Science Units

Nutritional Requirements of Microorganisms and Bacteria

Microorganisms require large amounts of macro elements and macronutrients to grow and survive. For example: C, O, H, N, S, P, K, Ca, Mg, and Fe. They also require trace amounts of micronutrients such as Mn, Zn, Co, Mo, Ni and Cu which are often found in water. Microorganisms can be placed into four nutritional types based on source of carbon, energy and electrons. The four groups are: photolithographic autotrophs, photoorganotrophic heterotrophs, chemolithotropic autotrophs and chemoorganotrophic heterotrophs. Prototrophs require light because they capture light energy with the use of chlorophyll pigments. Lithotrophs are capable of obtaining energy from oxidising inorganic chemicals and have the ability to grown and synthesise organic matter within mineral environments. Chemotrophs obtain energy from the oxidation of organic or inorganic compounds. Organotrophs use reduced organic compounds as their electron sources. Autotrophs are able to self-feed. Enzymes within the microorganism’s cells allow them to use the nutrients as long as they are provided in a specific form. Heterotrophs use reduced, preformed organic molecules (usually from other organisms) to gain carbon. Phototrops require the same nutrients as most naturally occurring members of their species. Auxotrophs are mutated and lack the ability to synthesise essential nutrients and obtain them from precursors in their environment. The basic nutrient requirements for bacteria are: carbohydrates, serum, whole blood, ascetic fluid, yeast extract, peptone and beef extract. Bacteria usually live inside their host so it is important to include elements they would have in their natural environment. Many microbes can grow in a simple solution of dry yeast extract powder dissolved in water. However, it has become clear that the chemical activities of bacteria can be affected by the presence or absence of certain nutrients in the growth medium. It is possible to use a synthetic media that is composed of pure chemicals. There are thousands of culture media recipes used for different bacteria’s nutritional needs to promote the best growth. Enrichment cultures can be used to isolate a specific type of bacteria, or to cause certain types to bloom, as the nutrients are better suited to their needs than the others. The bacteria receiving the best nutrients are able to crowd out the unwanted types of bacteria. For example: it is possible to find out which bacterias are able to obtain nitrogen gas from their environment by adding soil to the growth medium. The bacteria that are unable to obtain nitrogen gas this way do no survive, leaving only the types that can.

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Growth and Energy Generation

Microorganisms are grown in a pure culture medium. The medium contains agar which is a solidifying agent. The microorganism is isolated before being cultured. It will then grow if the medium contains the necessary nutrients and environmental conditions such as ph, oxygen, and temperature. Sterile conditions must be maintained to avoid contamination. The main components of the media are: nutrients, solidifying agent, water, additives or supplements depending on the specific microorganisms needs. Enrichment cultures are used to limit grown as they only provide nutrients and meet the environmental requirements for a specific microbe – all others would not be able to grow. Isolation mediums are used to kill undesired organisms while allowing the desired organisms to grow. Growth is limited by controlling the nutrients and conditions that the microorganism grows in. Chemical agents can be added or removed to affect growth because microorganisms react to the absence or presence of chemicals and it is possible to work out which are needed to ensure growth is limited where needed. Sterilisation limits growth by reducing the number of pathogens resulting in them posing no danger of disease. Disinfectants kill microbes that are on inanimate items but have little use on spores. Antiseptics control microbe growth in living tissue. Sanitizers reduce bacteria present on food-handling equipment and eating utensils. Microorganisms generate adenosine triphosphate (ATP) in various ways such as fermentation. Fermentation is used as a way to obtain energy by many anaerobes. Glucose molecules are fermented to alcohol and CO2 which leads to the creation of two ATP molecules. Anaerobes that use fermentation to obtain energy often end up with a lot of energy rich molecules that they are unable to use. Fermentation allows plants to transform solar energy into simple molecules enriched with energy (alcohol). Microorganisms use ATP to store energy for a time which they can then use later when it is needed, similar to storing cash in a savings account. Bacteria use two forms of photosynthesis to generate energy: oxygenic and non-oxygenic. The oxygenic form is found in microalgae and cyanobacteria. The non-oxygenic form is found in bacteria commonly referred to as purple bacteria. Both types of bacteria can use light as an energy source for growth, some species of both can fix molecular nitrogen, but it is the differences that result in important ecological differences. Purple bacteria can possess the ability to obtain growth energy in three ways: light, anaerobic fermentation of sugars, and aerobic respiration. Rapid growth can be sourced through the use of atmospheric N2 gas and CO2 (photoautotrophic) or through the use of simple organic compounds (photo heterotrophic). Photohetroptrophic is when light is only used as a source of energy for ATP regeneration and new cell materials are grown via organic substances within the growth medium. Oxygenic photosynthesis uses light energy to synthesise ATP and also uses light energy to furnish hydrogen atoms which are obtained from water and used to convert CO2 to organic compounds.

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Action and Effects

Many factors affect the activity of antimicrobial chemical and physical agents. Heat denatures proteins, refrigeration slows down enzymes, and freezing kills most bacteria (not all as some forms of bacteria can survive being frozen). UV light and ionizing radiation denatures DNA, membrane filtration physically removes cellular organisms, osmotic strength involves high concentrations of salt (or sugar) which deprive the cells of water which causes them to shrink or crenate. Chemical agents have effects on cell proteins, membranes, and the formation of cell walls, the structure of nucleic acid and metabolism or the cell. These are called germicides and are found in many forms. The effectiveness of chemical antimicrobial agents is affected by time, temperature, pH and concentration. It is possible to test their efficiency in three ways: compare their effectiveness to a traditional germicide, phenol. The phenol coefficient is the ratio of effective dilution rate which gives the same effect. The paper disc method involves saturating paper discs with the chemical agent and then placing the disc on an agar plate that has been inoculated with a test organism. If the chemical is effective clear zones will appear on the where the organism has been successfully inhibited. The use-dilution test involves adding a test microbe to dilutions of the chemical agent to see which dilutions remain clear after incubation, to know which dilution is most effective. Antibiotics work by attacking the microorganisms that are linked to causing disease or illness. It is important to note that they are produced to only attack specific targets rather than healthy cells. Antibiotics (derived from Latin – anti meaning against and biosis meaning life. So against life) attack problem causing bacteria. Antibiotic drugs are developed to target specific components of the microbial cells. For example: the cell wall or a metabolic pathway that would not occur in the human/animal host normally. Sulphonamides interfere with bacterial cells metabolism without damaging host body tissues. They inhibit the bacterial cells ability to create folic acid which halts nucleic acid synthesis and DNA replication within the cell and it eventually dies. Human cells cannot synthesis folic acid and therefore are not affected. One of the most common antibiotic mechanisms is blocking synthesis of the bacterial wall. Penicillin blocks carbohydrates in the peptidoglycan layer from crosslinking during cell wall formation leaving cells susceptible to swelling bursting. Important factor which limit the effectiveness of penicillin are allergic reactions and mutant cells that are now resistant to the effects of penicillin. The mutant cells are able to transform penicillin into a harmless penicilloic acid. Many strains of bacteria have become resistant to antibiotics. They have gained ways to avoid the negative effects antibiotics cause. Another factor which limits the effectiveness of antibiotics is the side effects that can be caused such as allergic reactions, organ damage, aches, rashes, and gastrointestinal distress. The disc diffusion test is used to measure how effective an antibiotic is. The effect of antibiotics on the growth of bacteria in a solid medium is measured. Test microorganisms are added to agar plates and discs with the antibiotic added to them are placed on the surface of the plate. The discs then release the antibiotic into the surrounding medium and zones of growth inhibition occur where the antibiotic proves to be successful. Standardised regression curves are used to correlate the size of the inhibition zone to the minimum inhibitory concentration of the antibiotic. It is important to test different concentrations and to keep correct records of findings. Growth patterns should be observed after at least 24-48 hours of growth time has been allowed.

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Cell Structure

The structure of Gram-positive and Gram-negative cell walls share many similar features and also have various differences. Both contain a cytoplasmic membrane and peptidoglycan but the peptidoglycan usually accounts for between 30-70% of the dry weight in Gram-positive while only accounting for less then 10% of the Gram-negative cell wall. The Gram-positive also contains teichoic acid, lipoteichoic acid and surface protein. The Gram-negative is a much more complex structure including periplasm, porin, LPS, phospholipid, and periplasmic protein. Capsules are found in certain pathogenic bacterial cells and offer protection against the cells found in the immune system. Endospores are dormant forms of cells that are produced by certain bacteria such as Bacillus and Clostridium. Endospores are highly resistant to temperature and pH extremes. They are also resistant to other negative environmental factors. They have the ability to germinate into new bacterial cells when conditions become more favourable to growth. Bacterial cells are generally very small and are usually either rod shaped, spherical or curved. They can be found to form groups such as pairs, chains, sheets or a packet of irregular aggregate shapes. It is important to use an electron microscope to be able to view the detailed structure of the cell. The typical cell contains a nucleoid (bacterial chromosome), ribosomes, and inclusions that are usually associated with the storage of nutrients. The cell wall gives the bacterial cell its shape and contains the plasma membrane which surrounds the cytoplasm. Cytoplasm is a thick soup like mixture of carbohydrates, proteins, lipids and inorganic salts. Ribosomes are found within the cell’s cytoplasm. They are made up of RNA and protein. Protein synthesises within the ribosomes. The plasma membrane is semipermeable and allows certain substances to travel in and out of the cell while holding the majority of the contents in their place within the cell. Ions and molecules can be transported across the lipid bilayer of the cell membrane. Transport systems tend to be highly specific which means that only one class of molecule, or a very similar class of molecules, can be transported at a time. Therefore there is a need for many different transport proteins to ensure that molecular transport between the inside and outside of the cell is regulated. Passive transport occurs when the membrane proteins move materials from a higher concentration area to a lower one. Hydrophilic (water loving) solutes are able to enter or leave a cell with no energy required by the cell for the move. Active transport membrane proteins act more like pumps and require energy to move molecules in and out of the cell against the concentration gradient. The larger molecule, such as protein and certain solutes, are moved using active transport as they are unable to freely diffuse across the membrane.

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Tests

The carbohydrate fermentation test is used to determine bacteria’s ability to ferment a particular carbohydrate. Acid production is identified by a colour change in a pH indicator (phenol red) included in the medium. A specific hydrolytic enzyme must be produced for the bacteria to utilise disaccharides like lactose and sucrose. After that resulting monosaccharide are fermented. The methyl red test (mixed fermentation) is used to determine bacteria’s ability to ferment glucose through mixed-acid fermentation. Significant amounts of organic acids are found in products of mixed-acid fermentation of glucose. The organic acids lower pH of the medium to below five and so the pH indicator, methyl red, remains red when the pH is 4.5 and lower which is a positive test. But it will change to orange or yellow (a negative test) when the pH level is higher. The oxidation-fermentation (OF) glucose test is used to determine if fermentation or aerobic respirations are used by a gram-negative bacterium to utilise a particular sugar to be used in sugar production.

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References Brock, Thomas D., Brock, Katherine M., Ward, David M. (1986). Basic Microbiology with Applications (Third Edition). New Jersey: Prentice-Hall.

Gest, Howard (2003). Microbes: An Invisible Universe. Washington: Asm Press.

Hogg, Stuart (2005). Essential Microbiology. England: John Wiley & Sons Ltd.

Sharma, Kanika (2007). Manual of Microbiology: Tools and 	Techniques. India: Ane Books.