Oxidative biochemical test

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Introduction to biochemical characteristics based
on carbohydrate metabolism
Oxidative fermentation test
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Presented to- Dr Yogesh Bhargav
(department of
applied Microbiology)

Presented by- Priyanshi Mishra
MSc 1st semester
Dr HariSingh
Gour University Sagar (M.P.)
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Biochemical test

• Biochemical test are the test used for
  identification of bacterial species based on
  differences in biochemical activities of
  different bacteria.
• The ability of bacteria to form organic compounds
  by metabolizing certain Carbohydrate and related
  compounds is widely used method for
  identification of microorganism.

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Oxidative fermentation test
Was first developed By hugh and leifson in
1953.They developed oxidative fermentation (OF)
test to differentiate between oxidative bacteria
(that produce acid from carbohydrate under
aerobic condition only) and fermentative bacteria
(that produce acid both under aerobic and
anaerobic conditions).
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Saccharolytic microorganism degrade glucose
either fermentatively or oxidatively. The end
product of fermentation are relatively strong
mixed acid can be detected in conventional
fermentation test medium. However the acid formed
in oxidative degradation of glucose are the
extremely weak and less and the more sensitive
oxidation fermentation of medium of Hugh and
leifsons OF medium is required for detection.
The medium was made by increasing the amount of
glucose about that found in medium used to detect
fermentation and by decreasing the amount of
peptone.
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Oxidative fermentation (OF) test
Objective- To differentiate
microorganism based on the ability to oxidze or
ferments specific carbohydrate.
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Principle of test

• Carbohydrate are organic molecule that contain
  carbon, hydrogen, oxygen (CH2O)n.
• Organism use enzyme differently depending upon
  their enzyme complement.
• The pattern of fermentation is characteristic of
  certain species, genera or groups of organism and
  for this reason this property has been
  extensively used as method of biochemical
  differentiation of microbes.
• Non fermentation bacteria are routinely tested
  for their ability to produce acid from six
  carbohydrates (glucose, xylose, mannitol lactose,
  gluclose and maltose).

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• Weather organism is oxidative or fermentative can
  be determined by using Hugh and Leifsons medium
  commonly called as OF medium which contain
  tryptone and bromothymol blue (an Indicator).
• Two tubes are required for interpretation of the
  (OF) test.
• Broth are inoculated, and one tube is overlaid
  with Mineral oil, producing an anaerobic
  environment. Growth of microorganism in this
  medium is either by utilising the tryptone which
  results in an alkaline reaction (dark blue
  colour) or by utilising glucose, which result in
  the production of acid (turning Bromothymol blue
  to yellow).
• Production of acid in the overlaid tube and open
  tube result in the colour change and is an
  indication of fermentation.
• Acid production in the open tube and colour
  change is the result of oxidation.

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Media used in OF test-

• Hugh and leifsons medium-
• Peptone - 2.0 gm/l
• Sodium chloride 5.0gm/l
• Dipotassium phosphate 0.30gm/l
• Glucose (dextrose) 10.0gm/l
• Bromothymol blue 0.030gm/l
• Agar- 3.0gm/l
• Final pH (at 25c) 7.1 0.2
  

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Microorganisms used-

• Escherichia coli ATCC 25922 fermentatIve,
• Pseudomonas aeruginosa ATCC 27853 oxidative.

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Procedure

• Inoculate two tubes of OF medium with organism by
  stabbing with a straight wire.
• Pour liquid paraffin over the medium to form a
  layer about one cm in depth into one of the tube.
• Incubate the tube at 35 37c for 24 to 48
  hours.
• Examine both open and closed tubes for the colour
  change.

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Result interpretation of OF test-
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Result interpretation of OF test-
Oxidative Yellow
colouration in open tube only. Fermentative -
Yellow colouration on both open
and closed tube.
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Limitation of OF test-

• OF medium is general purpose medium and may not
  support the growth of fastidious organisms.
• Slow growing organisms may not produce result for
  several days.

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The taxonomic significance of Fermentative versus
oxidative metabolism of Carbohydrates by various
gram-negative bacteria
By Rudolf Hugh and einar leifson
Publication January 2 1953
Some bacteria produced from carbohydrate only
under aerobic condition while other produce as
both under aerobic and anaerobic condition.
Studies of bacterial physiology have made it
increasingly evident that the bacterial
metabolism of Carbohydrates may be accomplished
by two apparently fundamentally different
mechanism.
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• One mechanism called fermentation- The glucose
  moleculeFirst is phosphorylated and then split
  into two triose molecule which undergoes further
  changes.
• Other mechanism called oxidation- Glucose is not
  a split into two triose molecule but the aldehyde
  group is oxidised to carboxyl group and forming
  gluconic acid for the oxidation, may take place
  to form various products such as key to gluconic
  acid.

Oxidation of Carbohydrates is aerobic process.
Degree of acidity is produced by oxidative
metabolism is usually lower than that produced by
fermentative metabolism.
Some bacteria apparently both ferments and
oxidize carbohydrate.
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Did you know?

• Two species described in sixth edition of
  bergeys manual as -
• Vibrio percolans,
• Vibrio cuneatus.
• Obtained from American type culture collection
  (ATCC).
• American type culture collection (ATCC)- Is a
  non profit organisation which collects, stores
  and distributes standard reference microorganism,
  cell lines and other material for research and
  development. Its present CEO is Raymond H.
  Cypress, founded in 1925, its headquarter in
  Manassa, Virginia and US.

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Vibrio percolons-
Was found to be fairly straight rods with
Liphotrichous flagella and with some detectable
action on carbohydrates.
Vibrio cuneatus-
found to be straight rod with polar
multitrichous flagella. The action on
carbohydrates was oxidative.
Several strains of organisms named bacterium
anitratum and written by schaub and hauber
(1948). This strain were all typically oxidizers
of Carbohydrates, such as, glucose and lactose.
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Peter jurtshuk JR and Donald N. MC. Quitting also
give a Publication on 12 December 1975 about use
of quantitative oxidase test for characterizing
oxidative metabolism in bacteria.
Modified oxidation fermentation medium for
detection of acid production from carbohydrates
by Nigeria species and Branhamella
Catarrhalis. Is an another Publication published
by joans knapp and King k. Holmes. Which was
received on 13 December 1982, and was accepted on
1 April 1983. It was developed as a practical
medium for highly sensitive specific detection of
acid production from carbohydrate by Nigeria
species and branhamella catarrhalis.
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Thank you.