Media

1
Media Biochemical Tests

• Laboratory Objectives

2
Tests To Know

• Case Study Tests
• Indole
• Methyl Red/Voges Proskauer
• Citrate
• H2S production in SIM
• Urea hydrolysis
• Motility
• Lactose fermentation
• Sucrose fermentation
• Glucose fermentation gas production
• Staphylococcus identification tests
• BAP
• MSA
• Mstaph broth
• Coagulase
• Stapyloside

3
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.

4
Indole
5
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.

6
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
7
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

8
Citrate
Left positive and right negative.
9
H2S Production in SIM

• 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.

10
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.

11
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.

12
Motility
From left to right
13
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.

14
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.

15
Glucose Fermentation Gas Production

• How to Perform Test Inoculate 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 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.

16
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
17
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.

18
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
19
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.

20
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.
21
Mstaph 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.

22
M-STAPH 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.
23
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.

24
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.

25
Staphyloside

• How to Perform Test Put one drop of Staphyloside
  reagent on one circle on the card. Take a small
  amount of culture from your slant and mix it with
  the Staphyloside reagent on the card. Break up
  any clumps and wait 5-10 minutes. Observe if
  clumping occurs.
• Property it tests for This tests for the
  presence of Staphylococcus aureus protein
  (antigen). If the sample clumps, it means the
  Staphylococcus aureus protein bound with the
  Staphyloside antibody. Staphylococcus epidermidis
  will not clump the antibodies
• Media and Reagents This is a card that contains
  Staphyloside antibodies attached to latex beads.

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Result of positive Staphyloslide test