Protocols

Microbiology Protocols. Molecular Biology and Genetics Protocols. Biology and Life Sciences Protocols for undergraduate and graduate students with diagrams.

Water Quality Analysis by Membrane Filter (MF) Technique

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Membrane Filter (MF) Technique

The Membrane Filter (MF) Technique was introduced in the late 1950s as an alternative to the Most Probable Number (MPN) procedure for microbiological analysis of water samples. It involves the use of membrane filters, which are thin porous sheet structures composed of cellulose esters or similar polymeric materials. They act essentially as two-dimensional screens and as such, all particles, both biological and non-biological, which exceed the pore size, are retained upon the surface of the filter from fluids passing through. … Read more

Oxygen Release in Photosynthesis Protocol

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Oxygen Release in Photosynthesis Protocol

Photosynthesis is the biological process that converts sunlight into chemical energy. It is employed by plants, algae and certain bacteria. It provides the basis for life on Earth and is the ultimate source of all fossil fuels and of the oxygen we breathe. There are a few key methods to calculate the rate of photosynthesis. These include: 1) Measuring the uptake of CO2                   2) Measuring the production of O2 3) Measuring the production of carbohydrates 4) Measuring the increase in … Read more

Brain Lipid Extraction Protocol

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Brain Lipid Extraction Protocol

“Brain lipid extracts” refers to a lipid mixture extracted from the brain tissue of animals. It has been found that such brain lipid extracts contain a mixture of long chain polyunsaturated fatty acids (PUFAs) that have four or more double bonds and closely resemble the mixture of PUFAs found in human breast milk. The mixture of PUFAs typically includes arachidonic acid (ARA), docosahexaenoic acid (DHA) and docosatetraenoic acid (DTA). These PUFAs are important structural lipids that are required for normal … Read more

Isolation of Mitochondria from Plants, Yeast Cells, Mice, Cell Culture

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Isolation of Mitochondria from Cell Culture

Mitochondria are central organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP and regulate a number of signaling cascades, including apoptosis. Investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers a full understanding of mitochondrial function. Mitochondria can be isolated from a variety of cells or tissues.  Isolation of Mitochondria from Plants (Potato Tubers) Materials and Reagents Mannitol 3-morpholinopropane-1-sulfonic acid (MOPS) Potassium hydroxide … Read more

Eukaryotic DNA Isolation Protocol

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Eukaryotic DNA Isolation Protocol

Deoxyribonucleic acid (DNA) extraction is the process by which DNA is separated from proteins, membranes, and other cellular material contained in the cell from which it is recovered. In eukaryotic cells, such as human and plant cells, DNA is organized as chromosomes in an organelle called the nucleus.  These cells also have a lipid bilayer outer membrane and cytoplasm containing proteins, sugars, lipids, and inorganic ions of various types and function. Eukaryotic cells also contain other membrane-enclosed compartments called organelles. … Read more

Isolation of Actinomycetes from Soil sample

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Procedure of Isolation of Actinomycetes

Actinomycetes are classified as a group of gram-positive bacteria that are unique for their spore forming abilities and formation of mycelia structures. They show marked chemical and morphological diversity but form a distinct evolutionary line of organisms that range from coccoid and pleomorphic forms to branched filaments. These bacteria have been noted to serve as rich reservoirs of medicinal antibiotics and are therefore extremely relevant to scientists, pharmaceutical industries and agricultural industries. Figure: Free living actinomycetes are ubiquitous in soil environments … Read more

Isolation of Bacillus thuringiensis (Bt) from Soil sample

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Isolation of Bacillus thuringiensis (Bt) from Soil sample

Bacillus thuringiensis (Bt) is an ubiquitous, Gram-positive and sporulating bacterium that synthesizes insecticidal proteins with specificity against a wide range of insects during sporulation (Cry and Cyt) and vegetative growth (Vip and Sip). These proteins have portrayed Bt as an environment-friendly alternative to chemical insecticides. Principle of Isolation of Bacillus thuringiensis Widely used methodologies to isolate Bt from soil consist of a thermal shock treatment followed by selective germination of spores. Thermal shock is employed to eliminate all bacteria incapable of producing endospores from … Read more

Bacterial Growth Curve Protocol

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Bacterial Growth Curve

When fresh liquid medium is inoculated with a given number of bacteria and incubated for sufficient period of time, it gives a characteristic growth pattern of bacteria. If the bacterial population is measured periodically and log of number of viable bacteria is plotted in a graph against time, it gives a characteristic growth curve which is known as growth curve. Measuring the growth rate of bacteria is a fundamental microbiological technique, and has widespread use in basic research as well as … Read more

RNA Isolation Protocol

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RNA Isolation Protocol

Total RNA isolation from Bacterial Cells Principle of RNA Isolation Total RNA is isolated and separated from DNA and protein after extraction with a solution called as Trizol. Trizol is an acidic solution containing guanidinium thiocyanate (GITC), phenol and chloroform. GITC irreversibly denatures proteins and RNases. This is followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total … Read more

Protocol of chloroplast isolation

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Protocol of chloroplast isolation

The chloroplast is an important organelle found in plant cells that conduct photosynthesis. It is enclosed by a pair of closely spaced membranes, the double-membrane envelope, consisting of the inner membrane bounding the matrix or stroma and the outer membrane in contact with the cytoplasm. Chloroplasts are the best starting material for studies of chloroplastic processes such as carbon assimilation, electron flow and phosphorylation, metabolic transport, or protein targeting. The chloroplast fraction can be further extracted to obtain membrane, stroma, … Read more