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  • 1. MICROBIOLOGICAL METHODS
  • 2. 5 BASIC TECHNIQUES  INOCULATION  INCUBATION  INSPECTION  ISOLATION IDENTIFICATION
  • 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. MEDIAprovides nutritional requirements for organisms SIMPLE - few inorganic compounds COMPLEX - inorganic & organic compounds
  • 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. LIQUID MEDIAWater based solutions, do not solidify at temps above freezing, flow freely in containers BROTHS, MILKS, INFUSIONSvarious solutes dissolved in distilled water
  • 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. 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. CHEMICAL CONTENTSYNTHETIC - Chemically defined media Highly pure organic & inorganic compoundsCOMPLEX - (Non synthetic) - one ingredient not chemically definable Of plant, animal or yeast extract
  • 10.  GENERAL PURPOSE MEDIAUsed for a broad spectrum of microbes, non syntheticExamples: Brain-heart infusion Tryptose soy agar Tryptose soy broth
  • 11.  ENRICHMENT MEDIUMcomplex organic substances : blood, serum, growth factorsUsed for FASTIDIOUS ORGANISMSStreptococcus pneumoniaeRequires blood - sterile horse, sheep or rabbit
  • 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. 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.  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. EXAMPLESMAcCONKEY AGAR - lactose + neutral redE. coli produces acid, metabolizes lactose RED-PINK coloniesSalmonella sp produce no acid OFF WHITE colonies
  • 16. E. coli & Salmonella sp. On MacConkey Agar
  • 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. E. coli & Salmonella sp. On XLD Agar
  • 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.  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.  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. INCUBATIONChamber (INCUBATOR) temperature & atmospheric gas controlledLAB INCUBATORS : 20 - 40oC Aerobic or AnaerobicINCUBATION PERIOD : hours-several weeks depending upon the organism
  • 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. Pseudomonas, Staphylococcus & Serratia on TSA plates
  • 25.  MICROSCOPICALLYindividual cells within a colony Evidence of cellular morphology: size, shape, details of structure allows for IDENTIFICATION
  • 26.  AIMSto provide adequate MAGNIFICATION, RESOLUTION and CLARITY of IMAGE
  • 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.  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. 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. 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. FIXED PREPARATIONS Advantage: Permanent mount, long term study Smear technique : Developed by Koch >100yrs ago Disadvantage: KILLS specimen
  • 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.  EXAMPLES:BASIC: Crystal violet, methylene blue, safraninACIDIC: Nigrosin, india ink
  • 34. POSITIVE STAINING +ve stain - sticks to specimen providing colour Bacillus cereus stained with carbol fuschin (1300x)
  • 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. 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. 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. Gram +ve Staphylococcus aureus Gram -ve Escherichia coli (1400x)
  • 39.  ACID FAST STAIN - Paul Ehrlichsimilar to Gram’s, used with resistant bacteriaAcid-fast Bacteria : PinkNon Acid-fast bacteria : BlueMycobacterium : tuberculosis
  • 40. Mycobacterium tuberculosis (300x)
  • 41. Mycobacterium marinum
  • 42. Mycobacterium leprae
  • 43.  ENDOSPORE STAINsimilar to Acid-fastDistinguishes between bacteria producing spores and those that do notFor Identification of Bacillus sp., Clostridium sp.
  • 44. Clostridium tetani (1400x)
  • 45. Gas Gangrene
  • 46. Anti gas serum - 1934
  • 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. Klebsiella pneumoniae
  • 49. Capsule Stain
  • 50. BACTERIAL SHAPES Characteristic Shapes - Bacteria Spherical - coccoid Cylindrical - rod Spiral - spirilla Pleomorphic - irregularly shaped
  • 51. BACTERIAL SHAPES
  • 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. Brightfield Microscope Extensively used: necessary to view stained specimens
  • 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. Staphylococci in blood - Epifluorescene
  • 56. Darkfield MicroscopeEliminates need for stainingAchieve contrast between specimen & background
  • 57. Treponema pallidum (syphilis)
  • 58. Phase Contrast MicroscopeStaining not requiredView structures & living organisms
  • 59. Paramecium caudatum (300x)
  • 60. Electron MicroscopesElectrons not light beamGreater resolutionHigher magnificationsTypes: Transmission EM Scanning EM
  • 61. Transmission EMElectrons pass through specimenView ultrastructure of organisms
  • 62. Influenza virus (360,000x)
  • 63. Scanning EMElectron beam scanned across surface of specimen: 3D image
  • 64. Candida albicans (2200x)
  • 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. IDENTIFICATION - BIOCHEMICAL Metabolic characteristics: substrates for growth
  • 67. BERGEY’S MANUAL OF DETERMINATIVE BACTERIOLOGY Bacteriologists BIBLE (Reference Text) Divided into sections by TAXONOMY & CLASSIFICATION