Antibiotic Sensitivity (1)

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Colonial morphology and biochemical
characterisation
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• Colonial morphology is the cultural
  characteristics of an organism on an agar plate.
• It includes
• the shape
• EDGE/MARGIN OF COLONY

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• ELEVATION OF COLONY
• CHROMOGENESIS (pigmentation) white, red, purple
  and so on
• OPACITY OF COLONY Is the colony transparent
  (clear), opaque (not transparent or clear),
  translucent (almost clear) or iridescent
  (changing colors in reflected light)
• SURFACE OF COLONY smooth, rough, glistening,
  rough, dull, rugose
• CONSISTENCY or TEXTURE butyrous, viscid,
  brittle/friable, mucoid

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Coagulase

• How to Perform Test Inoculate rabbit plasma with
  one single colony. Break up colony and stir
  until blended in plasma. Incubate at 37 degrees
  C for 24 hours.
• Property it tests for This tests for the
  bacterias ability to clot blood plasma using the
  enzyme coagulase. If the organism has coagulase
  it will clump rabbit plasma.
• Media and Reagents This media contains rabbit
  plasma dissolved in buffer.

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Coagulase Results

• Reading Results
• If the organism is has coagulase it will clump
  the plasma.
• If the organism does not have coagulase it will
  not clump the plasma.

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Catalase test
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• OXIDASE TEST
• Detects the presence of an enzyme oxidase
  produced by certain bacteria
• reagent tetramethyl-p-phenylene diamine
  dihydrochloride.
• Positive test is indicated by the development of
  a purple colour.

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• TRIPLE SUGAR IRON AGAR (TSI)

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Kliglers iron agar (KIA) tubes with
several reaction patternsA Acid/Acid, Gas B
Acid/Acid, No gas C Alkaline/Acid D Alkaline
/Acid, H2S E Alkaline/Alkaline.
The composition of Kliglers Iron Agar (KIA) is
identical to Triple Sugar Iron Agar (TSI) except
that 10gm sucrose is added in TSI. i.e no sucrose
in KIA
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• CITRATE UTILIZATION

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• UREASE TEST

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BIOCHEMICAL TEST REACTIONS

• They provide additional information for the
  identification of the bacterium.
• The tests include
• Catalase
• Coagulase
• Oxidase test
• Triple sugar iron agar (TSI)
• Citrate utilization
• Urease test

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• Biochemical characterisation of bacteria
• Catalase Test
• It is based on the principle that catalase, an
  enzyme, catalyzes the breakdown of hydrogen
  peroxide (H2O2) to water and oxygen
• Coagulase test
• It is based on the principle that S. aureus
  produce coagulase, an enzyme that reacts plasma
  causing it to clot.

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IMViC Test

• Case Study Tests
• Indole
• Methyl Red/Voges Proskauer
• Citrate
• H2S production in SIM -Sulfphide-Indole-Motility
  medium.
• Urea hydrolysis
• Motility
• Lactose fermentation
• Sucrose fermentation
• Glucose fermentation gas production
• Staphylococcus identification tests
• BAP-blood agar plate
• MSA-monitor salt agar
• Mstaph broth-monitor salt broth for
  staphylococcus
• Coagulase

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Indole Test

• How to Perform Test Inoculate Tryptone broth
  with inoculating loop.
• Property it tests for This test is performed to
  help differentiate species of the family
  Enterobacteriaceae. It tests for the bacteria
  species ability to produce indole. Bacteria use
  an enzyme, tryptophanase to break down the amino
  acid, tryptophan, which makes by-products, of
  which, indole is one.
• Media and Reagents Used Tryptone broth contains
  tryptophan. Kovacs reagentcontains
  hydrochloric acid, dimethylaminobenzaldehyde, and
  amyl alcoholyellow in color.
• Reading Results Kovacs reagent reacts with
  indole and creates a red color at the top part of
  the test tube.

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Indole
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Methyl Red/Voges Proskauer (MR/VP)

• How to Perform Tests Inoculate 2 glucose broths
  with inoculating loop. After 48 hours of
  incubation, add a few drops of MR to one tube,
  and VP reagents to the other tube.
• Properties they test for Both tests are used to
  help differentiate species of the family
  Enterobacteriaceae.
• MRtests for acid end products from glucose
  fermentation.
• VPtests for acetoin production from glucose
  fermentation.
• Media and Reagents Used
• Glucose Broth
• Methyl Red indicator for acid
• Voges Proskauer reagentsA 5 Alpha-Naphthol,
  ethanol, B Potassium Hydroxide, Deionized
  Water.

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MR/VP continued

• Reading Results
• MR a result is red (indicating pH below 6) and
  a result is yellow (indicating no acid
  production)
• VPA result is red after VP reagents are added
  (indicating the presence of acetoin) and a
  result is no color change.

VP left and right
Methyl Red left and right
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Citrate

• How to Perform Test Inoculate slant with
  inoculating loop.
• Property it tests for This test is used to help
  differentiate species of the family
  Enterobacteriaceae. It is selective for bacteria
  that has the ability to consume citrate as its
  sole source of carbon and ammonium as sole
  nitrogen source.
• Media and Reagents Used Simmons Citrate Agar
  contains sodium citrate (carbon source), ammonium
  ion (nitrogen source), pH indicatorbromthymol
  blue.
• Reading Results A result is blue (meaning the
  bacteria metabolised citrate and produced an acid
  end product) and a result remains green

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Citrate
Left positive and right negative.
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H2S Production in SIM -Sulfide-Indole-Motility
medium.

• How to Perform Test Stab SIM media with
  inoculating needle.
• Property it tests for This test is used to help
  differentiate species of the family
  Enterobacteriaceae. This test is used to
  determine the ability to reduce sulfur into H2S.
• Media and Reagents Used SIM media contains the
  sulfur containing amino acid, cysteine, sodium
  thiosulfate, peptonized iron or ferrous
  sulfate.
• Reading Results H2S will react with the iron or
  ferrous sulfate and produce a black precipitate.
  A positive result has a black precipitate present
  and a negative result has no black precipitate.

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Urea Hydrolysis

• How to Perform Test Inoculate Urea broth with
  inoculating loop.
• Property it tests for This test is done to
  determine a bacterias ability to hydrolyze urea
  to make ammonia using the enzyme urease.
• Media and Reagents Used Urea broth contains a
  yeast extract, monopotassium phosphate, disodium
  phosphate, urea, and phenol red indicator.
• Reading Results Urea broth is a yellow-orange
  color. The enzyme urease will be used to
  hydrolyze urea to make ammonia. If ammonia is
  made, the broth turns a bright pink color, and is
  positive. If test is negative, broth has no
  color change and no ammonia is made.

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Motility Test

• How to Perform Test Stab motility media with
  inoculating needle.
• Property it tests for This test is done to help
  differentiate species of bacteria that are
  motile.
• Media and Reagents Used Motility media contains
  tryptose, sodium chloride, agar, and a color
  indicator.
• Reading Results If bacteria is motile, there
  will be growth going out away from the stab line,
  and test is positive. If bacteria is not motile,
  there will only be growth along the stab line. A
  colored indicator can be used to make the results
  easier to see.

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Motility
From left to right
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Procedure for Triple Sugar Iron Agar (TSI) Test

• With a sterilized straight inoculation needle
  touch the top of a well-isolated colony
• Inoculate TSI Agar by first stabbing through the
  center of the medium to the bottom of the tube
  and then streaking on the surface of the agar
  slant. 
• Leave the cap on loosely and incubate the tube at
  35C in ambient air for 18 to 24 hours.

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Interpretation of TSI test

• If lactose (or sucrose) is fermented, a large
  amount of acid is produced, which turns the
  phenol red indicator yellow both in butt and in
  the slant. Some organisms generate gases, which
  produces bubbles/cracks on the medium.
• If lactose is not fermented but the small amount
  of glucose is, the oxygen-deficient butt will be
  yellow(remember that butt comparatively have more
  glucose compared to slant i.e. more media more
  glucose), but on the slant the acid (less acid as
  media in slant is very less) will be oxidized to
  carbon dioxide and water by the organism and
  the slant will be red (alkaline or neutral pH).
• If neither lactose/sucrose nor glucose is
  fermented, both the butt and the slant will be
  red. The slant can become a deeper red-purple
  (more alkaline) as a result of production of
  ammonia from the oxidative deamination of amino
  acids (remember peptone is a major constituent of
  TSI Agar).
• if H2S is produced, the black colour of ferrous
  sulphide is seen.

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Name of the organism Slant Butt Gas H2S
Escherichia, Klebsiella, Enterobacter Acid (A) Acid (A) Pos () Neg (-)
Shigella, Serratia   Alkaline(K) Acid (A)   Neg (-)   Neg (-)
Salmonella, Proteus Alkaline (K) Acid (A) Pos () Pos ()
Pseudomonas Alkaline (K) Alkaline (K) Neg (-) Neg (-)
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TSI
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IMViC Test results of Some Genera of
Enterobacteriaceae

• Escherichia coli
• 1. Indole Positive
• 2. Methyl-Red Positive
• 3. Voges-Proskauer test Negative
• 4. Citrate test Negative
• Enterobacter aerogenes
• 1. Indole Negative
• 2. Methyl-Red Negative
• 3. Voges-Proskauer test Positive
• 4. Citrate test Positive
• Proteus vulgaris
• 1. Indole Positive
• 2. Methyl-Red Positive
• 3. Voges-Proskauer test Negative
• 4. Citrate test Negative
• Citrobacter freundii
• 1. Indole Negative

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TSI Interpretation

• Alkaline slant/no change in butt (K/NC) i.e.
  Red/Red glucose, lactose and sucrose
  non-fermenter
• Alkaline slant/Alkaline butt (K/K) i.e. Red/Red
  glucose, lactose and sucrose non-fermenter
• Alkaline slant/acidic butt (K/A) Red/Yellow
  glucose fermentation only, gas ( or -), H2s (
  or -)
• Acidic slant/acidic butt (A/A)Yellow/Yellow
  glucose, lactose and/or sucrose fermenter gas(
  or -),H2s( or -)
• Composition of Triple Sugar Iron Agar
  (TSI)Lactose, Sucrose and Glucose in the
  concentration of 10101 (i.e. 10 part Lactose
  (1), 10 part Sucrose (1) and 1 part Glucose
  (0.1)). TSI is similar to Kliglers iron agar
  (KIA), except that Kliglers iron agar contains
  only two carbohydrates glucose (0.1) and
  lactose (1).

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Catalase test
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Tube Catalase Test-Procedure and Results 

• Add 4 to 5 drops of 3 H2O2 to in a test tube
• Using a wooden applicator stick, collect a small
  amount of organism from a well-isolated 18- to
  24-hour colony and place into the test
  tube (Note Be careful not to pick up any agar
  (especially if using Blood Agar).- Explanation in
  precaution below)
• Place the tube against a dark background and
  observe for immediate bubble formation (O2
  water bubbles) at the end of the wooden
  applicator stick.

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Procedure of Catalase test (Slide Test)

• Transfer a small amount of bacterial colony to a
  surface of clean, dry glass slide using a loop or
  sterile wooden stick
• Place a drop of 3 H2O2 on to the slide and mix.
• A positive result is the rapid evolution of
  oxygen (within 5-10 sec.) as evidenced by
  bubbling.
• A negative result is no bubbles or only a few
  scattered bubbles.
• Dispose of your slide in the biohazard glass
  disposal container.

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Procedure of Catalase test (Slide Test)
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Test Organisms Incubation Incubation Incubation Results
Test Organisms Time Temperature Atmosphere Results
Salmonella enterica ATCC  14028  18-24hr 35C Aerobic Growth Motility positive, H 2S positive (black colour along stab line) Indole negative 
Escherichia coli ATCC  25922  18-24hr 35C Aerobic Growth Motility positive, H 2 S negative Indole positive (Kovacs Reagent turns pink after adding three drops) 
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Lactose Fermentation

• How to Perform Test Inoculate lactose broth with
  inoculating loop.
• Property it tests for This tests for the
  bacterias ability to ferment lactose.
• Media and Reagents Used Lactose broth contains
  beef extract, gelatin peptone, and lactose. A
  phenol red indicator is added to indicate acid
  production from fermentation.
• Results
• A positive result is yellow after indicator is
  added (indicating lactose fermentation)
• A negative result will have no color change or
  will be redish.

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Sucrose Fermentation

• How to Perform Test Inoculate sucrose broth with
  inoculating loop.
• Property it tests for This test is done to help
  differentiate species of the family
  Enterobacteriaceae. This tests for the
  bacterias ability to ferment sucrose and
  production of acid end-product
• Media and Reagents Used Sucrose broth contains
  beef extract, gelatin peptone, and sucrose.
  Phenol red indicator is added to indicate an acid
  end-product.
• Results
• A positive result is yellow after indicator is
  added (indicating sucrose fermentation)
• A negative result has no color change or is
  reddish.

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Glucose Fermentation Gas Production

• How to Perform Test Inoculate broth with
  inoculating loop.
• Property it tests for This test is done to help
  differnetiate species of the family
  Enterobacteriaceae. This tests for the
  bacterias ability to ferment glucose and produce
  gas and/or an acid end-product..
• Media and Reagents Used Glucose broth contains
  beef extract, gelatine peptone, and glucose. A
  phenol red indicator is added to indicate an acid
  enproduct. A Durham tube is added to indicate
  gas production.
• Results
• A positive result for acid is yellow after
  indicator is added (indicating glucose
  fermentation)
• A positive result for gas is a bubble in the
  Durham tube.
• A completely negative result has no color change
  or reddish color and no bubble.

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Sugar Fermentation Tests
Tube 1 Negative acid /Negative gas Tube 2A
Must incubate longer (ambiguous result) Tube 2B
Positive acid /Negative gas Tube 3A Positive
acid/ Positive gas
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BAP with Novobiocin Test

• How to Perform Test Inoculate a BAP plate using
  streak plate method and incubate 24-48 hours.
  Place a novovbiocin disk in the first quadrant of
  the streak.
• Property it tests for This test is used to
  determine two things
• It determines if the organism is resistant or
  sensitive to Novobiocin
• It also determines if the organism can lyse red
  blood cells (hemolysis)
• Media and Reagents Media contains nutrient agar
  with 5 sheep's blood added. A Novobiocin
  antibiotic disk is added after inoculation.

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BAP with Novobiocin

• Possible test results
• Hemolysis
• Hemolysis occurs if the media surrounding the
  colonies turns translucent or green in color
• Novobiocin resistance
• Measure the zone of clearing around the disk. If
  the zone of clearing is smaller than the
  manufacturers recommendations for sensitivity
  (see package instructions) then the organism is
  resistant.

Non-hemolytic bacteria
No hemolysis Top streak is R for NB and bottom
is S
Hemolytic bacteria
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• Types of Haemolysis
• Alpha haemolysis
• Beta haemolysis
• Gamma haemolysis

Beta haemolysis
Alpha haemolysis
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Mannitol Salt Agar (MSA)

• How to Perform Test Inoculate an MSA plate using
  streak plate method and incubate 24-48 hours.
• Property it tests for This tests for the
  bacterias ability to tolerate 7 salt
  concentration and ferment mannitol. The media is
  selective because it selects for salt tolerant
  bacteria. The media is also differential because
  it differentiates the salt tolerant organisms on
  their ability to ferment mannitol.
• Media and Reagents MSA media contains nutrient
  agar, mannitol, 7 sodium chloride and phenol red
  indicator.

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MSA Results

• Reading Results
• If the organism is tolerant to salt it will grow.
• If the organism is not tolerant to salt it will
  not grow.
• If the salt tolerant organism can ferment
  mannitol then there will be yellow zones around
  the colonies.
• If the salt tolerant organism cannot ferment
  mannitol then the media will remain pink.

Growth with no mannitol fermentation.
Growth with mannitol fermentation.
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Mannitol Salt Broth

• How to Perform Test Inoculate broth and incubate
  for 24-48 hours.
• Property it tests for This tests for the
  bacterias ability to tolerate 7 salt
  concentration and ferment mannitol. The media is
  selective because it selects for salt tolerant
  Staphylococcus.
• Media and Reagents This media contains nutrients
  appropriate for growing Staphylococcus and 7
  salt.

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MSBstaph Results

• Reading Results
• If the organism is tolerant to salt it will grow.
• If the organism is not tolerant to salt it will
  not grow.

Tolerates Salt.
Does not tolerate salt.
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LF and NLF colonies in MacConkey Agar
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LF and NLF colonies in MacConkey Agar

• MacConkey agar was developed in 20th century by
  Alfred Theodore MacConkey.
• It was the first formulated solid differential
  media. 
• MacConkey agar is a selective and differential
  culture media commonly used for the isolation of
  enteric Gram-negative bacteria.
• It is based on the bile salt-neutral
  red-lactose agar of MacConkey.
• Crystal violet and bile salts are incorporated in
  MacConkey agar to prevent the growth of
  Gram-positive bacteria and fastidious
  Gram-negative bacteria, such as Neisseria and Past
  eurella. 
• Gram-negative enteric bacteria can tolerate bile
  salts because of their bile-resistant outer
  membrane

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Mixed growth of mucoid Lactose fermenting
colonies and NLF colonies in MacConkey agar
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Mixed growth of mucoid Lactose fermenting
colonies and NLF colonies in MacConkey agar

• Citrobacter spp. Late lactose fermenter
  therefore Non Lactose Fermenter (NLF) after 24
  hours Lactose fermenter (LF) after 48 hours
  colonies are light pink after 48 hours.
• Klebsiella spp. Mucoid lactose fermenter (MLF)
• Escherichia coli Lactose fermenter flat, dry,
  pink colonies with a surrounding darker pink area
  of precipitated bile salts.
• Serratia spp. Late lactose fermenter S.
  marcescens may be red pigmented, especially if
  the plate is left at 25C
• Non Lactose Fermenter (NLF) Organisms
• Proteus spp. NLF may swarm depending on the
  amount of agar in the medium characteristic foul
  smell
• Shigella spp. NLF Shigella sonnei produces flat
  colonies with jagged edges.
• Yersinia spp. NLF may be colorless to peach.
• Salmonella spp. NLF
• Other organisms showing colorless colonies on
  MacConkey agar are Edwardsiella spp, Hafniaspp., 
  Morganella spp., Providencia spp.

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Colonial morphology of E.coli, Salmonella and
Shigella in Salmonella-Shigella Agar
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Typical colonial morphology on
Salmonella-Shigella Agar is as follows

• Results
• Lactose fermenter If lactose fermentation
  occurs, the medium will turn red due to the
  acidic pH. e.g. Escherichia coli, Klebsiella
  pneumoniae gives red colonies.
• Non-Lactose fermenter Salmonella, Shigella, and
  other non-lactose fermenters appear as
  transparent or translucent colorless colonies.
  Colonies of Salmonella spp. may appear with or
  without black centers (depending on the species
  isolated).
• Typical colonial morphology on Salmonella-Shigella
  Agar is as follows
• E.coli ...Slight growth, pink or
  red
• Enterobacter/Klebsiella ....Slight growth,
  pink
• Proteus ...Colourless, usually with
  black centre
• Salmonella.colourless, usually with
  black centre
• Shigella ...colourless
• Pseudomonas ..Irregular, slight growth
• Gram-positive bacteriaNo growth

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Mixed Culture of E coli and Salmonella in Xylose
Lysine Deoxycholate (XLD) Agar
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Mixed culture of E coli and Salmonella in XLD agar

• Xylose lysine deoxycholate (XLD) Agar was
  formulated by Taylor for the isolation and
  differentiation of enteric pathogens including
  Salmonella Typhi from other Salmonella species.
• XLD Agar is both selective and differential
  medium for the isolation, cultivation and
  differentiation of gram-negative enteric
  microorganisms
• This media is primarily used for isolation and
  differentiation of Salmonella and Shigella from
  both clinical and non-clinical specimens.
• XLD agar contains sugars like Xylose, Lactose and
  Sucrose provide sources of fermentable
  carbohydrate.
• Yeast Extract provides sources of nitrogen,
  carbon, and vitamins required for organism
  growth.
• The indicator Phenol red imparts red colour to
  the prepared media which changes to yellow after
  sugar fermentation thus differentiating lactose
  fermenters from non-lactose fermenters .
• Most gut bacteria, including Salmonella, can
  ferment xylose to produce acid Shigella
  colonies cannot do this and therefore remain red.

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Xylose Lysine deoxycholate (XLD) Cultures. Left
Yellow Escherichia coli colonies Right
Red-pink Salmonella colonies (Some Proteus
species look identical)
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Colony characteristics in XLD Agar

• Enterobacter aerogenes ATCC 13048 Yellow
• Escherichia coli ATCC 25922 Yellow
• Proteus vulgaris ATCC 13315 grey with black
  centers
• Salmonella Paratyphi A ATCC 9150 Red
• Salmonella Paratyphi B ATCC 8759, Salmonella
  Enteritidis ATCC 13076  Salmonella Typhi ATCC
  6539 Red with Black centers
• Salmonella Typhimurium ATCC 14028  red  colonies
  with black centres (strong H2s producer)
• Shigella dysenteriae ATCC 13313, Shigella
  flexneri ATCC 12022  Shigella sonnei ATCC 25931
  Red
• Staphylococcus aureus ATCC 6538 No growth

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XLD CulturesLeft Red-pink black centered
colonies of Salmonella typhimurium Right
Red-pink Shigella colonies
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Xylose Lysine deoxycholate (XLD) Cultures. Left
Yellow Escherichia coli colonies Right
Red-pink Salmonella colonies (Some Proteus
species look identical)

• After exhausting the xylose supply Salmonella colo
  nies decarboxylate lysine, increasing the pH once
  again to alkaline and mimicking the
  red Shigella colonies.
• However, Salmonellae also metabolise thiosulfate
  to produce hydrogen sulphide, which leads to the
  formation of colonies with black centres and
  allows them to be differentiated from the
  similarly coloured Shigella colonies.
• Sodium Deoxycholate inhibits the growth of Gram
  positive organisms.
• Sodium Chloride maintains the osmotic balance in
  the medium.
• Agar is the solidifying agent.
• XLD Agar has been recommended for the
  identification of Enterobacteriaceae
  microbiological testing of foods, water and dairy
  products

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Yellow colonies of S. aureus in Mannitol Salt
Agar
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Mannitol Salt Agar (MSA)

• Mannitol Salt Agar (MSA) is a selective, 
  differential and indicator medium which is
  used to isolate and identify Staphylococcus
  aureus from the clinical specimen.
• Incorporation of  7.5 sodium chloride in the
  medium helps to select only those bacteria which
  can tolerate high salt concentrations. 
• MSA helps to demonstrate the ability of a
  bacterium to grow in a 7.5 salt environment
  (growth indicates tolerance for high salt
  environment no growth means intolerance).
• Species of staphylococci are able to tolerate
  this salt concentration but other pathogenic
  bacteria may not.
• This concentration inhibits the growth of most
  other gram-positive and gram-negative bacteria
• Thus MSA is a Selective media for Staphylococcus s
  pp

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Yellow colonies of S. aureus in Mannitol Salt
Agar (MSA)
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Eosin Mettaline Blue agar