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Nutritional requirement by microorganisms
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- 2. Nutrients are substancesused in biosynthesis and energy release and therefore are required for microbial growth INTRODUCTION
- 3. Common Nutrient Requirements The first six (C, O, H, N, S and P) : components of carbohydrates, lipids and proteins K+ : enzyme -protein synthesis Ca2+ : heat resistance of bacterial endospores Mg2+ : cofactors, stabilise the cell membrane and ribosomes Fe2+ and Fe3+ : part of cytochrome and cofactors for enzymes and ECP 95% cells dry weight Carbon Oxygen Nitrogen Phosphorous Calcium Hydrogen Potassium Magnesium Sulphur Iron Macroelements/ Macronutrients
- 4. Micronutrients/ Traceelements : Several nutrients which are required in small amounts Ubiquitous and normally part of enzymes and cofactors Aid in catalysis of reaction and maintenance of protein structure Mn2+: transfer of phosphate groups Mo2+ : nitrogen fixation Co2+ : component of vitamin B12 Hence microorganisms require a balanced mixture of nutrients Micro nutri ents Manganese Zinc Cobalt Molybdenum Nickel Copper
- 5. Requirements for Carbon,Hydrogen, Oxygen and Electrons All organisms need carbon, oxygen, hydrogen and source of electrons CARBON : Skeleton/backbone of all organic molecules from which the organism is built HYDROGEN & OXYGEN: Important elements found in the organic molecules ELECTRONS Electron transport chain and the oxidation reduction reactions- energy for use in cellular work Reduce molecules during biosynthesis (CO2 organic molecules) reduced
- 6. AUTOTROPHS : useonly CO2 as a sole source of carbon obtain energy from light or reduced inorganic molecule Because CO2 cannot supply their energy needs, they must obtain energy from other sources, such as light or reduced inorganic molecules HETEROTROPHS : organisms that use reduced, preformed organic molecules as their carbon source Heterotrophic microorganisms has extraordinary flexibility with respect to carbon sources Actinomycetes : common soil bacteria degrade the paraffin, amyl alcohol and even rubber Burkholderia cepacia : use 100 different carbon compounds Leptospira : use long chain fatty acids as the major source of carbon
- 7. Nutritional Types ofMicroorganisms Source of Carbon, Energy and Electrons CARBON SOURCES Autotrophs CO2 sole or principle biosynthetic carbon source Heterotrophs Reduced, performed, organic molecules from other organisms ENERGY SOURCES Phototrophs Light Chemotrophs Oxidation of organic or inorganic compounds ELECTRON SOURCES Lithotrophs Reduced inorganic molecules Organotrophs Organic molecules Microorganisms can be classified as autotrophs or heterotrophs on the basis of the preferred source of carbon. There are two sources of energy available to organisms Light energy Energy derived from oxidising organic or inorganic molecules PHOTOTROPHS : Use light as energy source CHEMOTROPHS : energy obtained from oxidation of chemical compound LITHOTROPHS (rock eaters) : Reduced inorganic substance as energy source ORGANOTROPHS : extract electrons from reduced organic source Majority of the microorganisms studied so far are Photolithotrophic autotrophs or chemoorganotrophic heterotrophs
- 8. Major nutritional typesof microorganisms NURTIONAL TYPE CARBON SOURCE ENERGY SOURCE ELECTRON SOURCE REPRESENTATIVE MICROORGANISMS Photolithography CO2 Light Inorganic e- donor Purple and green sulfur bacteria, cynobacteria Photoorganoheterophy Organic carbon Light Organic e- donor Purple nonsulfur bacteria, green nonsulfur bacteria Chemolithoautotrophy CO2 Inorganic chemicals Inorganic e- donor Sulfur, iron, hydrogen- oxidizing bacteria, methanogens Chemolithoheterotrophy Organic carbon Inorganic chemicals Inorganic e- donor Some sulfur oxidizing bacteria Chemoorganoheterotrophy Organic carbon Organic chemicals Organic e- donor Fungi, protists and many archaea
- 9. Phototrophic bacteria playimportant roles in aquatic ecosystems, where they can cause blooms (a) A cyanobacterial and an algal bloom in a eutrophic pond (b) Purple sulfur bacteria growing in a bog (c) A bloom of purple sulfur bacteria in a sewage lagoon Phototrophic Bacteria
- 10. Chemolithotrophic Bacteria (a)Transmission electronmicrograph of Nitrobacter winogradskyi, an organism that uses nitrite as its source of energy (b) Light micrograph of Beggiatoa alba, an organism that uses hydrogen sulfide as its energy source and organic molecules as carbon sources. The dark spots within the filaments are granules of elemental sulfur produced when hydrogen sulfide is oxidized
- 11. Requirements for Phosphorus,Nitrogen and Sulfur Nitrogen : synthesis of amino acids, purines, pyrimidines, some carbohydrates and lipids, enzyme cofactor Some incorporate ammonia directly through the action of enzymes such as glutamate dehydrogenase or glutamine synthetase and glutamine synthase Phototrophs and chemotrophic microbes Nitrogen Fixation : Variety of bacteria (Cyanobacteria, Symbiotic bacterium Rhizobium) Assimilate atmospheric nitrogen (N2) by reducing it to ammonium Nitrate Ammonia Incorporated Assimilatory nitrate reduction
- 12. Phosphorus Phosphorus presentin nucleic acid, phospholipids, nucleotides (ATP), cofactors, some proteins and other cell components Use of inorganic phosphate as phosphorous source which is directly incorporated Aquatic environments : Low phosphate levels limits microbial growth E.coli uses organic and inorganic phosphate Organophosphate Hexose 6-phosphate Taken up directly by the cell Others Inorganic Phosphate Transported across plasma membrane
- 13. Sulfur Sulfur Amino acids Biotin Carbohy drates Thiamine Microorganisms usesulfate as a source of sulfur Reduce it by assimilatory sulfate reduction Reduced form of sulfur such as cysteine is also used
- 14. Growth Factors Organiccompounds that are essential cell components or precursors of such components but cannot be synthesised by the organism Protein synthesis Nucleic acid synthesis All or some part of enzyme cofactors Vitamin Functions Examples Biotin One carbon metabolism, CO2 fixation Leuconostoc mesenteroids Saccharomyces cerevisiae Acanthamoeba castellanii Folic acid One carbon metabolism Tetrahymena spp. Enterococcus fecalis Riboflavin (B2) Precursor of FAD & FMN Caulobacter vibriodes Dictyostellium spp. Thiamine (B1) Aldehyde group transfer Bacillus anthracis Colpidium campylum Ochromonas malhamensis Pantothenic acid Precursor of coenzyme A Proteus morganii Paramecium spp. Functions of some common vitamins in microorganism
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- 16. A solid/liquidpreparation used to grow, transport, and store microorganisms Complex liquid media (urine, chicken/ meat broth)- Louis Pasteur Solid media (Potato surface, gelatin): Robert Koch • Gelatin melts at 24ºC • Microbes used it as a substrate Agar was first described for use in microbiology by Walter Hesse Culture Media
- 17. Culture Media Requirement s ofculture media Carbon source Energy source Nitrogen source Salts pH Growth factors Indicator s Inhibitor s Oxidatio n reductio n potential
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- 19. Liquid andsolidified media are routinely used in microbiology labs, solidified media are particularly important Both defined and complex media can be solidified with the addition of 1.0 to 2.0% agar; most commonly 1.5% is used Agar – Sulphated polymer (D-galactose, 3,6-anhydro-L-galactose, and D-glucuronic acid) Extracted from red algae Melting temperature- about 90°C and Solidifying temperature- 45°C Microbes growing on agar medium can be incubated at a wide range of temperatures Agar is an excellent hardening agent because most microorganisms cannot degrade it Other solidifying agents -silica gel is used to grow autotrophic bacteria Culture Media
- 20. Defined or Syntheticmedium • All chemical components are known in defined medium. • Can be in a liquid form (broth) or solidified by an agent such as agar • Widely used in research, as it is often desirable to know what the experimental microorganism is metabolizing • Culture photolithotrophic autotrophs (cyanobacteria and photosynthetic protists), chemoorganotrophic heterotrophs • All defined media are as simple, but may be constructed from dozens of components Culture Media Medium for Escherichia coli Amount (g/litre) Glucose 1.0 Na2HPO4 16.4 KH2PO4 1.5 (NH4)2SO4 2.0 MgSO4· 7H2O 200.0 mg CaCl2 10.0 mg FeSO4 · 7H2O 0.5 mg Final pH 6.8–7.0
- 21. Complex media Mediathat contain some ingredients of unknown chemical composition Single complex medium may be sufficiently rich to completely meet the nutritional requirements of many different microorganisms The nutritional requirements of a particular microorganism are unknown, and thus a defined medium cannot be constructed Undefined components like peptones, meat extract, and yeast extract Nutrient broth, tryptic soy broth, and MacConkey agar Culture Media Tryptic Soy Broth Amt (g/ltr) Tryptone (pancreatic digest of casein) 17 Peptone (soybean digest) 3 Glucose 2.5 Sodium chloride 5 Dipotassium phosphate 2.5
- 22. General purpose mediaor supportive media: they sustain the growth of many microorganisms. Ex: tryptic soy broth and tryptic soy agar Enriched media: Blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious microbes. These specially fortified media (e.g., blood agar) Selective media: favour the growth of particular microorganisms Differential media: are media that distinguish among different groups of microbes and even permit tentative identification of microorganisms based on their biological characteristics (e.g., blood agar: hemolytic and non-hemolytic bacteria) Functional types of media
- 23. Functional types ofmedia (a) Blood agar culture of bacteria from the human throat (b) Chocolate agar, an enriched medium used to grow fastidious organisms such as Neisseria gonorrhoeae The brown color is the result of heating red blood cells and lysing them before adding them to the medium It is called chocolate agar because of its chocolate brown color a b
- 24. Pure culture:a population of cells arising from a single cell, to characterize an individual species. Pure culture techniques were developed by Robert Koch Few common approach's to prepare the pure culture are The spread plate and streak plate The pour plate Isolation of Pure Cultures
- 25. The spreadplate is an easy, direct way of achieving this result. The dispersed cells develop into isolated colonies Isolation of Pure Cultures Spread-Plate Technique dilute microbial mixture (30 to 300 cells) transferred centre of an agar plate a sterile bent-glass rod spread evenly over the surface
- 26. Spread-Plate Technique (a)The preparationof a spread plate. (1) Pipette a small sample onto the centre of an agar medium plate. (2) Dip a glass spreader into a beaker of ethanol. (3) Briefly flame the ethanol-soaked spreader and allow it to cool. (4) Spread the sample evenly over the agar surface with the sterilized spreader. Incubate. (b)Typical result of spread-plate technique.
- 27. STREAK PLATE METHOD Pure colonies also can be obtained from streak plates The microbial mixture edge of an agar plate streaked out over the surface in one of several patterns Thus this is essentially a dilution process and single colonies are developed Isolation of Pure Cultures inoculating loop or swab transferred inoculating loop is sterilized streaking the third sector After the first sector is streaked Inoculum for the second sector is obtained from the first sector
- 28. A typical streakingpattern is shown
- 29. POUR PLATE Isolation ofPure Cultures Serial dilution of the originals sample Small volume of the serially diluted sample + liquid agar (45ºC) Mixture immediately transferred into the sterile culture dishes After the agar is hardened, each cell is fixed in a place and forms a individual colony Colonies growing on the surface can be taken to prepare pure cultures
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Editor's Notes
- #20 Solidified media can be used to isolate different microbes from each other in order to establish pure cultures
- #26 Because the number of colonies should equal the number of viable organisms in the sample, spread plates can be used to count the microbial population.
- #28 Eventually, very few cells will be on the loop, and single cells will drop from it as it is rubbed along the agar surface.
- #29 Extensively used with procaryotes and fungi, a pour plate also can yield isolated colonies. Plates containing between 30 and 300 colonies are counted. The total number of colonies equals the number of viable microorganisms in the sample that are capable of growing in the medium used.