unit-1-Nutritional-Requirements-raw-materials-used-for-culture.pptx

Nutritional Requirements

1. Nutritio n al Requireme n ts of Bacteria

2. Contents Nutritional Requirements C hemi c al Re q u ir e ments Phy s i c a l Re q u ir e ment s for Grow t h Temperature pH O s mo t i c P r e ss ur e G a s e ous R e quirement C u ltur e Med ia & Type Ra w mat e r i als u s ed f o r c u ltu r e me d ia Phy s i c a l p a ram e ters for grow t h Cultivation of anaerobes

3. What is m i crobi a l growth? Microbial growth refers to an increase in cell number(population), not in cell size. Bacteria grow and divide by binary fission , a rapid and relatively simple process. Requirement for optimum Microbial Growth are- Nutritional Requirement Favora bl e Physi c al C o nd iti o n

4. Nutritional Requirements: Nutrients are the chemical requirement essential for the growth of microbes. Extreme diversity is observer in bacteria and nutritional requirement varies widely. Therefore great difference in composition of culture media is there. All micro-organism require a source of energy , source of electron and source of carbon for their growth and development. E s sential chemica l re qu i red are carbon , nitro gen, sulphur, phos p horu s, ox y gen, m e tal ions an d vi t am ins.

5. Sou rce of Energy Li g ht Photo t roph In org a nic Photo lit h otrophs Organic Photo org a notrop h s Chemical c o m p o u nd Chemotroph In org a nic Chemo lithotrophs Organic Compound Chemo organotrophs S ou r c e of E le c tron Nutritional Requirements:

6. Nutritional Requirements: Autotrophs make their own food by using sunlight, carbon dioxide, and wa ter to fo r m s u gars w h ich they ca n use f or en er g y . Heterotrophs can not make their own food by using sunlight and therefore depend on other for carbon source. Chemoheterotrophs : Obtain carbon from their energy source form lipids, proteins, and carbohydrates. Chemoautotrophs and Photoautotrophs : Obtain carbon from carbon dioxide. Facultative Autotrophs Fa st i dious Heter o trophs

7. http://www.simplynotes.in/microbiology/nutritional-types-of-microorganisms/ Nutritional Requirements:

8. Chem i ca l Requ i rem e nt s - Carbon M a ke s up 50% of dry w e ight of ce l l and Struc t ura l backbone of a ll organic compounds. Obtain carbon from their energy source: lipids, proteins, and carbohydra tes or fro m carbo n dioxide.

9. Chem i ca l Requ i rem e nt s - Ni t rogen M a ke s up 1 4 % of dry ce l l w e ight. Use d to f o rm a min o acids, DN A , and RNA. Sources of nitrogen: Protein, Ammonium & Nitrogen gas (N 2 ) Important nitrogen fixing bacteria , live free in soil or associated with legumes (peas, beans, alfalfa, clover, etc.). Legume cultivation is used to fertilize soil naturally. Nitrates: Salts that dissociate to give NO 3 -

10. Chemica l Requiremen t s - Sulphu r a nd Phosphorus Sulfur : Used to for synthesis of amino acid, proteins and some vit a min s ( t hiamin and bio t in). So u rc e s of su lfu r is Protein, Hydrogen sulfide. Sulfates: Salts that dissociate to give SO4 2- Phosphorus : Used to for nucleotides in DNA, RNA, ATP, and phospholipids. Sources: Mainly inorganic phosphate salts and buffers Other Elements : Potassium, magnesium, and calcium are often required as enzyme cofactors. Calcium is required for cell wall synthesis in Gram positive bacteria.

11. Chem i ca l Requ i rem e nt s - Tr a c e Elem e nts Act as cofactors in various enzymes . Commonly found in tap water. Iron Copper Molybdenum Zinc Mn Ni B Co

12. Physical Requirements for Growth Temperature pH O s mo t ic P r es s ure Gaseous Re q uirement

13. Psychrophiles: “Cold-loving”- grow at o C or low. Optimum growth at 15 o C or below. Found in very cold environments (North pole, ocean depths). Seldom cause disease or food spoilage. Mesophiles : “ Middle loving”. Most of the bacteria lie in this group. Include most pathogens and common spoilage organisms. Optimum temperature commonly 37 o C. Many have adapted to live in the bodies of animals. Thermophiles : “Heat loving” Optimum growth temperature lie between 50 to 60 o C. Adapted to live in hot springs, compost piles, and sunlit soil. Some thermophiles form extremely heat resistant endospores. Extreme Thermophiles (Hyperthermophiles): can growth at 80 o C or higher. Archaebacteria. Most live in volcanic and ocean vents. Physical Requirements for Growth: Temperature

14. Physical Requirements for Growth: pH Most ba cteri a p re f e r neutral p H ( 6 . 5 - 7.5) Molds and yeast grow in wider pH range, but prefer pH between 5 and 6. 4 Acidity inhibits most microbial growth and is used frequently for food preservation (e.g.: pickling). Alkalinity inhibits microbial growth, but not commonly used for food preservation. Acidic products of bacterial metabolism interfere with growth. B u f f er s ca n be used to stabilize pH.

15. Acidophiles : “Acid loving”. Can grow at very low pH (0.1 to 5.4) Lactobacillus produces lactic acid, tolerates mild acidity. Neutrophiles : optimum pH for grow is 5.4 to 8.5. Includes all human pathogens. Alkaliphiles : “Alkali loving”. Can grow at alkaline pH (7 to 12 or higher) Vibrio cholerae and Alkaligenes faecalis optimal pH 9.Soil bacterium Agroba cteriu m grows a t p H 12. Phys i ca l Requi r emen t s fo r Growth: pH

16. Physical Requirements for Growth : Gaseous requirement Aerobic Bacteria- require oxygen for growth Ob l igate aerobe s - grow on l y in the pr e se n c e of oxygen Eg. Chol e ra bacillus. Anaerobic B acteria Facultative anaerobes- are ordinarily aerobic but can grow in the a b se n c e of ox y ge n . Obligate anaerobes ma y e ve n die on exposure to ox y ge n . Microaerophilic bacteria are those that grow best in the presence of lo w oxygen ten s ion.

17. Why are an a erobes k ill e d b y oxygen? Sing l et Oxygen: Extremely reactive f o rm of ox y ge n . Superoxide Free Radicals (O 2 - . ): is extremely toxic and reactive form of oxygen and can inactivate vital cell components. All organisms gro w ing have an enzym e supe r oxide dismu t a se (SOD), to get rid of them. SOD is made by aerobes, facultative anaerobes, and aerotolerant anaerobes, but not by anaerobes or microaerophiles. Reaction: SOD • O 2 - . + O 2 - . + 2H + ----- > H 2 O 2 + O 2

18. Why are anaerobes kille d by o x y g en? Hydrogen Peroxide (H 2 O 2 ): Peroxide ion is also toxic and the active ingredient of several antimicrobials (e.g. benzoyl peroxide). There are two diffe rent enzymes that brea k down hydro g en perox i de Catalase : converts hydrogen peroxide into water and O 2 . Commonly produced by humans, as well as many bacteria. Catalase 2 H 2 O 2 --------- - > 2H 2 O + O 2 . Peroxidase : Converts hydrogen peroxide into water. Peroxidase H 2 O 2 + 2H + --------- - > H 2 O

19. Physic al R equirem e nts fo r Growth: Osmot i c Pressure Halophiles: Require moderate to large salt concentrations. Ocean water contains 3.5% salt. Most bacteria in oceans. Extreme or Ob l igate Halo p hi l e s : R e quire v e ry hi g h salt concentrations (20 to 30%). Bacteria in Dead Sea, brine vats. Facultative Halophiles: Do not require high salt concentrations for gro w th, b u t t o l e ra t e 2 % sa l t or m o re.

20. Cultur e me d ia It is c h emically defined system containin g nutrients p r epared f or microbial growth in the laboratory. Cult u re medi a mus t be steri l e and s h ould co n t a in a p p ro p riate nutrients. It must be incubated at appropriate temperature at which microbe s that grow and m u ltip l y.

21. Cultur e me d ia: Types Chemically Defined Media - Nutrient material whose exact chemical composit i on is kno w n Complex Media -Nutrient material whose exact chemical composition is not known. Made of extracts from yeast, meat, plants, protein digests, etc. and composition may vary slightly from batch to batch. Widely used for heterotrophic bacteria and fungi. Energy, carbon, nitrogen, and sulfur requirements are primarily met by protein fragments (peptones). Vitamins and organic growth factors provided by meat and yeast extracts. Two forms of complex media: Nutrient broth: Liquid media Nutrien t agar: Soli d media

22. Cultur e me d ia: Types Nutrient Broth(Liquid Culture media) It is Liquid culture media Nutrient Agar(Solid culture media) It contains a solidifying agent agar for preparing agar plates, agar slants. Agar melts above 95 o C and once melted, does not solidify until it reaches 40 o C. It cannot be degraded by most bacteria. Selective Media : Used to encourage the growth of particular microbes and suppress the growth of unwanted bacteria. Saboraud’s Dextrose Agar: pH of 5.6 discourages bacterial growth. Used to isolate fungi. Brilliant Green Agar: Green dye selectively inhibits gram-positive bacteria. Used to i s ola t e gram - negative Sa l m o nella . Bismuth Sulfite Agar: Used to isolate Salmonella typhi. Inhibits growth of most other bacteria.

23. Cultur e me d ia: Types Differential Media : Used to distinguish colonies of a desired organism. Blood Agar : Used to distinguish hemolytic bacteria that destroy red blood cells (hemolysis)from nonhemolytic bacteria. Hemolysis appears as an area of clearing around c o lon y . Examp l e : Stre p t o c o c c u s p y o g e n es. Selective and Differential Media : Used for to distinguish colonies of a desired organism and also i n hibit the gro w th of other micro b es. Mannitol Salt Agar : Used to distinguish and select for Staphylococcus aureus. High salt (7.5% NaCl) discourages growth of other organisms. pH indicator changes color when mannitol is fermented to acid. MacConkey Agar : Used to distinguish and select for Salmonella . Bile salts and crystal violet discourage growth of grampositive bacteria. Lactose plus pH indicator: Lactose fermenters produce pink or red colonies, nonfermenters are colorless.

24. Cultur e me d ia: Types Enrichment Culture : favor the growth of a microbe that may be found in very small numbers. Unlike selective medium, does not necessarily suppress the growth of other microbes. Used mainly for fecal and soil samples. After incubation in enrichment medium, greater numbers of the organisms, increase the like l ihood of positiv e id e n ti f ic a tion. Assay Media -used to assay for vitamin, amino acid,antibiotics Media for Enumeration -used to determine bacterial count in water, milk etc Media for characterization Maintenance Me d ia

25. Culture Media for Anaerobic Bacteria : anaerobes are being killed in p r es e nce of ox y ge n . Reducing media contain ingredients that chemically combine with oxygen and r emove it fro m the medium . Exampl e : Sodium thioglycolate. Tubes are heated shortly before use to drive off ox y ge n . P l a t es m u st be grown in ox y gen fre e containers ( a naerobic chambers). Cultur e me d ia: Types

26. Ra w material s fo r cultur e me d ia N u trie n t Broth P e p t on e - 10 g m B e e f Extrac t - 10 g m So d i um Chlorid e - 5 g m Distilled Wated-1000 ml Nutrient Agar- Same composition as Nutrient broth but also contain agar (1-2%). Peptone contributes organic nitrogen in the form of amino acids and long-chained fatty acids. Beef Extract provides additional vitamins, carbohydrates, salts and other organic nitrogen compounds Sodium chloride is used to provide electrolytes and maintain the osmotic balance Water contributes as source of hydrogen and oxygen

27. Meat extract, yeast extract – Protein degradation products/carbohydrates/Inorganic salts/Growth factors. Blood – It e n ri c he s media Ra w material s fo r cultur e me d ia

28. Cultivatio n of Aerobic Bacteria: Smal l Scale Large Scale https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/G eneral_Biology_Labs/Biology_Labs_(under_construction)/Microbiology/Reading%3A_ Prokaryotes https://www.shutterstock.com/image-photo/flask-bacterial-culture- microbiology-laboratory-difference-564005815

29. Cultivation of Anaerobes: Pre-reduced Media Anaerobic Cha m ber Anae r obic J ars (or Gas P ak Anae r obic System) Oxyplates-

30. Cultivation of Anaerobes: Pre-reduced Media e l iminati o n of oxy g en fro m the c ult u re m e di um is the simp l est m e thod. The li q uid c u ltur e m e di um is b o il e d by hol d ing fo r 10 mi n utes to d r iv e off mo s t of the di s s olved oxygen R ed uc ing a g ent like c ys t eine 0.1%, a s c o rbic a c id 0.1%, sodium thioglyc o l l ate 0.1% etc is ad d ed to f u rth er lowe r t he oxygen c o nte nt. Oxygen - f r ee N 2 is b u b b l e d thr o u gh the m e di um to maintai n anae r o b ic c o n d ition. https://www.biologydiscussion.com/microorganisms/culture-media-for-cultivation-of-anaerobic-bacteria-4-types/55049

31. Cultivation of Anaerobes: Anaerobic Chamber Anaerobic chamber is an ideal anaerobic incubation system, wh i ch p rovide s ox y g e n - fr e e e nvironme nt It is a plastic anaerobic glove box that contains an a t m o s phere of H 2 , C O 2 , a nd N 2 Glove ports and rubber gloves are used by the operator to perform activities in the chamber. The re i s a n a i r - l o ck with i nn e r a nd outer door s . Air of the cha mb e r i s r e move d b y a v a cuum pump conne ct i on a nd r e p l a ced wi t h N 2 c i rcul a t o r f i t t ed i n t he m a i n ch a mbe r c i rcul a tes the g a s atmosphere pellets of palladium catalyst remove any residual O 2 present in the culture media by reaction with H 2

32. Cultivation of Anaerobes:Anaerobic Jars It is a cylin d rical vess e l mad e of glass or meta l with a meta l li d , which is held firmly in plac e by a clamp gauze sachet carr y palla d ium pellet s act as a catal y st fo r t h e conversio n of hy d rogen and ox y gen into wa t er. https://www.biologydiscussion.com/microorganisms/culture-media-for-cultivation-of-anaerobic-bacteria-4-types/55049

33. New tec h ni q ue used f o r iso l a t ion of anaerobes in labora t o ries Us e of Oxyra se enzym e that reduce O 2 into water There is a sealing ring in the lid of the plate Cultivatio n of Anaerob e s: Oxyplates

34. References Pelczar, M. J., E. C. S. Chan, and N. R. Krieg. "Microbiology. International edition." 1996 ;Tata McGraw Hill Inc. Page No-133-148 Kar, A; “Pharmaceutical Microbiology” New Age International Publisher,2008 Page no-149 Carter, S. J. "Tutorial Pharmacy. Cooper and Gunn’s." (1999);CBS Publis h er and Distrib u ti o n.