Lab 10 Microbial metabolism part I

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Lab 10 Microbial metabolism part I
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Biochemical characteristics of bacteria

• Many of the detectable differences between
  bacteria are biochemical
• We take advantage of these principals by
  performing biochemical test on bacteria
• These test give us the ability to distinguish
  between different bacteria
• Over these 5 days you will perform various
  biochemical test that are used to distinguish
  between different bacteria.

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Flow chart for bacteria identification (example)
http//www.kcom.edu/faculty/chamberlain/Website/la
b/idlab/4gram-.jpg
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Objective 1Carbohydrate fermentation

• Carbohydrate are big molecules
• Require enzymes to be broken down in smaller
  molecules
• If the bacteria does not have the enzyme it can
  not break down/use the carbohydrate
• Fermentation is noted by acid production

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Objective 1 Carbohydrate fermentationA. Lactose
broth

• Escherichia coli
• Proteus vulgaris
• Positive result yellow color change with or
  without gas production
• Negative result no color change
• Gas production ? bubble in the durham tube

http//www2.austin.cc.tx.us/microbugz/images/26suc
.jpg
http//biosci.usc.edu/courses/2002-fall/documents/
bisc300-lab_Carbohydrate_Fermentation_Sucrose.jpg
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Objective 1 Carbohydrate fermentationB. OF test
(oxidation-fermentation)

• Contains a Glucose
• pH indicator bromothymol blue ? turn yellow
  under acidic condition
• Determine whether a bacterium has the enzymes
  necessary for the aerobic breakdown of glucose
  (oxidation) and/or for the fermentation of
  glucose (anaerobic).

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OF glucose test Use the needle
oil
1 no oxidation, no fermentation 3
Fermentation 2 oxidation but no fermentation
http//www.jlindquist.net/generalmicro/DMimages/ne
wglucof2.jpg
Escherichia coli Pseudomonas aeruginosa
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Objective 2 Starch hydrolysis

• Starch is a big sugar molecule
• Is broken into smaller molecules by the enzyme
  amylase
• Area of starch hydrolysis becomes apparent on the
  media following the addition of iodine
• Positive result clear halo around the colonies
• Negative result areas of dark blue

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Starch hydrolysis
Bacillus subtilis
Escherichia coli
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Add Gram Iodine
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Objective 3 MR-VP

• Methyl red
• Identify bacteria that produce stable acids as
  end-products when fermenting glucose
• pH indicator added after incubation
• After 24 hours,Add Methyl Red 5 6 drops
• Positive Red
• Negative yellow

E. Coli E. Aerogenes
http//www.marietta.edu/spilatrs/biol202/labresul
ts/methyl20red.jpg
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Objective 3 MR-VP

• Voges-Proskauer
• Identify bacteria that produce butanediol as
  end-products of fermentation
• Add 15 drops of reagent 1
• Add 5 drops of reagent 2
• Color change can take up to 30 60 minutes
• Positive red color
• Negative yellow/translucent color

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Objective 4 Catalase test
TSA

• Aerobic respiration results in the formation of
  hydrogen ions (H) which are converted into
  H2O2.n (hydrogen peroxide).
• The Catalase enzyme breaks down the peroxide into
  non toxic water

Streptococcus faecalis
Staphylococcus aureus
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Catalase test
24 hours later add hydrogen peroxide on the
colony
Bubble positive result
http//biology.ucok.edu/Microbiology/images/catala
se.jpg
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Objective 5 Oxidase

• Cytochrome oxidase is an enzyme involved in the
  reduction of oxygen at the end of the electron
  transport chain
• React with oxygen and the reagent to produce a
  purple (dark almost black) color change
• Positive result purple color change
• Negative result no color change

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Oxidase test
TSA

• Aerobic respiration results in the formation of
  hydrogen ions (H) which are converted into
  H2O2.n (toxic).
• The Catalase enzyme breaks down the peroxide into
  non toxic water

P. aeruginosa
E. coli
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Oxidase
2-3 drops oxidase reagent
Purple color change Positive result
http//biology.ucok.edu/Microbiology/images/oxidas
e2.jpg
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Objective 6 Citrate utilization

• Test the ability of the bacteria to use citrate
  as their sole carbon source
• Test whether they possess the citrase enzyme or
  not
• It test for the presence of alkaline (basic)
  products as a result of citrate utilization
• Bromothymol blue ? detect pH change

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Citrate utilization

• Inoculate 1 slant
• E. coli
• Enterobacter aerogenes
• Incubate
• Positive media change from green to blue
  (citrate was used)
• Negative no color change

http//www.marietta.edu/spilatrs/biol202/labresul
ts/citrate.jpg
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Objective 7 Nitrate reduction test

• It test the ability of some bacteria to reduce
  Nitrate to nitrite.
• Media contains peptone and beef extracts to
  support growth as well as potassium nitrate
• ? if the organism can reduce nitrate it will
  react with the reagents
• 1) sulfanillic acid
• 2) naphthalamine acetate
• 3) Addition of zinc dust might be necessary if
  the nitrate has been reduced all the way to
  elemental nitrogen

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Nitrate reduction test ? positive

In slant Staphylococcus aureus Streptococcus
faecalis Incubate at 37

http//www.cdc.gov/std/Gonorrhea/lab/images/NO3pos
.GIF
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Objective 8 TSI

• Triple Sugar iron
• Used for identification of Gram-negative enteric
  rods
• measures a bacterium's ability to utilize three
  sugars
• Glucose 0.1
• Sucrose 1
• Lactose 1
• pH indicator in the media detect acid production
  ? if acid turn yellow
• Also measure the reduction of sodium thiosulfate
  2 tests in one tube

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TSI

• Inoculation is a two step procedure
• First, a loopful of bacteria is spread across the
  surface of the agar using a needle
• Second, the needle is stabbed into the bottom
  (butt) of the tube.
• Acid production only in the butt ? indicative of
  glucose fermentation only.
• the small amount of glucose in the medium is
  fermented within the first hours of incubation.
• After, the bacteria get their energy by oxidizing
  peptone ? media on the slant turn red (alkaline
  conditions)
• Peptone can not be used in the butt because there
  is no O2 ? butt remains yellow

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TSI

• Acid production in the slant and butt ? sucrose
  and/or lactose fermentation
• high concentrations of these sugars ? extensive
  acid production.
• Reduction of sodium thiosulfate to hydrogen
  sulfide.
• Hydrogen sulfide production will turn parts of
  the agar black
• Production of gas
• cracks in the agar
• air bubbles trapped at the bottom of the tube

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TSI
K alkaline A acid
Escherichia coli Pseudomonas aeruginosa Proteus
vulgaris
http//users.stlcc.edu/kkiser/TSIreact.jpg