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METABOLIC ACTIVITIES OF

MICROORGANISMS
Group 1
Bernas, Boris et.al
INTRODUCTION
Microorganisms grow and multiply by using raw materials
found in their environment. The available nutrients in the
environment may consist of simple to complex molecules. They
can oxidize these nutrient to obtain energy and modify then to
build precursors for the synthesis of necessary cellular
components. Microorganisms utilize nutrients in a variety of
ways and the metabolism often produces by-products that can
be used in the differentiation of microorganisms. The
production of the visible pigments is often used in
distinguishing groups of microorganisms particularly bacteria.
Objectives
• Perform the biochemical tests used in identification of
bacteria
• Explain the principle behind the production of
metabolic by-products
• Emphasize the importance of fermentation reactions
in the identification of bacteria
• Become aware on the importance of enzyme
production by bacteria
Materials
• inoculating loop
• Clean test tube
• Disinfectant
• 24 hr-old broth cultures of
• E.coli
• P.vulgaris
• E.aerogenes
• S.epidermidis
• M.luteus
• S.aureus
• P.aeruginosa
• S.Faecalis
 Culture media
 Basal medium
› Glucose
› Maltose
› Sucrose
› Lactose
› Mannitol
• Reagents
– 10% ferric chloride
– Methyl red
– 40%KOH
– 5% alpha naphthol
– Oxidase reagent
– Catalase reagent
– Kovac’s reagent
– Methyl red voges proskauer broth (MRVP)
– SIM
– LDC
– DC
– PA
– SCA
– CUA
– NA
Results and Discussion
Table 1.1

Bacteria BMglu BMmal BMlac BMsuc BMman


E. coli AG N AG N A
M. luteus N N N V N
P. vulgaris AG AG N AG A
S. aureus A N A A A
Unknown A N N N N
Fermentation Test

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
Results of Fermentation test
Results of Fermentation test
Results of Fermentation test
Results of Fermentation test
Principle of Fermentation of sugar

 To determine the ability of an organism to


ferment a specific carbohydrate (sugar)
incorporated in a medium producing acid
or acid with gas.
Type of fermentation

Explain the type of fermentation of the unknown.

-the unknown bacteria is a bacteria that belongs


to the family of Enterobacteriaceae because that
bacteria ferments glucose. One characteristic of
the family Enterobactericeae is they are glucose
fermenters.
Methyl red and Voges Proskauer
Table 1.2
Bacteria Methyl Red Voges Proskauer

E.coli + -
E. aerogenes - +
Unknown - +
Methyl red (MR)

 this test detects the production of sufficient


acid during fermentation of glucose by
bacteria and sustained maintenance of a
pH below 4.5.
 MR—tests for acid end products from
glucose fermentation.
Methyl Red (MR)

Tube A.  Methyl red negative


Tube B.  Methyl red positive
Positive control: E. coli, Listeria monocytogenes
Negative control: Klebsiella, Enterobacter spp.
E. Coli group (+)
 Ferments glucose
 Produce acid by & end products
 Low pH (4.5 and below)
 Methyl red turns to red
Enterobacter klebsiella group (-)
 Ferments glucose
 Produce non-acidic by & end products (alcohol,
acetion & acetyl methyl carbinol
 High pH 6.2
 Methyl red turns to yellow
Voges Proskauer

 This test depends on the production of acetyl


methyl carbinol (acetoin) from pyruvic acid in
the media. In the presence of alkali and
atmospheric oxygen, acetoin is oxidized to
diacetyl which reacts with α-naphthol to give
a red color.
 VP—tests for acetoin production from
glucose fermentation.
Voges Proskauer

Tube A.  Voges-Proskauer


positive
Tube B.  Voges-Proskauer
negative

 Positive control: Klebsiella sp., Enterobacter sp.,


Staphylococcus.
 Negative control: E. coli, Micrococcus.
Based on the table, is it possible to
have both MR and VP positive?

 Based on the table, it is not possible to have both


MR and VP. It is because these two tests aims to
differentiate one species of microbes from
another by their end-products which are acetoin
for VP and acid for MR.
Catalase and Oxidase test for
bacteria
Table 1.3

Bacteria Catalase Oxidase


E.coli + + - -
P. flourescens + + -
S. epidermidis + + -
S. faecalis - -
Unknown + -
Principle of Catalase and Oxidase

 certain bacteria have an enzyme catalase


which acts on hydrogen peroxide to release
oxygen.
Positive test: Immediate bubbling easily
observed
Negative test: No bubbling
Catalase

Positive control: Staphylococcus,


Micrococcus, all members of
Enterobacteriaceae except Shigella dysentriae.
Negative control: Shigella dysenteriae typeI,
Streptococcus, Clostridium.
Catalase

Bubbles appear
indicating a
positive result
Oxidase

 To determine the presence of an enzyme


cytochrome oxidase which catalyses the
oxidation of reduced cytochrome by
molecular oxygen.
Positive: deep purple within 10 secs.
Negative: – No color change
Oxidase

Positive control: Pseudomonas


sp., Vibrio sp., Alcaligenes sp.
Negative control: All members
of Enterobacteriaceae
Utilization of amino acids of the
test bacteria
Bacteria Indole Sulfide Lysine Phenylalanine
Production Production Production Production
E.coli - + - - - + - -
E. aerogenes - - - - - - -
C. freundii - + - - - - -
P. vulgaris + + + + - - + +
unknown - + + -
Indole
 Testing for indole production is important in the
identification of Enterobacteria. Most strains of E.
coli, P. vulgaris, Povidencia species break down the
amino acid tryptophan with release of indole.
 The test organism is cultured in a medium which
contains tryptophan. Indole production is detected
by Kovac’s or Ehrlich reagent which contains
4(p)-dimethylaminobenzaldehyde. This reacts with
the indole to produce a red colored compound.
Indole
Positive indole control: E. coli.
Negative indole control: Klebsiella
pneumoniae, Enterobacter aerogenes.
Sulfide Production

 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.
Sulfide production

 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
Citrate and Urea Utilization of test
bacteria
Bacteria Citrate Urea
E. coli - - - -
E.aerogenes + + - -
P. vulgaris - +/- + +
Unknown - -
Urea
 This test is done to determine a bacteria’s ability to
hydrolyze urea to make ammonia using the enzyme
urease.
 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.
IMViC reaction of E. coli and
E.aerogenes
Bacteria Indole Methyl red Voges Citrate
production Proskauer concentration

E.coli + + - -

E. aerogenes - - + -
Identity of unknown bacteria
 Salmonella typhii
Conclusion and Recommendation
 Different tests are needed to be conducted for us
to better understand to what certain chemicals
and enzymes they might react for easier
determination of the different reactivity and
identity of the bacteria.
 Based on the unknown bacteria that was
assigned to us we have identified it as Salmonella
typhii
Conclusion and Recommendation
 We recommend that practicing of these tests
regularly will ensure the accuracy and
preciseness of the tests being conducted in the
perspective of being a microbiologist.

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