reaction is evidenced by no growth to trace growth with no change in color (medium remains dark green).


Amino Acid Decarboxylation

Enterobacteriaceae contain decarboxylases with substrate specificity for amino acids, and are detected using Moeller decarboxylase broth overlayed with mineral oil for anaerobiosis.

Moeller broth contains:

glucose for fermentation,

peptone and beef extract,

amino acid, pyridoxal,

pH indicator bromcresol purple.

134 Dr. Khoramrooz


If an Enterobacteriaceae contains amino acid decarboxylase, amines produced by decarboxylase action cause an alkaline pH, and bromcresol purple turns purple.

Lysine, ornithine, and arginine are utilized.

A base broth without amino acid is included in which glucose fermentation acidifies the broth, turning the bromcresol purple yellow.

135 Dr. Khoramrooz

Amino Acid Decarboxylation1

Lysine → Cadaverine

Ornithine → Putrescine

Arginine → Citrulline → Ornithine → Putrescine

1Conversion of arginine to citrulline is a dihydrolase reaction

136 Dr. Khoramrooz


Decarboxylation patterns are essential for the genus identification of Klebsiella, Enterobacter, Escherichia, and Salmonella.

Decarboxylation patterns are also essential for the species identification of Enterobacter aerogenes, Enterobacter cloacae, Proteus mirabilis, and Shigella sonnei.

137 Dr. Khoramrooz


Lys Orn Arg

Klebsiella + – –

Enterobacter +/– + +/–

Escherichia + +/– –/+

Salmonella + + +

138 Dr. Khoramrooz


Lys Orn Arg

E. aerogenes + + –

E. cloacae – + +

P. mirabilis – + –

P. vulgaris – – –

Shigella D – + –

Shigella A-C – – –

139 Dr. Khoramrooz

Phenylalanine Deaminase Reaction

Enterobacteriaceae utilize amino acids in a variety of ways including deamination.

Phenylalanine is an amino acid that forms the keto acid phenylpyruvic acid when deaminated.

Phenylpyruvic acid is detected by addition of ferric chloride that forms an intensely dark olive-green colored complex when binding to phenylpyruvic acid.

The deamination of phenylalanine is an important biochemical property of Proteus, Morganella, and Providencia.

140 Dr. Khoramrooz

Intended Use

Decarboxylase media are used in the biochemical differentiation of gram-negative enteric bacilli based on the production:

Arginine dihydrolase

Lysine decarboxylase

Ornithine decarboxylase

Decarboxylase Medium Base, with added arginine, lysine or ornithine is used for the same purpose.

Lysine Decarboxylase Broth is used for differentiating microorganisms based on lysine decarboxylation.



Moeller introduced the decarboxylase media for detecting the production of lysine and ornithine decarboxylase and arginine dihydrolase.

These media are a useful adjunct to other biochemical tests for the speciation and identification of the Enterobacteriaceae and other gram-negative bacilli.

The production of OD is particularly useful for differentiating Klebsiella and Enterobacter species.

Klebsiella species are non-motile and, except for K. ornithinolytica, do not produce ornithine decarboxylase, while most Enterobacter species are motile and, except for E. agglomerans, usually produce this enzyme.



Pyridoxal is an enzyme co-factor for the amino acid decarboxylase.

Dextrose is a fermentable carbohydrate.

Bromcresol purple and cresol red are pH indicators.

The amino acids lysine, ornithine or arginine are added to the basal medium at a concentration of 10.0 g/L to detect the production of the enzyme specific for these substrates.


When the medium is inoculated with a bacterium

that is able to ferment dextrose, acids are produced

that lower the pH of the medium and change the

color of the indicator from purple to yellow.

The acidic condition also stimulates decarboxylase

activity.

If the organism produces the appropriate enzyme,

the amino acid in the medium is degraded, yielding

a corresponding amine.


Decarboxylation of lysine yields cadaverine.

while decarboxylation of ornithine yields putrescine.

Arginine is first hydrolyzed to form ornithine, which is then decarboxylated to form putrescine.

The production of these amines elevates the pH of the medium, changing the color of the indicator from yellow to purple or violet.

If the organism does not produce the appropriate enzyme, the medium remains acidic (yellow).


Each isolate to be tested must also be inoculated into a tube of the basal medium that does not contain the amino acid.

If this tube becomes alkaline, the test is invalid.

To obtain the appropriate reactions, the inoculated tubes must be protected from air with a layer of sterile mineral oil.

Exposure to air may cause alkalinization at the surface of the medium, which could cause a decarboxylase-negative organism to appear positive.


Expected Results

Compare the color of tubes of media containing the specific amino acids with the color of control tubes of basal media (without amino acid) that have been inoculated with the same isolate.

If inoculated control tubes show an alkaline reaction, the test is invalid; i.e.,

Improperly performed or the test organisms

Degrade the peptone sufficiently to produce an alkaline reaction in the absence of a specific amino acid.

The medium becomes purple to violet if the reaction is positive (alkaline).

A yellow color indicates a negative test; i.e., the organism does not produce the appropriate enzyme.


The lysine iron agar (LIA) test is a tubed agar slant.

It contains the amino acid lysine, glucose, ferric ammonium

citrate, and sodium thiosulfate.

The pH indicator is bromcresol purple.

LIA is used primarily to determine whether the bacteria

decarboxylate or deaminate lysine.

H2S production is also detected in this medium.

LIA is inoculated in the same manner as a TSI agar slant.

LIA is most useful in conjunction with TSI in screening stool

specimens for the presence of enteric pathogens, differentiating

Salmonella spp. (lysine-positive) from Citrobacter spp. (lysine-

negative).


Decarboxylation occurs anaerobically only; the

presence of a dark purple butt is positive for lysine

decarboxylation.

The production of H2S can mask the purple color

in the butt of the tube.

Because H2S production in LIA occurs only in an

alkaline environment, a black precipitate indicating

H2S is also a positive result for decarboxylation.


LIA is also useful in differentiating Proteus,

Morganella, and Providencia spp. from most other

members of Enterobacteriaceae

This group of enterics deaminates (attacks the NH2

group instead of the carboxyl group) amino acids.

In the LIA slant, deamination of lysine turns the

original light purple color slant to a plum or reddish

purple color; the butt turns yellow because of glucose

fermentation.


IPViC Reactions for Initial Grouping of the Enterobacteriaceae

Indole

Phenylalanine deaminase

Voges-Proskauer

Citrate

155 Dr. Khoramrooz

Urease Test

Urea agar contains urea and phenol red

Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3

Ammonia combines with water to produce ammonium hydroxide, a strong base which ↑ pH of the medium.

↑ in the pH causes phenol red r to turn a deep pink. This is indicative of a positive reaction for urease

Urea Urease

CO2 + NH3 H2O

NH4 OH ↑ in pH

Phenol Red

Pink Positive test

Streak a urea agar tube with the organism

incubate at 37°C for 24 h

Method

Principle

156 Dr. Khoramrooz


Urease Test

If color of medium turns from yellow to pink indicates positive test.

Proteus give positive reaction after 4 h while Kelebsiella and Enterobacter gave positive results after 24 h

Result

Positive test Negative test

157 Dr. Khoramrooz

Intended Use

Urea Agar and Urease Test Broth are used for the

differentiation of organisms, especially the

Enterobacteriaceae, on the basis of urease

production.



The urea medium of Rustigian and Stuart is particularly suited for the differentiation of Proteus species from other gram negative enteric bacilli capable of utilizing urea.

Unable to do so in Urease Test Broth because of limited nutrients and the high buffering capacity of the medium.

To provide a medium with greater utility, Urea Agar was devised by Christensen with peptone and dextrose included and reduced buffer content to promote more rapid growth of many of the Enterobacteriaceae and permit a reduction in incubation time.


The complete Urea Agar contains 15.0 g/L of agar

in addition to the ingredients in the base

medium.

When organisms utilize urea, ammonia is formed

during incubation which makes the reaction of

these media alkaline, producing a red-pink color.

Consequently, urease production may be detected

by the change in the phenol red indicator.


Urease medium

Dehydrated Product

BBL™ Urea Agar Base

1. Dissolve 29 g of the powder in 100 mL of purified water. Mix thoroughly. Sterilize by filtration.

2. Suspend 15 g of agar in 900 mL of purified water.

3. Autoclave at 121°C for 15 minutes.

4. Cool to 50°C and add 100 mL of the sterile Urea Aga Base.

5. Mix thoroughly and dispense aseptically in sterile tubes.

6. Cool tubed medium in a slanted position so that deep butts are formed.

7. Do not remelt the complete medium.

8. Test samples of the finished product for performance using stable, typical control cultures.


Procedure

Using a heavy inoculum (2 loopfuls) of growth from

an 18- to 24-hour pure culture (TSI Agar or other

suitable medium), inoculate the broth or agar

(streaking back and forth over the entire slant

surface).


Do not stab the butt since it serves as a color control.

For broth, shake tubes gently to suspend the bacteria.

Incubate tubes with loosened caps at 35 ― 2�‹C in an

incubator or water bath.

Observe reactions after 2, 4, 6, 18, 24 and 48 hours.

For agar, continue to check every day for a total of 6

days; even longer incubation periods may be necessary.


The production of urease is indicated by an intense

pink-red (red-violet) color on the slant or

throughout the broth.

The color may penetrate into the agar (butt); the

extent of the color indicates the rate of urea

hydrolysis.


A negative reaction is no color change.

The agar medium remains pale yellow to buff; the

broth remains yellowish orange.


Urea Agar Base

1. 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 (especially in slant agar by Pseudomonas aeruginosa, for example) or other proteins which 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.

2. Do not heat or reheat the medium because urea decomposes very easily.


3. Urea Agar detects rapid urease activity of only the urease

positive Proteus species.

For results to be valid for the detection of Proteus, the results

must be read within the first 2-6 hours after incubation.

Urease-positive Enterobacter, Citrobacter or Klebsiella, in

contrast, hydrolyze urea much more slowly, showing

only slight penetration of the alkaline reaction into the butt

of the medium in 6 hours and requiring 3-5 days to

change the reaction of the entire butt.


Urea Broth

1. To rule out false positives due to protein

hydrolysis (as opposed to urea hydrolysis) that

may occur in the medium after prolonged

incubation, perform a control test with the same

test medium without urea.

2. Do not heat or reheat the medium because urea

decomposes very easily.


3. The high buffering system in this medium masks urease activity in organisms that are delayed positive.

This medium is therefore recommended for the detection of urease activity in all Proteus spp., Providencia rettgeri and urease-positive Providencia stuartii.

M. morganii slowly hydrolyzes urea and may require approximately a 36 hour incubation for a strong urease-positive reaction to occur.

If in doubt as to a result, compare with an uninoculated tube or incubate for an additional 24 hours.

4. Variations in the size of the inoculum can affect the time required to reach positive (alkaline, pH 8.1) results.


Motility From left to right: + – +

172 Dr. Khoramrooz

SIM Sulfide, Indole, Motility

Dr. Khoramrooz 173

Initial Grouping of the Enterobacteriaceae

(VP=Voges Proskauer, PDA=Phenylalanine

Deaminase)

GENERA VP PDA

Klebsiella POSITIVE NEGATIVE

Enterobacter POSITIVE NEGATIVE

Serratia POSITIVE NEGATIVE

Hafnia POSITIVE NEGATIVE

Pantoea POSITIVE NEGATIVE

175 Dr. Khoramrooz

Initial Grouping of the

Enterobacteriaceae

GENERA VP PDA

Proteus1 NEGATIVE POSITIVE

Morganella NEGATIVE POSITIVE

Providencia NEGATIVE POSITIVE

1Proteus mirabilis: 50% of strains VP positive176 Dr. Khoramrooz


Enterobacteriaceae

GENERA VP PDA

Escherichia NEGATIVE NEGATIVE

Shigella NEGATIVE NEGATIVE

Edwardsiella NEGATIVE NEGATIVE

Salmonella NEGATIVE NEGATIVE

Citrobacter NEGATIVE NEGATIVE

Yersinia NEGATIVE NEGATIVE

177 Dr. Khoramrooz


Enterobacteriaceae1

GENERA INDOLE CITRATE

Escherichia POSITIVE NEGATIVE

Shigella

Yersinia

POSITIVE2

POSITIVE3

NEGATIVE

NEGATIVE

Edwardsiella POSTIVE NEGATIVE

1VP negative, PDA negative 2Shigella groups A, B, and C variably positive

for indole production (25-50%), group D

Shigella negative. 3Yersinia enterocolitica 50% positive

178 Dr. Khoramrooz


Enterobacteriaceae1

GENERA INDOLE CITRATE

Salmonella NEGATIVE POSITIVE2

Citrobacter NEGATIVE POSITIVE

1VP negative, PDA negative 2Salmonella serotype Paratyphi A and Typhi

negative

179 Dr. Khoramrooz

Key Characteristics of the

Enterobacteriaceae

TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

E

coli

A/A + + + + + +/

/

+ Shi

A-

C

Ak/

A /

+

Shi

D

Ak/

A + + Ed Ak/

A + + + + + + Sal Ak/

A + + + + + + +/

Cit A/A

Ak/

A

+ + + + +/

+ /

+

+/

Yer A/A

+ +/

+/

RT

(1) +

(1) RT=room temperature 180 Dr. Khoramrooz


Enterobacteriaceae


Kle

pne

A/A + + + + + +

Kle

oxy

A/A + + + + + + +

En

aer

A/A + + + + + + +

En

cloa

A/A + + + + +/ + + +

Serr

(1)

A/A + + + + + + +

Haf Ak/

A + + + + + + Pan A/A

Alk/

A

+ /+ +/ /+ +/ /+ /+

(1) Produces DNase, lipase, and gelatinase 181 Dr. Khoramrooz


Enterobacteriaceae


Prot

mir

a

Ak/

A + + +/ +/ + + +s +

Prot

vulg

A/A +/ + + /+ + + +s

Mor Ak/

A + + + + + + Pro

v

Ak/

A + + + + +

s = swarming motility

182 Dr. Khoramrooz

Biochemical Characteristics of

Escherichia coli and Shiglla

E. coli E. coli O157:H7 Shigella

TSI A/Ag A/Ag Alk/A

Lactose + + –

ONPG + + –/+1

Sorbitol + – +/–

Indole + + +/–

Methyl red + + +

VP – – –

Citrate – – –

Lysine + + –

Motility + + –

1Shigella sonnei (group D) ONPG +

183 Dr. Khoramrooz

Biochemical Characteristics of

Salmonella

Most Serotypes Typhi Paratyphi A

TSI Alk/A Alk/A Alk/A

H2S (TSI) + + (weak) –

Citrate + – –

Lysine + + –

Ornithine + – +

Dulcitol + – +

Rhamnose + – +

Indole – – –

Methyl red + + +

VP – – –

184 Dr. Khoramrooz

185 Dr. Khoramrooz

The End

186 Dr. Khoramrooz

t

Dr. Khoramrooz 187

Dr. Khoramrooz 188

Intended Use

Malonate Broth is used for differentiating Enterobacter from Escherichia based on malonate utilization.


Malonate Broth contains ammonium sulfate, which is the sole source of nitrogen in the medium;

Sodium malonate is the sole source of carbon.

Increased alkalinity resulting from malonate utilization causes the indicator, bromthymol blue, to change color from green to blue.


Procedure

1. Inoculate tubes with a loopful of test organism.

2. Incubate at 35 ― 2�‹C for 18-48 hours.

3. Examine tubes for a change in the color of the medium from green to blue.

Expected Results

Malonate utilization is indicated by a change in the color of the medium from green to blue:

Positive: Blue

Negative: Green


Intended Use

SS Agar and Salmonella Shigella Agar are

moderately selective and differential media for the

isolation of pathogenic enteric bacilli, especially

those belonging to the genus Salmonella.

This formulation is not recommended for the

primary isolation of Shigella.



SS Agar and Salmonella Shigella Agar are designated as moderately selective media based upon the degree of inhibition of gram-positive microorganisms that they inhibit due to their content of bile salts, brilliant green and citrates.

Differentiation of enteric organisms is achieved by the incorporation of lactose in the medium.

Organisms that ferment lactose produce acid which, in the presence of the neutral red indicator, results in the formation of red colonies.

Lactose nonfermenters form colorless colonies.


The latter group contains the majority of the

intestinal pathogens, including Salmonella and

Shigella.

The sodium thiosulfate and ferric citrate enable

the detection of hydrogen sulfide production as

evidenced by colonies with black centers.


Procedure

A nonselective medium should also be streaked to increase the chance of recovery when the population of gram-negative organisms is low and to provide an indication of other organisms present in the specimen.

Incubate plates, protected from light, at 35 ± 2°C for 18-24 hours.

If negative after 24 hours, reincubate an additional 24 hours.


Expected Results

Typical colonial morphology on Salmonella

Shigella Agar is as follows:


Limitation of the Procedure

Due to the relatively high level of selectivity, some

Shigella strains may not grow on SS Agar and

Salmonella Shigella Agar and, therefore, these

media are not recommended for the primary

isolation of Shigella.

Media recommended for the isolation of Shigella are Hektoen Enteric and XLD agars.


Intended Use

Selenite Broth (Selenite-F Broth) is used as an enrichment medium for the isolation of Salmonella from feces, urine, water, foods and other materials of sanitary importance.


The peptone provides essential nitrogenous and carbon compounds.

The lactose in the medium serves to maintain a uniform pH.


When selenite is reduced by the growth of bacteria, alkali is produced, and such increase in pH would lessen the toxicity of the selenite and result in overgrowth of extraneous bacteria.

The acid produced by lactose fermentation serves to maintain a neutral or slightly decreased pH.

The function of the phosphate is two-fold; it serves to maintain a stable pH and lessens the toxicity of the selenite, thus increasing the capacity of the medium.

Sodium selenite inhibits many species of grampositive and gram-negative bacteria including enterococci and coliforms.


Procedure

For feces and other solid materials, suspend 1-2 g of the specimen in the broth (approximately 10-15% by volume) and emulsify with an inoculating needle, if necessary.

Incubate tubes with loosened caps at 35 ± 2°C for up to 24 hours.

Subcultures should be made after 12-18 hours of incubation, if possible.

Coliforms will tend to overgrow the pathogens if incubated longer than 24 hours.


Expected Results

After incubation, there should be an increase in the

number of pathogens that the medium is designed

to select for and enrich.

Subculture onto appropriate selective and

differential media (e.g., MacConkey Agar,

Hektoen Enteric Agar, XLD Agar, XLT4 Agar,

CHROMagar™ Salmonella) to isolate

pathogens for identification.


Limitation of the Procedure

Enrichment broths should not be used as the sole

isolation medium.

They are to be used in conjunction with selective

and nonselective plating media to increase the

probability of isolating pathogens, especially when

they may be present in small numbers.


The End

208 Dr. Khoramrooz

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