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This is based on New Zealand Qualification Authority Unit 8023 entitled "Demonstrate knowledge of microbial metabolism."



The nutritional requirements of microorganisms.

Some of micro-organisms uses inorganic as sole nutritional requirements and some uses higher complex organic compounds. They use carbon, oxygen, nitrogen, and water in one way or other way for their nutrition. Eg hydrogen and oxygen forming organic & inorganic compound which is good source of nutrient.


Image taken by Radheyshyam N Bhagat UCOL National Diploma in Science 2008.


How microorganisms can be subdivided into metabolic catagories depending on electron source and carbon source ?.

  • Microbs divides in to metabolic classses which they use as source of energy.

1) Heterotrops or Chemoorganotrops which utilizes organic molecules as source of energy.

2) Phototrops which obtain energy from light.

3) Lithotrops which obtain energy from inorganic compounds and carbon for cell synthesis is obtained from organic molecules.

4) Chemolithotrops

These are the organisms that obtain energy by oxidation of inorganic compounds. They are lithotropic in nature and thus they fix carbon dioxide .some of them are heterotrophic in nature. most of them uses Calvin cycle to fix carbon dioxide. To fix 1 mol of carbon dioxide by this pathway, 3 mol of ATP and 2 mol of NADPH+ H+ are required. This is obtained from oxidation of in organic molecules. A less amount of energy is available from this oxidation than from the oxidation of glucose to carbon dioxide. Electron cannot be donated directly from production of NADPH+ H+ to overcome this. ATP is required to drive the revised electron transport for the production of NADPH+ H+.





The greater yield of energy is achieved in presence of oxygen from oxidation of pyruvate to carbon dioxide via TCA cycle. Before entering to this pathway it is converted acetyl COA and reaction is catalysed by pyruvate dehydrogenase.

(PYRUVATE + NAD+ + COA -> Acetyl COA + NADH+ H+)


Electron during glucose oxidation enter the electron transport chain through NADH+ H+ OR FADH2. These electron are transported via number of carriers like pyridine, nucleotides, proteins, iron, cytochromes etc. Some of them are proton carrier with progressively more redox potential and reaches the oxygen. The energy liberated during this process is partially converted in synthesis of ATP.


Production of ATP in Fermentation

The major product of catabolic pathways is NADH+ H+. The is NADH+ H+ and FADH2 formed in TCA cycle, NADH+ H+ produced during glycolysis can be oxidized by electron transport pathway. However in absence oxygen, they oxidized back to NAD+. This leads to increase in ATP synthesis and which is important factor for organisms growing in absence of oxygen.


Electron during glucose oxidation enter the electron transport chain through NADH+ H+ OR FADH2. These electron are transported via number of carriers like pyridine, nucleotides, proteins, iron, cytochromes etc. Some of them are proton carrier with progressively more redox potential and reaches the oxygen. The energy liberated during this process is partially converted in synthesis of ATP.



Enzyme activity-Nitrate reduction

Nitrate in organic nitrogen easily available for microbial use. The assimilation of nitrogen from nitrate into protein and cellular molecules called as assimilatory nitrate reduction. In initial reaction nitrate is reduce to nitrite by enzyme nitrate reductase and nitrite then further reduced by a second enzyme nitrite reductase and finally reduced to ammonia. This happens through the involving the addition electron through series of reactions.


Catabolic activity

Glycogen is formed and stored in liver and muscles. This is also found in human vagina after puberty. Vaginal bacteria such as lactobacillus degrade this polymer into acidic. The end products that contribute to acidic PH of vagina. The glycogen storage found in bacteria such as Escherichia coli serves as food reserves for these microbial organisms. Bacteria uses endogenous glycogen as a carbon and energy by phosphorylysis and do not require expenditure of energy.


Phototrophy are the organisms which uses the light as for the primary source of energy. They use the radiant energy from the sun and convert into the chemical energy from the sun and convert into the chemical energy in the form of carbohydrate and other molecules.

Simple diffusion is where nutrient molecules can freely enter and exit a cell through the cytoplasmic membrane. Molecules from outside the cell will move inwards until the concentration is equal both in side and outside the cell. The concentration of the nutrient molecules will not be higher inside the cell then out. The molecule move from high concentration to lower concentration. More of water in side the cell than out side and the carbon dioxide moves out by simple diffusion. Metabolic energy is not needed for simple diffusion.



The membrane bound vesicle in the cytoplasm fuses with the plasma membrane, to move the things from inside to the outside the cell. This material accumulates in the plasma membrane and this way parts of the plasma membrane lost during endocytosis is replaced. This process provides the routes to membranes impermeable molecules such as proteins hormones that are synthesized by the cell which can be released into the extracellualr materials. This process is use to continue the increase in cystolic calcium concentration which in turn activates proteins essential for vesicle membrane to fuse with the plasma membrane and continuing the process to maintain the holes from the prior process.

This is based on New Zealand Qualification Authority Unit 8024 entitled "Demonstrate knowledge of Bacterial structure."



1. Heat resistant spores are formed by some species(Endospores). No
2. Poly unsaturated fatty acids or sterols are rare in the membrane. Common.
3. Some species fix atmospheric nitrogen and able to dissimilate Nitrate to nitrogen gas. No.
4. Some species uses inorganic compound as sole energy source. No.
5. Ribosomes are dispersed through out cytoplasm. To endoplasmic reticulum.
6. Mitochondria and chloroplast is absent. Present.


Gram stains are used to enhance the features of specific cell constituents such as endospores, nuclear regions and granules. In Gram stain dyes like crystal violet, iodine solution and safarnin are used .They are positively charged and combine with negatively charged and fixes dyes inside the cell. The cell components such as nucleic acid and acidic polysaccharides acidic dye have an affinity for positively charged constituents like proteins etc.

1.3 CAPSULE :-

The composition and biological properties of major external structures.

Bacteria secrete extracellular polymers outside of their cell walls. These polymers are usually composed of polysaccharides and sometimes protein. Capsules are relatively impermeable structures that cannot be stained with dyes such as India ink. They are structures that help protect bacteria from phagocytosis and desiccation. India ink (or Indian ink in British English), also called Chinese ink since it may have been first developed in either China or India, is a simple black ink once widely used for writing and printing, and now more commonly used for drawing, especially when inking comics and comic strips. Indian ink generally will readily clog the fountain pens if not used for long time, its then necessary to use water to unclog it. An exception to this is Pelikan Fount India, which does not contain shellac, the substance which can cause clogging.

A capsule made up of polysaccharides (complex carbohydrates). Capsules play a number of roles, but the most important are to keep the bacterium from drying out and to protect it from phagocytosis (engulfing) by larger microorganisms. The capsule is a major virulence factor in the major disease-causing bacteria, such as Escherichia coli and Streptococcus pneumoniae. Nonencapsulated mutants of these organisms are avirulent, i.e. they don't cause disease.



The description outlines the composition and function of bacterial structures.


Ribosome’s consist of a small and large subunit made up of a complex proteins and RNAs . These are the sites of protein synthesis in the cell. The Ribosome’s in prokaryotic cells although similar in shape and function to those of eukaryotic cells are in different in the nature of the proteins and RNAs that make up their structure. This have provided to be very useful to the human population as antibiotics that acts by inhibiting protein synthesis in bacteria are not effective against eukaryotic protein synthesis, thus allowing selective toxicity.Archaebacterial ribosome’s are the same as those of the eubacteria but, in some features they are similar to eukaryotic ribosome’s in that they are resistant to antibiotics like streptomycin and chloramphenicol, and sensitive to the action of diphtheria toxin.


The cytoplasm is the contents of a cell that is enclosed within the plasma membrane. In eukaryotic cells the cytoplasm contains organelles, such as mitochondria, that are filled with liquid kept separate from the cytoplasm by cell membranes. The part of the cytoplasm that is not held within organelles is called the cytosol. The cytosol is a complex mixture of cytoskeleton filaments, dissolved molecules, and water that fills much of the volume of a cell. The cytosol is a gel, with a network of fibers dispersed through water. Due to this network of pores and high concentrations of dissolved macromolecules, such as proteins, an effect called macromolecular crowding occurs and the cytosol does not act as an ideal solution. This crowding effect alters how the components of the cytosol interact with each other.

. 1 Constituents

. 1.1 Cytosol

. 1.2 Organelles

. 1.3 Cytoplasmic inclusions


Proteins in different cellular compartments and structures tagged with green fluorescent protein. The cytosol is the portion of a cell that is not enclosed within membrane-bound organelles. The cytosol is a translucent fluid in which the other cytoplasmic elements are suspended. Cytosol makes up about 70% of the cell volume and is composed of water, salts and organic molecules. The cytoplasm also contains the protein filaments that make up the cytoskeleton, as well as soluble proteins and large structures such as ribosome’s, proteasomes, and the mysterious vault complexes. The inner, granular and more fluid portion of the cytoplasm is referred to as endoplasm.


Organelles are membrane-bound compartments within the cell that have specific functions. Some major organelles that are suspended in the cytosol are the mitochondria, the endoplasmic reticulum, the Golgi apparatus, lysosomes, and in plant cells chloroplasts.

Cytoplasmic inclusions

The inclusions are small particles of insoluble substances suspended in the cytosol. A huge range of inclusions exist in different cell types, and range from crystals of calcium oxalate or silicon dioxide in plants,[3][4] to granules of energy-storage materials such as starchs, glycogen, or polyhydroxybutyrate. A particularly widespread example are lipid droplets, which are spherical droplets composed of lipids and proteins that are used in both prokaryotes and eukaryotes as a way of storing lipids such as fatty acids and sterols. Lipid droplets make up much of the volume of adipocytes, which are specialized lipid-storage cells, but they are also found in a range of other cell types.


Plasmids are tenth or variable the size of prokaryotic chromosomes and are stable. These stable extra chromosomal DNA molecules exist as closed loops containing 5 to 100 genes .there can be one or more plasmids in cell and contain similar or different genes. Some cells no plasmids. Plasmids replicate independently of chromosomes and can transfer between cells during recombination. They are not essential for cellular growth but provide a level of genetic flexibility.



The formation of Bacterial endospores.

When a bacterium detects environmental conditions are becoming unfavorable it may start the process of sporulation, which takes about eight hours. The DNA is replicated and a membrane wall known as a spore septum begins to form between it and the rest of the cell. The plasma membrane of the cell surrounds this wall and pinches off to leave a double membrane around the DNA, and the developing structure is now known as a forespore. Calcium dipicolinate is incorporated into the forespore during this time. Next the peptidoglycan cortex form between the two layers and the bacterium adds a spore coat to the outside of the forespore. Sporulation is now complete, and the mature endospore will be released when the surrounding vegetative cell is degraded.


The chemical and structural properties to the resistance of endospores.

Endospores are resistant to most agents which would normally kill the vegetative cells they formed from. Household cleaning products generally have no effect, nor do most alcohols, quaternary ammonium compounds or detergents. Alkylating agents however, such as ethylene oxide, are effective against endospores. Whilst resistant to extreme heat and radiation, endospores can be destroyed by burning or autoclaving. Exposure to extreme heat for a long enough period will generally have some effect, though many endospores can survive hours of boiling or cooking. Prolonged exposure to high energy radiation, such as x-rays and gamma rays, will also kill most endospores.

This is based on New Zealand Qualification Authority Unit 8034 entitled "Culture microorganisms and control."



The nutrient requirements of bacteria.

Bacteria also use the elemental traces such as ZN, CU, CO, MO. These are in the form of water, inorganic ions, small molecules, macromolecules.they take up these substance by membrane transport process.


The types of media used to culture bacteria.

Microbiological assay media

Some of microorganisms can be used for the concentration of substances like antibiotics, minerals, vitamins etc. Many of antibiotics are resistant to blood serum or tissue fluids. Than in this type of assay is involves the measurement of growth inhibition caused by the antibiotics within the given time level of inhibition is proportional to the amount of drugs. Eg persons exposed to penicillin for long time unknowingly and treated with same antibiotics.



Use of a closed system to grow micro organisms.

They are incubated in a closed culture vessel (Petri dish) with a batch of medium. Because no fresh medium is provided during incubation, nutrient concentration decline and concentration of waste increase.


The culture of microorganisms under appropriate condition in accordance with laboratory protocol.

Pipette should be filled with use of pipette aids and operated in such away to avoid creating aerosols. Hypodermics syringe and needle should be used whenever necessary and then with care. Aqueous aerosol is a gaseous suspension of liquid or solid particles should be away from flames and, removal of closures from shaken culture tubes and contaminated loop should be away from contaminating materials.




The use of chemical agents to limit microbial growth.

Antiseptics-are the chemical agents apply to tissue or fluids to prevent the infection by killing or inhibiting pathogenic growth. These are temporary prevention and not so toxic. Disinfectants- are agents used to carryout disinfection. They kill, inhibit or remove the microorganisms that cause disease and mostly used on inanimate objects. These chemicals also kill or inhibit the growth of non pathogenic organisms as well.



6) Temperature at which microorganisms are exposed to microbial agent: At higher temperature one can kill large amount of bacteria.

7) Characteristic of microorganisms : Gram positive bacteria are more resistant to heat than gram negative and some time gram negative bacteria are more resistant to heat than gram positive.



Mode of action of antibiotics on Cytoplasmic Membrane

Antibiotic such as Polymyxins have ability to damage the cytoplasmic membrane of bacteria. Polymyxins are bactericidal because they disrupt the phospholipids that make up the structure of the membrane. This destroys normal permeability characteristics of cell membrane. So that essential substances leak from cells, resulting in cell death.

'Antibiotics and Mode of action'

Antibiotics Mode of action Active against
Penicillins, Cephalosporins, Monobactams (Beta-Lactams) Inhibit cell wall synthesis Gram positive bacteria,Gram negative bacteria causing respiratory, intestinal and urinary infections
Streptomycin, Spectinimycin, Neomycin, Kanamycin, Gentamicin (Aminoglycosides) Induce abnormal protein synthesis Tuberculosis infection Penicillin resistant Neisseria gonorrhoeae, Inhibits intestinal bacteria,Gram negative bacteria and Gram positive
Tetracyclines Interfere with protein synthesis Broad spectrum bacteria
Erythromycin, Lincomycin (Macrolides) Interferes with protein synthesis Gram positive bacteria
Chloramphenicol Interferes with protein synthesis Broad spectrum bacteria
Vancomycin Interferes with protein synthesis Gram positive including penicillinase producing staphylococci and enterococci
Viomycin Interferes with protein synthesis Tuberculosis infection
Rifamycin Interferes with protein synthesis Tuberculosis infection
Polyenes Nystatin (Antifungal) Damage Cell membrane Fungal infections, particularly oral, skin, intestinal and vaginal lesions
Amphotericin B (Antifungal) Interferes with membrane function Deep seated mycotic infection
Griseofulvin (Antifungal) Damage Cell membrane Fungal infections


Tuberculosis Treatment and complience.


Effectiveness of an Antibiotic.

Antibiotics are the molecules produced by microbes that inhibit the growth or kill, the other microbes. They are normally used to treat the bacterial infection those that are selectively toxic to bacterial cells and do not harm the host cells. They target the sites such as Peptidoglycan, ribosomes, nucleic acid synthesis which are different for prokaryotic and eukaryotic cells. The effectiveness of antibiotic is reduced by development of bacterial antibiotic resistant.


Microbiology by J.Nicklein, K.graeme-Cook, T.Paget &R. Killington


Alcomo's Fundamentals of Microbilogy

Prescott Microbiology

Microbiology concepts and application by Chan,Kreig,Pelczar