ENVIRONMENTAL MICROBIOLOGY -LABORATORY
MANUAL-PREPARED FOR ENVIRONMENTAL MICROBIOLOGY
IV
BIOCHEMICAL ACTIVITY OF MICROORGANISM
E N V I R O N M E N T A L M A N A G E M E N T T E C H N O L O G Y
Terms
Metabolism
Metabolism refers to all chemical
reactions that occur within a living
organism.
Anabolic and Catabolic Reactions are Linked by ATP in
Living Organisms
Catalyst = an agent that accelerates
chemical reaction without itself being
destroyed or used up
Enzyme
Organic catalyst
(elaborated by living
cell)
Protein mollecular
Thermolabile
(denaturated by heat)
Precipitated by ethanol and high
concentration inorganic salts
Specifcity
Enzymes are highly specifc and usually catalyze only one or a few closely related reactions
Effectivity
Speed up reaction up to 10 billion times more than without enzyme.
Enzymes are
Energy of Activation
Energy of activation:
The amount of energy
required to trigger a
chemical reaction.
Enzymes speed up
chemical reactions by
decreasing their energy
of activation without
increasing the
Extracellular Enzyme Altering nutrient in vicinity of cell
Exoenzymes
works outside cell
Intracellular Enzyme Endoenzymes
Synthesizing cellular material to provide required energy
Enzyme Components
Some enzymes consist of protein only.
Others :
Holoenzyme = Apoenzyme + Cofactor
Enzyme cofactors
may be a metal ion, an organic
molecule, or derived from vitamins.
Examples:
NAD+:
Nicotinamide adenine dinucleotide
NADP+:
Nicotinamide adenine dinucleotide phosphate
are both cofactors derived from niacin (B vitamin).
Mechanism of Enzymatic Action
Surface of enzyme contains an
active site
that binds
specifcally to the substrate.
1. An
enzyme-substrate complex
forms.
2. Substrate molecule is
transformed
by:
Rearrangement of existing atoms
Breakdown of substrate molecule
Combination with another substrate molecule
3.
Products
of reaction no longer ft the active site
and are
released
.
Factors that Affect Enzyme Activity: pH,
Denaturation: Loss of three-dimensional protein structure. Involves breakage of H and noncovalent bonds.
Denaturation of a Protein Abolishes its Activity
REDOX
Redox Reactions:
Reactions in
which both oxidation and reduction
occur.
OXIDATION
Associated with loss of energy Removal of electrons or H atoms Addition of oxygen
REDUCTION
Aerobic Respiration is a Redox
Reaction
C
6H
12O
6+ 6 O
2--->
6 CO
2+ 6 H
2O +
ATP
Hydrolysis
Hydrolysis is a chemical reaction during
which molecules of water (H2O) are split
into hydrogen cations (H+)
(conventionally referred to as protons)
and hydroxide anions (OH−) in the
Carbohydrate Catabolism
Most microorganisms use glucose or other
carbohydrates as their primary source of
energy.
Lipids and proteins are also used as
energy sources.
Two general processes are used to obtain
energy from glucose:
cellular respiration
Cellular Respiration :
AEROBIC
ATP generating process in which food molecules are
oxidized.
Final electron acceptor is oxygen.
Aerobic Respiration
C
6H
12O
6+ 6 O
2---> 6 CO
2+ 6H
2O + ATP
Glucose oxygen oxidized reduced
Aerobic Respiration occurs in three stages
:
1. Glycolysis
2. Kreb’s Cycle
Fermentation
Releases energy from sugars or other
organic molecules.
Does
not
require
oxygen
, but may occur
in its presence.
Does not require an electron transport
chain.
Final electron acceptor is
organic
molecule.
Inefcient
: Produces a small amount of
ATP for each molecule of food.
End-products are energy rich organic
compounds:
CELLULAR RESPIRATION : ANAEROBIC
Final electron acceptor is not oxygen.
Instead it is an
inorganic
molecule:
Nitrate (NO
3-):
Pseudomonas
and
Bacillus
. Reduced to
nitrite (NO
2-):, nitrous oxide, or nitrogen gas.
Sulfate (SO
42-):
Desulfovibrio
. Reduced to hydrogen
sulfde (H
2S).
Carbonate (CO
32-):
Reduced to methane.
Inefcient (2 ATPs per glucose molecule).
Only part of the Krebs cycle operates without oxygen.
Not all carriers in electron transport chain participate.
When identifying a suspected organism, you inoculate a series of differential media
Y
The results of these tests on the suspected microorganism are then compared to known results for that organism to confrm its identifcation.
Exp. M.O. Reagent Reaction Enzyme
18.A Starch Hydrolysis E. coli, B.subtilis Lugol Polysaccharide monosaccharide Amilase 18.B Lipid Hydrolysis - - Tryglyseride glycerol + fatty acid Lipase 18.C Casein Hydrolysis E. coli, B.subtilis Lugol Proteinpeptide amino acid Protease 19.AFermentation of
Carbohydrates E.coli, P. Aeruginosa Red Phenol e.g.Glucose
pyruvic acid Various 19.B Reduction of
Nitrate E.coli, P. aeruginosa Sulfanilic Acid,Alphanaphtyla mine
Nitrate Nitrite Nitrate reductase 19.C Catalase Reaction S.aureus, S.lutea,
B.subtilis 3 % H2O2 H2O2
H2O + O2 Catalase,
superoxide dismutase 19.D Oxidation Test E.coli, P. Aeruginosa,
A.faecalis
p-aminodimethylanyl ine oxalate
Form of oxydase cytochrome Oxydase
20.A1 Indol Reaction
Test E.coli, B.cereus Kovac Tryptophane
indole + pyruvic acid
+ ammonia Tryptophanase 20.A2 Methyl Red
Reaction
E.coli, E.aerogenes Methyl red Glucose + H2O acid + CO2 + H2 various 20.A3 Vogus-Proskauer
Reaction E.coli, E.aerogenes Barrit Glucose + O2
acetic acid
acetymethylcarbinol + CO2 + H2
20.A4 Use of Citrate E.coli, E.aerogenes Bromthymol blue
Citrate oxaloacetate acid + acetat
pyruvic acid + CO2
Citrate permease 20.B Triple Sugar Iron
Agar Test E.coli, P. Aeruginosa, E.aerogenes Phenol red Carbohydrate + O2 acid 21.A Litmus Milk
Reaction E.coli, P. Aeruginosa, A.faecalis, S.lactis, E.aerogenes
Litmus Lactose glucose + galactose
pyruvic acid lactate acid B-galactosidase 21.B Urease Test E.coli, P.vulgaris Phenol red Amide substances ammonia urease
21.C H2S Production E.coli, P.vulgaris Ferrous sulphate
Cysteine pyruvic acid + H2S + ammonia
Hydrolysis of Starch and Casein
Hydrolysis of Starch
Starch
dextrin /
monohydrates
Amylase
Starch Agar
the
presence of starch in
medium
Iodine
indicator, if
Hydrolysis of Casein
Protein various amino acids
linked together in long chains by means of peptide bonds
Protein peptide amino acid Prior use as nutrition material
needs to be degraded into simpler substances through peptonization or proteolysis process using protease
enzyme (breaking CO-NH bonding)
Hydrolysis of Starch &
Casein
E. coli, B.subtilis
Carbohydrate
Streak
Incubate 37
Milk Agar
Streak
19.A. Fermentation of Carbohydrates
19.B. Reduction of Nitrate
19.C. Catalase Reaction
19.D. Oxidation Test
19.A. Fermentation of Carbohydrates
A wide variety of carbohydrates may be
fermented in order to obtain energy and the
types of carbohydrates which are fermented by
a specifc organism can serve as a diagnostic
tool for the identifcation of that organism.
End products of fermentation.
acid end products.
acid and gas end products.
Red phenol
red in normal pH, yellow in acid
19.A. Fermentation of Carbohydrates
E.coli, P. Aeruginosa
Lactose broth tube + Durham
Inoculate, control Inoculate,
19.B. Reduction of Nitrate
Bacteria can reduce nitrate
Anaerobic condition
Nitrate reductase enzyme
NO3- + 2e- + 2H-
NO2- + H2O
Reagent A Sulfanilic acid + Reagen B
alphanaphtylamine, if nitrite is presence
(+), red
If (-), zinc will reduce nitrate, bring red
19.B. Reduction of Nitrate
E.coli, P. Aeruginosa
nitrate broth tube
inoculate
19.C. Catalase Reaction
Aerobic reaction
hydrogen peroxide,
reactive, destructing enzyme
Catalase
preventing damage, turning
H
2O
2into free H
2
0 and O
219.C. Catalase Reaction
S.aureus, S.lutea, B.subtilis
Nutrition Agar plate
streak
19.D. Oxidase Test
Oxidase enzyme
Electron transport
system in aerobic resp.
p-aminodimethylanyline oxalate
artifcial substrate, donating electrone
and be oxidized into black substances if
oxydase and free oxygen are present
(+)
pink – maroon-black
19.D. Oxidase Test
E.coli, P. Aeruginosa, A.faecalis, A, B
Trypticase soy in plate
streak
20.A.1 Indol Reaction Test
20.A.2 Methyl Red Reaction
20.A.3 Vogus-Proskauer Reaction
20.A.4 Use of Citrate
20.B. Triple Sugar Iron Agar Test
A. IMVic Test
Enterobactericeae
G.I tract
Identifcation is important in preventing
contamination to food and water supply
Pathogenic, sometimes pathogenic,
20.A.1 Indol Reaction Test
Indol =a component of
tryptophane, an essential
amino acid
Will not occur if carbohydrate
that needs to be degraded is
exsist
low pH
Specifc characteristic of
intestinal bacteria
Indol + Kovac reagent
red
20.A.1 Indol Reaction Test
E.coli, B.cereus
Trypton 1 % broth tube
streak
Incubate 37
Trypton 1 % + glucose 1 % broth tube
Streak
20.A.2 Methyl Red Reaction
Glucose
primary energy source for
enteric
Some turn glucose into acid (glucose
fermentation)
low pH
Important in differentiating
E.coli
and
E.aerogenes
Methyl red
red : pH 4
20.A.2 Methyl Red Reaction
E.coli, E.aerogenes
MR-VP broth tube
inoculate
Incubate 37
20.A.3 Vogus-Proskauer Reaction
Some fermentative organisms do not
produce enough stable acids to lower the
pH of the medium.
m.o. culture
MR-VP broth tube
inoculate
Incubate 37
Voges-Proskauer Test
Left: uninoculated control
Right: negative (copper color)
Left: uninoculated control
20.A.4 Use of Citrate
When carbohydrate does not present, citrate is used
as carbon source in providing energy
Citrate permease
facilitating citrate transport
Citrate
oxalatoacetate acid + acetate
pyruvic
acid + CO
2
during this reaction medium will turn to
alkaline condition
Bromthymol blue indicator
will turn from green at
neutral pH (6.9) to blue when a pH higher than 7.6 is
reached (basic or alkaline).
If the citrate is utilized, the resulting gowth will produce
E.coli, E.aerogenes, A, B
Simmons citrate slant agar
Stab or streak
Incubate 37
Citrate Utilization
Enterobacter cloacae: positive
Eschericia coli: negative
20.B. Triple Sugar Iron Agar Test
To differentiate
Enterobactericeae (bacil, gram
-, fermenting glucose, producing acid) vs non
Enterobactericeae
TSI
glucose + lactose + sucrose + phenol red
Phenol red as pH indicator
red (alkaline),
yellow (acid)
Surface : red, bottom : yellow
glucose
fermentation only
Surface and bottom : yellow
lactose and sucrose
fermentation also occur
20.B. Triple Sugar Iron Agar
Test
E.coli, P. Aeruginosa, E.aerogenes
TSI slant agar
Stab and streak
21.A. Litmus Milk Reaction
21.B. Urease Test
21.C. H
2S Production
21.A. Litmus Milk Reaction
Several milk substrate reactions using
litmus
in
media:
Glucose fermentation
litmus act as pH indicator
purple : normal pH, pink : acid, formation of gas
Litmus reduction
litmus act as acceptor to bond
hydrogen ion
purple : oxidized, white : reducted
Curd formation
acid type (solid) and rennet type
(semi solid)
Proteolysis
forming of ammonia
litmus act as
pH indicator
POSSIBLE
(arrow denotes
gas pocket)
C. Uninoculated
Control
D. Acid Formation
E. Proteolysis of
casein
21.A. Litmus Milk Reaction
E.coli, P. Aeruginosa, A.faecalis, S.lactis, E.aerogenes
Litmus milk broth tube
inoculate
21.B. Urease Test
Identifying
P.vulgaris
with urease
enzyme
Amide substances
ammonia
21.B. Urease Test
E.coli, P.vulgaris
Urea broth tube
inoculate
21.B. H
2
S Production
Some bacteria are capable of breaking down
sulfur or reducing inorganic sulfur-containing
compounds to produce
hydrogen sulfde
(H
2S).
For identifying
Proteus
and
Salmonella
.
a medium with a sulfur-containing compound
and iron salts
If the sulfur is reduced and
hydrogen sulfde is produced, it will combine
with the iron salt to form a visible
black
21.B. H
2
S Production
E.coli, P.vulgaris
H
stab
