Biochemical Testing: July 2009

What is a Biochemical Test?

Biochemical test are tests which are carried out to study the chemical processes and substances which occur in living things. These test helps to identify the main biologically important chemical compounds.

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Why are biochemical tests are essential for laboratory work of microbiologists?

Biochemical test are important in the aspect of microbiology because both of them goes hand in hand. It is essential to have a basic working knowledge of biochemistry to understand the working or growth, metabolism and even the defences of micro organism. Aerobic and non aerobic respiration are both important in microbiology and they are highly important biochemical reaction of life and not just the bacteria.

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METABOLIC PATHWAYS
USES OF CARBOHYDRATE AS ENERGY SOURCE
SELECTIVE & DIFFERENTIAL MEDIA
OXIDASE TEST

MacConkey's Agar

It is selective for gram-negative bacteria.
Grow of gram positive bacteria is inhibited by the crystal violet dye and bile salts in the media.

It is differential for lactose fermentation. It contains neutral red pH indicator.

When the bacterial waste products generated, organic acid metabolites, change the pH indicator in MSA from red to yellow. Pathogenic staph, such as Staphylococcus aureus, are mannitol fermenters, and when growing on Mannitol Salt Agar, their wastes turn the MSA a bright yellow color.

In contrast, nonpathogenic staph such as Staphylococcus epidermidis (aka Staph epi), the normal flora that grows on human skin, does not ferment mannitol. When Staph epi grows on Mannitol Salt, the naturally orange-pink color of the agar doesn’t change, since S. epidermidis doesn’t ferment mannitol or produce the resulting organic acid wastes.

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Catalase Reaction

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Some bacteria and macrophages can reduce diatomic oxygen to hydrogen peroxide or superoxide. Both of these molecules are toxic to bacteria. Some bacteria, on the other hand, possess a defense mechanism which can minimize the harm done by the two compounds. These resistant bacteria use two enzymes to speed up the conversion of hydrogen peroxide and superoxide back into diatomic oxygen and water. One of these enzymes is catalase and its presence can be detected by a simple test. The catalase test involves adding hydrogen peroxide to a culture sample or agar slant. If the bacteria in question produce catalase, they will convert the hydrogen peroxide and oxygen gas will be evolved. The evolution of gas causes effervescence and is indicative of a positive test.

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MSA (Mannitol Salt Agar)

Mannitol salt agar Picture.sources

MSA is selective for gram-positive Staphylococci bacteria.

It contains: 7% salt in the medium which inhibits the growth of most gram-positive and gram-negative bacteria.

MSA is also differential for it’s mannitol fermentation

It contains: phenol red pH indicator

The indicator will turns yellow if there are presence of acid by products of mannitol fermentation.

The bacterial waste products generated, organic acid metabolites change the pH indicator in MSA from red to yellow. Pathogenic staph, such as Staphylococcus aureus, is mannitol fermenters, and when growing on Mannitol Salt Agar, their wastes turn the MSA a bright yellow color.

In contrast, nonpathogenic staph such as Staphylococcus epidermidis (aka Staph epi), the normal flora that grows on human skin, does not ferment mannitol. When Staph epi grows on Mannitol Salt, the naturally orange-pink color of the agar doesn’t change, since S. epidermidis doesn’t ferment mannitol or produce the resulting organic acid wastes.

Figure 1

A Mannitol Salt plate(figure 1) with two separate samples on same plate. Yellow differential reaction showing pathogenic, mannitol-fermenting Staph present. Opposite side of same plate remains pink, with nonpthogenic Staph growing on it.

Metabolic Pathways

A metabolic pathway is a series of chemical reactions occurring within a cell. Enzymes catalyze reactions often require dietary minerals, vitamins, and other cofactors in order to function properly. Because of the many chemicals that may be involved, pathways can be quite elaborate. This collection of pathways is called the metabolic network. Pathways are important to the maintenance of homeostasis within an organism.

Metabolism is a step-by-step modification of the initial molecule to shape it into another product. The result can be used in one of three ways:

To be stored by the cell
To be used immediately, as a metabolic product
To initiate another metabolic pathway, called a flux generating step.

A molecule called a substrate enters a metabolic pathway depending on the needs of the cell and the availability of the substrate. An increase in concentration of anabolic and catabolic end-products would slow the metabolic rate for that particular pathway.

Glycolysis was the first metabolic pathway discovered:

1. As glucose enters a cell, it is immediately phosphorylated by ATP to glucose 6-phosphate in the irreversible first step. This is to prevent the glucose from leaving the cell.
2. In times of excess lipid or protein energy sources, glycolysis may run in reverse (gluconeogenesis) in order to produce glucose 6-phosphate for storage as glycogen or starch.

Metabolic pathways are often regulated by feedback inhibition, or by a cycle wherein one of the products in the cycle starts the reaction again, such as the Krebs Cycle (see below).
Anabolic and catabolic pathways in eukaryotes are separated either by compartmentation or by the use of different enzymes and cofactors.

Several distinct but linked metabolic pathways are used by cells to transfer the energy released by breakdown of fuel molecules to ATP. These occur within all living organisms in some forms:

1. Glycolysis
2. Anaerobic respiration
3. Krebs cycle / Citric acid cycle
4. Oxidative phosphorylation

Other pathways occurring in (most or) all living organisms include: