Environmental Microbiology -Laboratory Manual- prepared for Environmental Microbiology IV Biochemical Activity of microorganism

  ENVIRONMENTAL MICROBIOLOGY _{PREPARED FOR ENVIRONMENTAL MICROBIOLOGY}

• LABORATORY MANUAL-

  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  Anabolism  Catabolism  Catalytic  Catalyst}  _{Enzyme  Apoenzyme  Holoenzyme  Coenzym  Endoenzyme}  _{Exoenzyme  Enzyme activity  Enzyme system  Substrate  Active Site}  _{Oxidation  Reduction  Hydrolysis  Glycolysis  Fermentation}  _{Aerobic respiration  Anaerobic respiration  Peptide bonding  Peptonization  Starch}  _{Carbohydrate  Amino acid  Lipid  Fatty acid  Protein}  _{Casein  Pyruvic acid  ATP, ADP}

  Metabolism 

  

Metabolism refers to all chemical

reactions that occur within a living organism.

  Catabolic: Anabolic: Click icon to add picture

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 Enzyme 

  Organic catalyst (elaborated by living cell)

   Protein mollecular

   Thermolabile (denaturated by heat)

   Precipitated by ethanol and high concentration inorganic salts

  

Non-dialyzable (does not go through

semi-permeable membrane)

CHARACTERISTIC OF ENZYMES

  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 extremely efcient. 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 temperature or pressure inside the cell.

  Endoenzymes Altering nutrient in

  Synthesizing cellular vicinity of cell Extracellular Enzyme Intracellular Enzyme material to provide

  Exoenzymes required energy works outside cell works inside cell 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).

   Coenzyme A is derived from panthotenic acid. Components of a Holoenzyme

  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 .

  4. Unchanged enzyme is free to bind to more substrate molecules. Mechanism of Enzymatic Action

  Factors that Affect Enzyme Activity: pH,

Temperature, and Substrate Concentration

  Click icon to add picture

Denaturation of a Protein Abolishes its Activity

Denaturation : Loss of three-dimensional protein structure.

  Involves breakage of H and noncovalent bonds. 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 Gain of electrons or H atoms Associated with gain of energy

  Oxidation-Reduction Reactions Aerobic Respiration is a Redox Reaction C H O + 6 O ----->

6 CO + 6 H O + ATP

  6

  12

  6

  2

  2

  2 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 process of a chemical mechanism.

  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 H O + 6 O ----->

6 CO + 6H O + ATP

  6

  12

  6

  2

  2

  2 Glucose oxygen oxidized reduced Aerobic Respiration occurs in three stages :

  1. Glycolysis

  2. Kreb’s Cycle

  3. Electron Transport & Chemiosmosis

  

Three Stages of Aerobic Respiration 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 Aerobic Respiration versus Fermentation

  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 ):, nitrous oxide, or nitrogen gas.

  2- 

  Sulfate (SO 42- ): Desulfovibrio. Reduced to hydrogen sulfde (H S).

  2  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.

   Anaerobes tend to grow more slowly than aerobes.

  EXPERIMENTS

BASIC PRINCIPLES

  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.

  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

  19.D Oxidation Test E.coli, P. Aeruginosa, A.faecalis ^{p- aminodimethylanyl} _{ine oxalate} Form of oxydase cytochrome Oxydase

  Phenol red Carbohydrate + O2  acid

  20.B Triple Sugar Iron Agar Test E.coli, P. Aeruginosa, E.aerogenes

  20.A4 Use of Citrate E.coli, E.aerogenes Bromthymol blue Citrate  oxaloacetate acid + acetat _ pyruvic acid + CO ₂ Citrate permease

  20.A3 Vogus-Proskauer Reaction

E.coli, E.aerogenes Barrit Glucose + O2  acetic acid 

acetymethylcarbinol + CO ₂ + H ₂

  20.A2 Methyl Red Reaction E.coli, E.aerogenes Methyl red Glucose + H ₂ O  acid + CO ₂ + H ₂ various

  20.A1 Indol Reaction Test E.coli, B.cereus Kovac Tryptophane  indole + pyruvic acid

  19.C Catalase Reaction S.aureus, S.lutea, B.subtilis 3 % H ₂ O ₂ H ₂ O ₂ ^ H ₂ O + O ₂ Catalase, superoxide dismutase

  Exp. M.O. Reagent Reaction Enzyme

  19.B Reduction of Nitrate E.coli, P. aeruginosa Sulfanilic Acid, Alphanaphtyla mine Nitrate  Nitrite Nitrate reductase

  19.AFermentation of Carbohydrates E.coli, P. Aeruginosa Red Phenol e.g.Glucose  pyruvic acid Various

  E. coli, B.subtilis Lugol Proteinpeptide amino acid Protease

  18.C Casein Hydrolysis

  18.B Lipid Hydrolysis - - Tryglyseride  glycerol + fatty acid Lipase

  18.A Starch Hydrolysis E. coli, B.subtilis Lugol Polysaccharide monosaccharide Amilase

• ammonia Tryptophanase

  

Exp. 18 – Acivities of Extracelullar

Enzymes Hydrolysis of Starch and Casein

  

Hydrolysis of Starch



  Starch  dextrin / monohydrates 

  Amylase 

  Starch Agar  the presence of starch in medium

   Iodine  indicator, if starch is still present, blue (-), if does not present, transparent (+)

  

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)

   Proteolytic zone  transparent Hydrolysis of Starch & Casein E. coli, B.subtilis

  Carbohydrate Milk Agar

  Streak Streak

  

Exp. 19 – Acivities of Intracelullar Enzymes :

Fermentation Test and Oxidation

  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

condition

19.A. Fermentation of Carbohydrates

  

E.coli, P. Aeruginosa

  Sucrose Glucose Mannitol Lactose broth broth broth broth tube tube+ tube+ tube+

• Durham Durham Durham Durham Inoculate, Inoculate, Inoculate, Inoculate, control control control control

  Incubate 37 Incubate 37 Incubate 37 Incubate 37

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 color,indicates that nitrate did not reduced before, if transparent (+)

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

2 H O into free H 0 and O

  2

  2

  2 

  Superoxide dismutase  in species which has no catalase

19.C. Catalase Reaction

  S.aureus, S.lutea, B.subtilis Nutrition Agar plate streak

  Incubate 37

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

   (-)  no color change

19.D. Oxidase Test

  E.coli, P. Aeruginosa, A.faecalis, A, B

  Trypticase soy in plate streak Incubate 37

  

Exp. 20 – Acivities of Intracelullar Enzymes :

  IMViC and TSI Test

  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

  N

   Enterobactericeae  G.I tract

  o F t e

   Identifcation is important in preventing f

  r e m

  contamination to food and water supply

  N r e m o

  

  E n

  Pathogenic, sometimes pathogenic,

  n e ti n E n

  normal fora

  n t ti n g e n t ri l g e a c ri l c a c t c o t s o e

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

cherry on the surface of the

test tube

20.A.1 Indol Reaction Test

  E.coli, B.cereus Trypton 1 % broth tube streak

  Incubate 37 Trypton 1 % + glucose 1 % broth tube

  Streak Incubate 37

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

   Yellow : pH6

20.A.2 Methyl Red Reaction

  E.coli, E.aerogenes MR-VP broth tube

  The Methyl Red Test: Left to Right:

  inoculate

  positive, positive,

20.A.3 Vogus-Proskauer Reaction

   Some fermentative organisms do not produce enough stable acids to lower the pH of the medium.

   To detect the ability of m.o. In producing non acid substance acetymethylcarbinol

   Characteristic of E. aerogenes m.o. culture MR-VP broth tube

  Voges-Proskauer Left: uninoculated

  Test

  inoculate

  control Left: uninoculated Right: positive (red control

  Incubate 37

  color) Right: negative

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  during this reaction medium will turn to

  2 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

alkaline products (pH >7.6), changing the color of the

medium from green to blue.

  E.coli, E.aerogenes, A, B Simmons citrate slant agar

  Citrate Utilization Stab or streak

  Enterobacter cloacae:

  positive Incubate 37

  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

   Surface and bottom : red  no carb fermentation

  

20.B. Triple Sugar Iron Agar

Test

  E.coli, P. Aeruginosa, E.aerogenes

  TSI slant agar Stab and streak

  Incubate 37

  

Exp. 21 – Other Biochemical Activities

  21.A. Litmus Milk Reaction

  21.B. Urease Test

  21.C. H S Production

  2

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 

  Alcaline reaction  litmus act as pH indicator

  POSSIBLE REACTION A. 

Acid/Reduction/Cu

rd B.  Reduction/Curd (arrow denotes gas pocket) C.  Uninoculated Control

D.  Acid Formation

E.  Proteolysis of casein F.  Alkaline Reaction

21.A. Litmus Milk Reaction

  E.coli, P. Aeruginosa, A.faecalis, S.lactis, E.aerogenes

  Litmus milk broth tube inoculate Incubate 37

21.B. Urease Test

   Identifying P.vulgaris with urease enzyme

   Amide substances  ammonia

   Phenol red as pH indicator

21.B. Urease Test

  E.coli, P.vulgaris Urea broth tube inoculate

  Incubate 37

21.B. H S Production

  2 

  Some bacteria are capable of breaking down sulfur or reducing inorganic sulfur-containing compounds to produce hydrogen sulfde (H S).

  2  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 ferric sulfde (FeS) in the tube.

  21.B. H

  2 S Production

  E.coli, P.vulgaris H stab

  Incubate 37 Why all these efforts ? “To identify bacteria, we must rely

heavily on biochemical testing. The

types of biochemical reactions each organism undergoes act as a "thumbprint" for its identifcation.”