Methods

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Methods

  1. 1. MICROBIOLOGICAL METHODS
  2. 2. 5 BASIC TECHNIQUES  INOCULATION  INCUBATION  INSPECTION  ISOLATION IDENTIFICATION
  3. 3. INOCULATIONIntroduction of small sample of cells (INOCLUM) into a container of nutrient mediumCLINICAL SAMPLE - blood, urine , CSF, feces, etcHABITAT SMAPLE - soil, water, sewage, food, etcCONTAINERS(individual) test tube, flask, agar plate (petri dish)(industry) large scale fermenters
  4. 4. MEDIAprovides nutritional requirements for organisms SIMPLE - few inorganic compounds COMPLEX - inorganic & organic compounds
  5. 5. PHYSICAL CHEMICAL FUNCTIONAL STATE COMPOSITION TYPE (Purpose) Liquid Synthetic General Purpose (Chemically) Semi-solid EnrichedSolid (Liquid) Selective Solid Non-synthetic Differential (not chemical) Anaerobic Growth Specimen transport Assay Enumeration
  6. 6. LIQUID MEDIAWater based solutions, do not solidify at temps above freezing, flow freely in containers BROTHS, MILKS, INFUSIONSvarious solutes dissolved in distilled water
  7. 7. SEMI SOLID MEDIAclot like consistency, contain solidifying agent (agar/gelatin - 0.3-0.5%)Used to determine motility, localize reaction at specific sites
  8. 8. SOLID MEDIA firm surface, allows cells to form discrete colonies Advantageous for ISOLATION/SUBCULTURING2 Forms:LIQUEFIABLE : reversible solid, agar, thermoplasticNON LIQUEFIABLE : NOT thermoplastic, cooked meat, potato slices, egg mediaTHERMOPLASTIC - solid at RTP/incubation temps liquid at 100oC resolidifies at 42oC
  9. 9. CHEMICAL CONTENTSYNTHETIC - Chemically defined media Highly pure organic & inorganic compoundsCOMPLEX - (Non synthetic) - one ingredient not chemically definable Of plant, animal or yeast extract
  10. 10.  GENERAL PURPOSE MEDIAUsed for a broad spectrum of microbes, non syntheticExamples: Brain-heart infusion Tryptose soy agar Tryptose soy broth
  11. 11.  ENRICHMENT MEDIUMcomplex organic substances : blood, serum, growth factorsUsed for FASTIDIOUS ORGANISMSStreptococcus pneumoniaeRequires blood - sterile horse, sheep or rabbit
  12. 12.  SELECTIVE & DIFFERENTIAL MEDIAdesigned for isolation & identification of specific groups of microbes from mixed populations SELECTIVE - contains 1 or more inhibitory agents DYES, ACID, ANTIMICROBIAL AGENTSExample: growth of A, B and C INHIBITED, but selective growth of D
  13. 13. Examples:MANNITOL SALT AGAR - 7.5% NaCl, inhibitory [ ] to human pathogen’sMAcCONKEY AGAR/DEOXYCHOLATE CITRATE AGAR - High Bile salt [ ], inhibitory to Gram +ve bacteriaSABOURAUD’S AGAR (Fungi) - pH 5.6 (acid), inhibits bacteria
  14. 14.  DIFFERENTIAL MEDIUMallows for growth of several typesBUT highlights differencesColony size, colour, formation of gas, pptDYES (differential agents) - act as pH indicatorscolour change due to production of acid or base
  15. 15. EXAMPLESMAcCONKEY AGAR - lactose + neutral redE. coli produces acid, metabolizes lactose RED-PINK coloniesSalmonella sp produce no acid OFF WHITE colonies
  16. 16. E. coli & Salmonella sp. On MacConkey Agar
  17. 17. XYLOSE LYSINE DEOXYCHOLATE AGAR (XLD)contains xylose, lysine, iron, thiosulphate, bile + phenol redE.coli acid production RED-PINK coloniesSalmonella sp convert thiosulphate to H2S gas (SMELL) forms a black ppt with iron
  18. 18. E. coli & Salmonella sp. On XLD Agar
  19. 19. OTHER MEDIA REDUCING - thioglycollic acid or cystine absorbs oxygen/slows penetration of oxygenTHUS reducing availabilityREQUIRED for growing ANAEROBIC BACTERIA CARBOHYDRATE FERMENTATION - sugars for fermentation, conversion to acids, pH indicatorREQUIRED for BIOCHEMICAL/IDENIFICATION TEST
  20. 20.  TRANSPORT - required for maintaining and preserving specimens for a period of timeExamples: STUART’S + AMIEScontains salts, buffers & absorbantsPrevents cell destruction, pH changes, toxic substances NO GROWTH
  21. 21.  ASSAY - tests effectiveness of antimicrobial agents, i.e., disinfectants, antiseptics, cosmetics etc. ENUMERATION - used in industry allows enumeration of organisms in milk, water, food and soil samples
  22. 22. INCUBATIONChamber (INCUBATOR) temperature & atmospheric gas controlledLAB INCUBATORS : 20 - 40oC Aerobic or AnaerobicINCUBATION PERIOD : hours-several weeks depending upon the organism
  23. 23. INSPECTION Observable growth on or in the medium (CULTURE) at various stages of incubation (EVALUATE GROWTH)MACROSCOPICALLY - naked eyeLIQUID MEDIA - cloudiness, sediment, scum or colour changeAGAR PLATE - discrete isolated colonies, mass of clinging cells (fungi)
  24. 24. Pseudomonas, Staphylococcus & Serratia on TSA plates
  25. 25.  MICROSCOPICALLYindividual cells within a colony Evidence of cellular morphology: size, shape, details of structure allows for IDENTIFICATION
  26. 26.  AIMSto provide adequate MAGNIFICATION, RESOLUTION and CLARITY of IMAGE
  27. 27.  TOTAL POWER OF MAGNIFICATION Power of Power of Total Objective Ocular Magnification 40x high (dry) 10x 400x 100x oil imm 10x 1000x 10x low 20x 200x power
  28. 28.  SUB-CULTURE common microbiological procedureallows for a pure STOCK-CULTURE of organism DISPOSAL OF CULTURESmost important - if presents a biological hazardAutoclaving - steam sterilizationIncineration - burningRadiation - X or raysDisinfection - chemical
  29. 29. PREPARATION OF SPECIMENS MOUNT - a sample on a glass slidesits between condenser and objective lens3 FACTORS1. Condition of specimen (Living or Preserved)2. Aims of examiner3. Type of microscope available
  30. 30. LIVING SPECIMENS Appear as near natural state as possible Media - suspended in water, broth, saline Allows for motility Temperature - to maintain viability Advantages: quick & easy to prepare Disadvantage: no cover slip, susceptible to drying out, free to contamination
  31. 31. FIXED PREPARATIONS Advantage: Permanent mount, long term study Smear technique : Developed by Koch >100yrs ago Disadvantage: KILLS specimen
  32. 32. STAINING PROCEDURES Any process in which coloured chemicals (DYES) are applied to specimens DYES - impart colour to cell or cell parts - become affixed through chemical reaction 2 types:BASIC (cationic) +ve charge ACIDIC (anionic) -ve charge PRINCIPLE : “opposites attract”
  33. 33.  EXAMPLES:BASIC: Crystal violet, methylene blue, safraninACIDIC: Nigrosin, india ink
  34. 34. POSITIVE STAINING +ve stain - sticks to specimen providing colour Bacillus cereus stained with carbol fuschin (1300x)
  35. 35. NEGATIVE STAINING -ve stain - (reverse) settles around specimen boundary forms a silhouette (stains the glass slide) Escherichia coli stained with India ink (1300x)
  36. 36. SIMPLE & DIFFERENTIAL STAINING +ve staining methods (classification) Simple - only 1 dye, uncomplicated procedure Differential - 2 coloured dyes, primary and counterstain, complex procedure Distinguishes cell types and parts
  37. 37. TYPES OF DIFFERENTIAL STAIN GRAM’S STAIN - Hans Christian GramDifferential - colour reaction with cellsGram +ve bacteria : purple/blueGram -ve bacteria : red/pinkBasis for IDENTIFICATION Diagnosis
  38. 38. Gram +ve Staphylococcus aureus Gram -ve Escherichia coli (1400x)
  39. 39.  ACID FAST STAIN - Paul Ehrlichsimilar to Gram’s, used with resistant bacteriaAcid-fast Bacteria : PinkNon Acid-fast bacteria : BlueMycobacterium : tuberculosis
  40. 40. Mycobacterium tuberculosis (300x)
  41. 41. Mycobacterium marinum
  42. 42. Mycobacterium leprae
  43. 43.  ENDOSPORE STAINsimilar to Acid-fastDistinguishes between bacteria producing spores and those that do notFor Identification of Bacillus sp., Clostridium sp.
  44. 44. Clostridium tetani (1400x)
  45. 45. Gas Gangrene
  46. 46. Anti gas serum - 1934
  47. 47. SPECIAL STAINS CAPSULE STAIN - specificundetected by conventional stainsCryptococcus sp. - fungal infection in AIDS patients FLAGELLAR STAIN - specificundetected by microscope due to limited resolving power
  48. 48. Klebsiella pneumoniae
  49. 49. Capsule Stain
  50. 50. BACTERIAL SHAPES Characteristic Shapes - Bacteria Spherical - coccoid Cylindrical - rod Spiral - spirilla Pleomorphic - irregularly shaped
  51. 51. BACTERIAL SHAPES
  52. 52. MICROSCOPES Magnifies size of imageVarious types: basic toolMagnification: enlargement of objectResolution: degree to which detail is maintained in magnified imageResolving power: closest spacing between 2 points where can be clearly seen as separate entities
  53. 53. Brightfield Microscope Extensively used: necessary to view stained specimens
  54. 54. Epifluorescence MicroscopeSpecimen illuminated at one wavelength of light,observed by light at another wavelengthUses fluorescent stainingNo condenser.Objective lens focuses lightUseful diagnostic procedures:Identify microorganisms
  55. 55. Staphylococci in blood - Epifluorescene
  56. 56. Darkfield MicroscopeEliminates need for stainingAchieve contrast between specimen & background
  57. 57. Treponema pallidum (syphilis)
  58. 58. Phase Contrast MicroscopeStaining not requiredView structures & living organisms
  59. 59. Paramecium caudatum (300x)
  60. 60. Electron MicroscopesElectrons not light beamGreater resolutionHigher magnificationsTypes: Transmission EM Scanning EM
  61. 61. Transmission EMElectrons pass through specimenView ultrastructure of organisms
  62. 62. Influenza virus (360,000x)
  63. 63. Scanning EMElectron beam scanned across surface of specimen: 3D image
  64. 64. Candida albicans (2200x)
  65. 65. Various Types of MicroscopesTYPE Max Useful Magnification ResolutionBrightfield 1500 x 100-200nmDarkfield 1500 x 100-200nmFluorescence 1500 x 100-200nmPhase Contrast 1500 x 100-200nmTEM 500,000 – 1,000,000 x 1-2nmSEM 10,000 – 1,000,000 x 1-10nm
  66. 66. IDENTIFICATION - BIOCHEMICAL Metabolic characteristics: substrates for growth
  67. 67. BERGEY’S MANUAL OF DETERMINATIVE BACTERIOLOGY Bacteriologists BIBLE (Reference Text) Divided into sections by TAXONOMY & CLASSIFICATION

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