1 . C A R B O H Y D R A T E C A T A B O L I S M

2 . F E R M E N T A T I O N O F C A R B O H Y D R A T E S

3 . P R O T E I N C A T A B O L I S M

4 . R E S P I R A T I O N

5 . R A P I D I D E N T I F I C A T I O N M E T H O D S

MICROBIAL METABOLISM

MICROBIAL METABOLISM

Metabolism is the sum of the chemical reactions in an organism.

Catabolism is the energy-releasing processes.

Anabolism is the energy-using processes.

MICROBIAL METABOLISM

A metabolic pathway is a sequence of enzymatically catalyzed chemical reactions in a cell.

Metabolic pathways are determined by enzymes.

Enzymes are encoded by genes.

The majority of enzymes function inside a cell-that is, they are endoenzymes.

Many bacteria make some enzymes, called exzoenzymes, that are released from the cell to catalyze reactions outside of the cell.

MICROBIAL METABOLISM

1. CARBOHYDRATE CATABOLISM

Most bacteria catabolize carbohydrates for carbon and energy.

Carbohydrates can be classified based on size: monosaccharides, oligosaccharides, polysaccharides.

Exzoezymes are mainly hdyrolytic enzymes that leave the cell and break down, by the addition of water, large subrates into smaller components, which can be transported into the cell.

Amylase hydrolyzes the polysaccharide starch into smaller carbohydrates.

In the laboratory, the presence of an exzoenzyme is determined by looking for a change in the substrate outside of a bacterial colony.

starch glucose+glucose+

1. CARBOHYDRATE CATABOLISM

H2O

Amylase

Procedure of Starch Hydrolysis Test

1. With a marker, divide the petri into three sector.

2. Streak the bacteria (Bacillus, E.coli and Pseudomonas)

3. Incubate the plate for 24h.

4. After the incubation, flood the plate with Grams iodine. Areas of starch hydrolysis will appear clear , while unchanged starch will stain dark blue.

1. CARBOHYDRATE CATABOLISM

Releases energy from fermentation of organic molecules

Does not require oxygen

Does not use the Krebs cycle or ETC

Most bacteria can ferment the glucose without using oxygen.

The metabolic end-products of fermantation are small organic molecules, usually organic acids. Some bacteria produce gases from the fermantation of carbohydrates.

2. FERMANTATION OF CARBOHYDRATES

2. FERMANTATION OF CARBOHYDRATES

2. FERMANTATION OF CARBOHYDRATES

A fermantation tubes (duraham tube) is used to detect acid and gas production from carbohydrates.

Acid-base indicator phenol red inverted tube trap gas

3. PROTEIN CATABOLISM (urease)

Urea Ammonia (NH3) + CO2

H2O

urease

3. PROTEIN CATABOLISM (urease)

Bacteria can hydrolize the peptides or polypeptides to release amino acids. They use the amino acid as carbon and energy sources. Urea is waste product of protein digestion

Tryptophan hydrolysis-Some bacteria split tryptophan into indole and pyruvic acid using the hydrolase called tryptophanase.

Indole can be detected with Kovac's reagent (Indole reagent).

3. PROTEIN CATABOLISM (indole)

4. Respiration

Catalase Test

Microorganisms able to live in oxygenated environments produce enzymes which neutralize toxic forms of oxygen.

One such enzyme is catalase, which breaks hydrogen peroxide into water and molecular oxygen. Organisms which produce catalase will bubble when placed into hydrogen peroxide.

Oxidase Test

The oxidase test is a test used in microbiology to determine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent.

4. Respiration

Gram Test (3% KOH)

The KOH test is based on differences in the chemistry of the bacterial cell wall. The cell wall of gram-negative bacteria is easily disrupted when exposed to dilute alkali solutions.

When the cell walls are disrupted, the suspension in KOH becomes viscous due to the release of relatively unfragmented threads of deoxyribonucleic acid.

5. Rapid Identification Methods

Identify the bacteria quickly and accurately.

Provide large number of results from one inoculation.

Api 20E strip Biolog Vitek 2 Compact

Experiments in this lab

Exp 1: Carbohydrate Catabolism

Starch agar (E.coli, Bacillus, Pseudomonas)

Exp 2: Protein Catabolism 1

Urea agar (Pseudomonas and Proteus vulgaris)

Exp 3: Protein Catabolism 2

Indole Test (E.coli, Proteus vulgaris)

Exp 4: Respiration Oxidase (E.coli, Pseudomonas) Catalase (Bacillus, Staphylococcus) Exp 5: Rapid Identification Methods Citrate (E.coli and Enterobacter) Exp 6: Gram test by KOH 3% KOH (E.coli and Bacillus)