Module 3a culture and staining

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Module 3a culture and staining

  1. 1. Bacterial Staining & Culture Stains & Types of Dyes Cultivating MicroorganismsMicrobiology - HTL
  2. 2. Microbiology - HTL
  3. 3. What Organisms need to grow s Nutrition s Carbon s Oxygen s Nitrogen s Phosphorus s Sulfur s Trace elements s Organic growth factorsMicrobiology - HTL
  4. 4. Chemical requirements: s Carbon – structural backbone of living matter s Nitrogen – form the amino group of the amino acids of proteins • Nitrogen fixation – Cyanobacteria, Rhizobium s Sulfur – Synthesize sulfur containing amino acids and vitamins. • Eg. Thiamine and Biotin s Phosphorus – synthesis of Nucleic acids and the phospholipids of the cell membranesMicrobiology - HTL
  5. 5. More chemicals: s Other elements: s Trace elements • Potassium • Iron • Magnesium • Copper • Calcium • Molybdenum • Zinc s Used as cofactors for enzymesMicrobiology - HTL
  6. 6. Microbiology - HTL
  7. 7. Culture Media s Anything that possesses nutritional and environmental requirements for bacterial growth s Culture -- is a group of organisms obtained in a culture media s Colony – is a culture containing group of bacteria forming on a solid culture medium as a result of separated division of 1 or a few organismsMicrobiology - HTL
  8. 8. Media preparation s materials s weigh out s dissolve in solvents s filter to clarify s adjust pH s place in containers s sterilize & control s store & refrigerateMicrobiology - HTL
  9. 9. Ingredients of a culture media: s protein s nitrogen s carbohydrate s solidifying agents /agar & gelatin • Agar – polysaccharide extracts of seaweeds and are most commonly used as base medium s chemical substance s dyes & indicators s enriching substance – e.g. chocolate, blood, glycerine, egg, albuminMicrobiology - HTL
  10. 10. Microbiology - HTL 1
  11. 11. Types of media (consistency) s Liquid / broth - motility, transport , enrichment, biochemical tests • Eg. Thioglycolate broth, BHI, TSB, Nutrient broth s semi-solid - 0.25% agar + liquid motility, anaerobic culture, stock culture, microaerophiles, & biochemical tests • SIM, Fletcher s solid - 1-2% agar used for studying colonies • BAP, CAP, MSA, EMB, TSI, SCAMicrobiology - HTL 1
  12. 12. Types of Culture – ( specie ) s Pure culture – made up of only one pure specie s Mixed culture – made up of organisms belonging to different specie s Stock Culture – pure culture of organism used as a source of supply for industry, research or academic uses.Microbiology - HTL 1
  13. 13. Microbiology - HTL 1
  14. 14. Types of media – (composition) s Synthetic Culture Medium – exact composition is known or ingredients are known s Non-synthetic – exact composition is not known s Tissue culture medium – used for culturing living cells • Eg. Human cancer cell linesMicrobiology - HTL 1
  15. 15. Type of media – (method of dispensing or distributed) s Plated Medium – dispensed in petridishes s Tube medium – dispensed in test tubes • Slant • Butt • Butt/slantMicrobiology - HTL 1
  16. 16. Types of Media – (B ased on use) s Simple medium – supports the growth of fastidious microorganisms • Used for routine cultivation and maintenance of microorganisms – Eg. Nutrient broth, Nutrient agar s Enrichment medium – containing nutritive suplements needed for some microbes to growth – Eg. Peptone water – growth of V. choleraMicrobiology - HTL 1
  17. 17. Continued… s Enriched medium – containing nutritive supplements for growth of some microorganisms • Eg. BAP – contains Factor V ( Coenzyme – heat labile factor  Nicotinamide dinucleotide) & Factor X (Hemin  heat stable factor)Microbiology - HTL 1
  18. 18. More… s Differential Medium – distinguishes organism growing together by differences in their cultural characteristic • Eg. EMB, MCA, MSA, TCBS (Trypticase Citratr Bile Salt Agar ), SSA s Selective Medium – promotes growth of desirable organism but at the same time inhibiting the growth of others. • Used for culture of specific organismMicrobiology - HTL 1
  19. 19. Last na… s Special/Specific culture medium • Same as the purpose of selective culture medium • Used to isolate hard to isolate or grow strains • Eg. Petrognani, Lowenstein, Petroffs – for Mycobacterium tuberculosis • Thayer Martin medium – Neisseria • McBride Agar – Listeria monocytogenesMicrobiology - HTL 1
  20. 20. Microbiology - HTL 2
  21. 21. Microbiology - HTL 2
  22. 22. Types of media ( function) s Defined - Glucose s Complex - egg, blood, beef, yeast, milk s Selective - SPS agar (Clostridium) s Differential - Blood agar s Selective/differential - MacConkey agar Crystal violet / lactose s Enrichment - Nitrogen free mediaMicrobiology - HTL 2
  23. 23. Bacterial Colony: s Colonies – groups of bacteria forming on certain solid media as a result of several divisions of one or several specific type of organism s Only one type of bacteria will be found in a bacterial colonyMicrobiology - HTL 2
  24. 24. Types of colonies: s S or smooth colonies • Uniform texture and homogenecity • Forms glistening texture • Easily emulsified in Normal saline solution • Usually associated with virulent organisms • Eg. Gram negative organisms ( Neisseria )Microbiology - HTL 2
  25. 25. M or mucoid colonies s Associated with capsulated & virulent organisms s Exhibits slimy or watery confluent appearance and are positive to string test which indicates presence of Gm – organisms like K. pneumoniae in EMB agar – demonstrated with 3% KOHMicrobiology - HTL 2
  26. 26. R or rough colonies s Granulated in appearance and hard to emulsify in NSS s Eg. Corynebacterium diptheriaeMicrobiology - HTL 2
  27. 27. Possible descriptions of bacterial growth on agar slants: s Arborescent- branched s Beaded s Echinulate – pointed s Filiform – even s Rhizoid – rootlike s SpreadingMicrobiology - HTL 2
  28. 28. Providing a suitable environment s Oxygen Requirement s Temperature s pH s Osmotic Pressure s Hydrostatic Pressure s Salt ConcentrationMicrobiology - HTL 2
  29. 29. Oxygen & Temperature requirements : s Obligate aerobes s Thermophilic s Facultative aerobe • above 50 s Obligate anaerobe s Mesophilic s Facultative • best at 37 anaerobe s Psychrophiles s Microaerophiles • below 5 s Capnophiles s MGT, mGT , OGT s HyperthermophilesMicrobiology - HTL 2
  30. 30. Microbiology - HTL 3
  31. 31. pH, Hydrostatic & Osmotic pressure s pH scale 1-14 s Buffers: chemicals • Acidophiles that is used to • Alkaliphiles neutralize the acids and maintain the s wide range but proper pH internally usually neutral 6.5-7.5 s Methods • colorimetric • electrometricMicrobiology - HTL 3
  32. 32. Osmotic pressure: s Osmoprotectants – concentration of solutes > solvent • High conc  Plasmolysis • Low conc  Plasmoptosis s Halophiles – high salt concentration s atmospheres - barotolerantMicrobiology - HTL 3
  33. 33. Microbiology - HTL 3
  34. 34. Measuring Numbers of Microorganisms s direct microscopic • Petroff Hauser counter s electronic count s plate count – Standard agar Plate s MPN – Most probable number s viable count – Trypan blue s Filtration - s Turbidity – indirect way of extimating s dry weight – for filamentous organisms e.g. molds s metabolic activity – reduction Test e.g. oxygen uptakeMicrobiology - HTL 3
  35. 35. Microbiology - HTL 3
  36. 36. Microbiology - HTL 3
  37. 37. Growth of Microorganisms s Population - microbial growth s Doubling time/Generation Time • Time interval until the completion of next bacterial division s Growthrate s Exponential growthMicrobiology - HTL 3
  38. 38. Microbiology - HTL 3
  39. 39. Bacterial Growth Curve s Latent phase ( Lag 8000 phase) 7000 6000 s Logarithmic phase 5000 (Log phase) 4000 3000 s Stationary phase 2000 1000 s Death Phase 0 (Phase of Decline)Microbiology - HTL 3
  40. 40. The way microrganisms die s Rate of microbial death - temperature, type of microbe, physiologic state, presence of other substances that might protect s Decimal reduction time - D value (time in minutes -- 90% population) s Thermal death point - TDP s Thermal death time - TDTMicrobiology - HTL 4
  41. 41. Bacterial Death s Death is due to • Lack of food • Accumulation of toxins & dead debris • Development of unfavorable conditions s Death is the complete ceasation of multiplicationMicrobiology - HTL 4
  42. 42. Bacterial relationships s Free living s Symbiosis s Commensalism s Parasitism s Synergism s AntagonismMicrobiology - HTL 4
  43. 43. Common Types of Staining s Simple Stain s Types of Dyes: • methylene blue • Basic -Safranin , carbol fuchsin, s Differential stain Methylene blue • Gram’s Stain • Acidic - Eosin, • Acid Fast Stain acid fuchsin, congo red s Special Stain s Mordants • Wirtz Conklin s Decolorizers • LeifsonMicrobiology - HTL 4
  44. 44. Microbiology - HTL 4
  45. 45. Microbiology - HTL 4
  46. 46. Gram Stain & Acid Fast Stain s Crystal Violet s Carbol Fuchsin s Gram’s Iodine s Heat s 95% Alcohol s Acid Alcohol s Safranin s Methylene Blue • (+) Violet to • (+) Pink to Red Purple • (-) Blue to Violet • (-) Pink to RedMicrobiology - HTL 4
  47. 47. Microbiology - HTL 4
  48. 48. Microbiology - HTL 4

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