Nutritional requirement and growth factors.pptx

detailed knowledge of nutritional requirements and growth factors and culture medias for microorganisms

1. UNIT I 1.3 Nutritional requirement and Growth factors Presented by: Mohammad Abuzar( M. Pharm ) Assistant Professor School of Pharmacy AIKTC, New Panvel .

2. CONTENTS 2

3. 3 Introduction Nutrients are substances used in biosynthesis and energy production and therefore are required for microbial growth Environmental factors such as temperature, oxygen levels, and the osmotic concentration of the medium are critical in the successful cultivation of microorganisms Common nutritional requirements

4. 4 Element % Source Function Carbon 50 Organic compounds or CO2 Main constituent of cellular material Oxygen 20 H2O, organic compounds, CO2 and O2 Constituent of cell material and cell water, O2 is electron acceptor in aerobic respiration Nitrogen 14 NH3, NO3, organic compounds, N2 Constituent of amino acids, nucleic acids nucleotides, and coenzymes Hydrogen 8 H2O, organic compounds, H2 Main constituent of organic compounds and cell water Phosphorus 3 inorganic phosphates (PO4) Constituent of nucleic acids, nucleotides, phospholipids, LPS, teichoic acids Potassium 1 Potassium salts Main cellular inorganic cation and cofactor for certain enzymes Magnesium 0.5 Magnesium salts Inorganic cellular cation, cofactor for certain enzymatic reactions Sulphur 1 Organic sulfur compounds Constituent of cysteine, methionine, glutathione, several coenzymes Calcium 0.5 Calcium salts Inorganic cellular cation, cofactor for certain enzymes and a component of endospores Iron 0.2 Iron salts Component of cytochromes and certain non-heme iron-proteins and a cofactor for some enzymatic reactions

5. 5 Nutritional Types of Microorganisms

6. 6 Growth factors There are three major classes of growth factors: Amino acids Needed for protein synthesis Purines and pyrimidines Needed for nucleic acid synthesis Vitamins Needed as coenzymes and functional groups of certain enzymes Some bacteria (e.g. E. coli) do not require any growth factors: they can synthesize all essential purines, pyrimidines, amino acids and vitamins, starting with their carbon source, as part of their own intermediary metabolism. Certain other bacteria (e.g. Lactobacillus) require purines, pyrimidines, vitamins and several amino acids in order to grow. These compounds must be added in advance to culture media that are used to grow these bacteria.

7. 7 The physical factors affects the growth of microorganism. 1) Temperature Temperature is the most important factor that influences the rate of enzyme catalysed reactions and rate of growth. For every organisms there is an optimum temperature for growth and minimum temperature for inhibiting the growth. Most extreme the microbes need liquid water to grow. (330C). some algae and fungi grow at 55-60 degree C. Prokaryotes are grow at 100 degree.

8. 8 i ) Psychrophiles They are grow at zero degree celsius . They isolated from arctic and antarctic habitats. Because 90% is ocean. The cell membrane of psychrophilic microorganisms have high levels of unsaturated fatty acid and remain semifluid when cold. If temperature is more than 15 degree celcius the cell membrane will disrupts. Eg : Pseudomonas vibrio ii) Mesophiles They are grow at 20-45 degree C. Almost all human pathogens are mesophiles. Eg : cyanobacteria (Nostoc, Anabaena, Oscillatoria etc.) iii)Thermophiles They are grow at 45-65 degree C. They have more heat stable enzymes and protein synthesis system able to function at high temperature. Eg : Thermus aquaticus. iv)Hyper thermophiles They are grow at 80-100 degree celcius . They found in the hot areas of sea floor. Eg : Pyrococcus abysi

9. 9 2) Oxygen requirements i ) Aerobic bacteria → they require O2 for growth. Eg : pseudomonas. ii)Anaerobic bacteria → they do not need O2 for growth. Eg : Streptococcus pyogenes iii) Facultative anaerobic bacteria → they can grow in presence or absence of oxygen. Eg : Yeast. iv) Microaerophilic bacteria → they require low level of oxygen. The high level of O2 is toxicant to the microbes. Eg : campylobacter 3)PH Based on pH microorganisms are classified into three. i ) Acidophile They can survive at pH 0 and 0.5 Eg : picrophilus . ii) Neutrophile They can survive at Ph 5.5 & 8.0 Eg : E. Coli iii) Alkalophile They can survive at Ph 8.5 & 11.5 Eg : nitrobacteria.

10. 10 4) Light The photosynthetic microorganisms such as cyanobacteria, microalgae, and photosynthetic bacteria need light for energy production. 5) Salt concentration & water activity Based on these factors the microbes are classified into 2. Halphile They require high level of Nacl to grow. Eg : halobacterium 2) Osmophile They require wide range of water activity. Eg : Stephallococcus aureus Culture media A culture medium is a solid or liquid preparation used to grow, transport, and store microorganisms Specialized media are essential in the isolation and identification of microorganisms, the testing of antibiotic sensitivities, water and food analysis, industrial microbiology, and other activities Knowledge of a microorganism’s normal habitat often is useful in selecting an appropriate culture medium

11. 11 Defined and complex media Synthetic or Defined Media A medium in which all components are known is a defined medium or synthetic medium. Many chemoorganotrophic heterotrophs also can be grown in defined media with glucose as a carbon source and ammonium salt as a nitrogen source. Defined media are used widely in research, as it is often desirable to know what the experimental microorganism is metabolizing Complex Media Contain some ingredients of unknown chemical composition May be sufficiently rich and complete to meet the nutritional requirements of many different microorganisms and contain undefined components like peptones, meat extract, and yeast extract Three commonly used complex media are Nutrient broth, Tryptic soy broth, MacConkey agar

12. 12 Types of media General purpose media tryptic soy broth and tryptic soy agar are called general-purpose media because they support the growth of many microorganisms Enriched media Blood and other special nutrients may be added to general-purpose media to encourage the growth of fastidious heterotrophs. These specially fortified media (e.g., blood agar) are called enriched media. Enrichment media If the sample contains more than one bacteria, the undesired bacterial growth can be reduced by the addition of certain media components . The desired organism is facilitated to grow.

13. 13 Selective media Favor the growth of particular microorganisms Bile salts or dyes like basic fuchsin and crystal violet favor the growth of gram-negative bacteria by inhibiting the growth of gram-positive bacteria without affecting gram-negative organisms. Examples: Endo agar, eosin methylene blue agar, and MacConkey agar Differential media Media that distinguish between different groups of bacteria Permit tentative identification of microorganisms based on their biological characteristics Examples – Blood agar is both a differential medium and an enriched one MacConkey agar is both differential (crystal violet & bile salts) and selective (lactose & neutral red dye) Blood agar media

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15. 15 Microbial growth   Growth is defined as an increase in cellular constituents and may result in an increase in a microorganism’s size, population number, or both Techniques can be used to study microbial growth - following changes in the total cell number, the population of viable microorganisms, or the cell mass In the natural environment, growth is often severely limited by available nutrient supplies and many other environmental factors. The growth   Growth may be defined as an increase in cellular constituents. It leads to a rise in cell number when microorganisms reproduce by processes like budding or binary fission.

16. 16 Batch culture and continuous culture   Batch culture When microorganisms are grown in a closed system, population growth remains exponential for only a few generations and then enters a stationary phase due to factors such as nutrient limitation and waste accumulation. Continuous culture In an open system with continual nutrient addition and waste removal, the exponential phase can be maintained for long periods

17. 17 The growth curve   The growth of microorganisms reproducing by binary fission can be plotted as the logarithm of the number of viable cells versus the incubation time. The resulting curve has four distinct phases

18. 18 Bacterial growth curve Lag Phase When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs Cell division does not take place right away and there is no net increase in mass, the cell is synthesizing new components   Reasons for lag phase The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized before growth can begin The medium may be different from the one the microorganism was growing in previously. Here new enzymes would be needed to use different nutrients Injured microorganisms require time to recover The cells retool, replicate their DNA, begin to increase in mass, and finally divide Duration of lag phase depends on the condition of the microorganisms and the nature of the medium.

19. 19 Exponential Phase Microorganisms are growing and dividing at the maximal rate possible Depends on their genetic potential, the nature of the medium, and the conditions under which they are growing Rate of growth is constant Each individual divides at a slightly different moment, the growth curve rises smoothly rather than in discrete jumps Uniform in terms of chemical and physiological properties Stationary Phase Population growth ceases and the growth curve becomes horizontal The total number of viable microorganisms remains constant Balance between cell division and cell death, or the population may simply cease to divide though remaining metabolically active Reasons for stationary phase Nutrient limitation - if an essential nutrient is severely depleted, population growth will slow Aerobic organisms often are limited by O2 availability Accumulation of toxic waste products.

20. 20 Death Phase Detrimental environmental changes † Nutrient deprivation and the buildup of toxic wastes † Decline in the number of viable cells † Death phase The death of a microbial population, is usually logarithmic The total cell number remains constant because the cells simply fail to lyse after dying The only way of deciding whether a bacterial cell is viable is by incubating it in fresh medium; if it does not grow and reproduce, it is assumed to be dead. Death is defined to be the irreversible loss of the ability to reproduce

21. 21 The Continuous Culture of Microorganisms Growing microorganisms in an open system, a system with constant environmental conditions Continual provision of nutrients and removal of wastes A microbial population can be maintained in the exponential growth phase and at a constant biomass concentration for extended periods Two major types of continuous culture systems commonly are used: (1) Chemostat method In a chemostat, the nutrients are continuously supplied at a constant flow rate, and the density of the cells is adjusted according to the supplied essential nutrients for growth. In a chemostat, the growth rate is determined by adjusting the concentration of substrates like carbon, nitrogen, and phosphorus (2) Turbidostat method Cell growth is controlled and remains constant in this system, but the flow rate of fresh media varies. Cell density is controlled based on the set value for turbidity, which is created by the cell population while fresh media is continuously supplied.

22. 22 Chemostat Turbidostat

23. 23 Nutrients are substances used in biosynthesis and energy production and therefore are required for microbial growth Major elements required for growth - carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, and iron. Minor elements - manganese, zinc, cobalt, molybdenum, nickel, and copper Nutritional types of bacteria – Autotrophs, Heterotrophs, Phototrophs, Chemotrophs, Lithotrophs, Organotrophs Growth factors - Amino acids, Purines and pyrimidines, Vitamins A culture medium is a solid or liquid preparation used to grow, transport, and store microorganisms Growth may be defined as an increase in cellular constituents. It leads to a rise in cell number when microorganisms reproduce by processes like budding or binary fission. Organisms grown in a closed culture with limited nutrients – batch culture Continuous supply of nutrients and removal of waste – continuous culture Phases of bacterial growth curve – lag, log, stationary, and decline Continuous culture can be obtained using chemostat or Summary

24. 24 W.B. Hugo and A.D. Russel: Pharmaceutical Microbiology, Blackwell Scientific publications, Oxford London. Prescott and Dunn., Industrial Microbiology, 4th edition, CBS Publishers & Distributors, Delhi. Pelczar , Chan Kreig , Microbiology, Tata McGraw Hill edn . Malcolm Harris, Balliere Tindall and Cox: Pharmaceutical Microbiology. Rose: Industrial Microbiology. Probisher , Hinsdill et al: Fundamentals of Microbiology, 9th ed. Japan Cooper and Gunn’s: Tutorial Pharmacy, CBS Publisher and Distribution. Peppler : Microbial Technology. I.P., B.P., U.S.P.- latest editions. Ananthnarayan : Text Book of Microbiology, Orient-Longman, Chennai Edward: Fundamentals of Microbiology. 12. N.K.Jain : Pharmaceutical Microbiology, Vallabh Prakashan , Delhi REFERENCES

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