Bacterial metabolism - biochemical tests, immuno-assays, mass spectrometry and nucleic acid-based assays, immuno-assays, enzyme immunoassays

bacteria take up nutrients from the environment and

transform them into energy and cellular components

enzymes, proteins with catalytic activity, are required to

carry out many of the reactions necessary for metabolism

metabolic processes and enzymatic characteristics vary between

bacterial strains and species

these differences are exploited by differential or selective media and biochemical tests

closely related organisms can be differentiated using

a series of tests that identify various metabolic properties of different bacterial species

main drawback of biochemical identification methods is

they can only be used to organisms that can be cultivated in the laboratory

catalase tests

• the enzyme catalase catalyzes the reaction of hydrogen peroxide into oxygen and water

• hydrogen peroxide is a harmful byproduct of respiration

• catalase protects the cell from oxidative damage - it is present in most aerobic bacteria

• the presence of catalase can be determined by the production of bubble of gas when bacterial culture is added to hydrogen peroxide

catalase positive vs catalase negative

bubbles vs no bubble

oxidase test

• cytochrome c oxidase is an enzyme present in many aerobic bacteria

• catalyzes the transport of electrons from electron donors donor compounds to electron acceptors in the energy-producing electron transport chain

• in the oxidase test, the enzyme oxidises reagents that are colorless (or light pink) in the reduced state and blue in the oxidized state

presence of cytochrome c oxidase will show up as

a blue color

urease test

• urease catalyzes the reaction of urea into ammonia, water, and carbon dioxide

• the presence of the enzyme can be determined by adding urea to the medium and detecting ammonia through associated increase in pH

Indole test

determines the ability of bacteria to produce indole from tryptophan by various enzymes

can be detected in a bacterial culturing by adding it to kovac’s reagent, which results in a pink to red color or p-dimethylaminocinnamaldehyde (DMACA) which results in a blue color

antibodies are proteins that specifically bind to

particular molecules, such as proteins, peptides or hormones

immuno-assays are

biochemical assays that take advantage of the characteristics of antibody-antigen interaction to detect or measure biological molecules

immuno-assays use different labels to detect antibodies and antigens, most commonly

enzymes (as in ELISA), but radioactive, fluorescent, and DNA probes are also widely used

due to highly specific antibodu-antigen binding

immuno-assays have high specificity and sensitivity

the classical methods of microbe identification are

relatively laborious and tend to rely on being able to culture bacteria in the lab

identification using

molecular methods has greatly improved the speed and specificity of the identification process

in molecular methods

nucleic acid sequences (DNA and RNA) from unknown species and known species are compared to determine their similarities

two main types of molecular identification

genotypic methods and protein-based methods

genotypic methods involve

extraction of genomic DNA from an unknown microbe species, which is further analysed with…

Southern blot

Polymerase chain reaction (PCR)

Restriction Fragment Length Polymorphism (RFLP)

Protein based methods include

Western blot

immunoprecipitation

Enzyme Linked Immunosorbent Assay (ELISA)

all use antibodies to detect specific proteins that are unique to an organism