Identification And Differentiation Of Microorganisms
- 1. IDENTIFICATION AND DIFFERENTIATION OF MICROORGANISMSMicrobiology
- 2. Processes InvolvedStainingMicroscopyMicrobial CultureColony MorphologyBiochemical CharacteristicsImmunological MethodsUltrastructure Studies
- 3. MicroscopyThe human eye cannot resolve objects much less than 0.1 – 0.2 mm.It cannot detect objects with diameters less that about one thousandths of an inch (30 µm).Bacterial size range from 0.2 – 2 µm.Much of what we know today about microorganisms was made possible with the aid of the microscope.
- 4. Parts of a Microscope
- 5. Types of MicroscopesBright-Field MicroscopeDark-Field MicroscopePhase-Contrast MicroscopeFluorescent MicroscopeDissection MicroscopeScanning Electron MicroscopeTransmission Electron Microscope
- 6. StainingUsed to increase visibility of microorganisms being studiedUsed to identify the shape of bacteriaUsed to determine the morphological features of microorganismsUsed to detect contamination Used to differentiate and classify microorganisms (differential stains)Used to detect bacterial parts such as capsule, spores, flagella or inclusion bodies (special stains)
- 7. FixationThe process by which the internal and external structures of microorganisms are preserved and fixed in place.Heat fixation – fixation by means of application of heatThe prepared smear of microorganisms is gently heated and air-dried.Chemical fixation – involves the use of chemicals such as ethanol and formaldehyde.
- 8. Simple Staining TechniqueOnly one stain is usedCrystal Violet, CarbolFuchsin, and Methylene Blue are examples of basic dyes used in simple staining technique.
- 9. Differential StainingUsed to classify bacteria into separate groups based on their distinct staining propertiesMakes use of a primary stain, a decolorizer, and a counterstainGram Stain and Acid-Fast Stain are examples
- 10. Gram StainDifferentiates bacteria into two groups: Gram positive and Gram negativeStain mechanism is generally related to the thickness of the cell wall, pore size and permeability properties of intact cell envelopeMakes use of a primary stain (Crystal Violet), a mordant (Gram’s Iodine) a decolorizer (Alcohol or Acetone) and a counterstain (Safranin).Gram positive organisms stain blue while gram negative organisms stain red.Most cocci are gram positive; a third of all cocci, half of the bacilli, and all the spiral bacteria are gram negative.
- 11. Examples of Gram Positive BacteriaStaphylococcus aureusStreptococcus pyogenesClostridium perfringensListeriamonocytogenes
- 12. Examples of Gram Negative BacteriaEscherichia coliHaemophilusinfluenzaeVibriocholeraeNeisseriameningitidis
- 13. Acid-Fast StainUsed to differentiate Mycobacteria (M. tuberculosis) from other bacteriaMycobacteria are lipophilic or difficult to stain.Once stained, Mycobacteria are resistant to counterstain.Nocardia are partially acid fastMakes use of a primary stain (KinyounCarbolFuchsin), decolorizer (Acid Alcohol), and counterstain (Methylene Blue).Acid fast bacteria appear as bright red bacilli against a light blue background.
- 15. Special Staining TechniquesNegative Staining using India Ink or Negrosine is used to reveal the presence of capsuleSchaefer-Fulton using Malachite Green and Safranin is used to stain endosporesThe Leifson Method (pararosalanine), and Gray Method (basic fuchsin) are used to stain flagella.
- 16. Microbial CultureMakes use of a growth environment called culture mediumMicroorganisms form culture when inoculated in or on the medium.May be purely chemical (defined or synthetic), may contain organic materials, or may consist of living organisms.
- 17. Types of Culture MediumNutrient broth and Nutrient agar – used to cultivate bacteria. Both contain proteins, salts, and growth enhancers. Selective Medium – enhances the growth of a desired organism while retards the growth of unwanted organisms.Differential Medium – does not retard growth but provides environment where different bacteria can be differentiated from each other.
- 18. Selective MediumMacConkey Agar retards the growth of Gram + bacteria and enhances the growth of Gram - bacteria
- 19. Selective MediumThayer-Martin Medium favors the growth of Neisseria gonorrhea only
- 20. Selective MediumLowenstein-Jensen medium is used for the detection of M. tuberculosis
- 21. Differential MediumBLOOD AGAR PLATE (BAP)
- 22. Differential MediumMacConkey Agar is used to differentiate lactose fermenters from non-lactose fermenters
- 23. Colony MorphologyS. Pneumoniae in BAPP. aeroginosa
- 25. Biochemical CharacteristicsRefers to the characteristic patterns of substrate utilization, metabolic product formation and sugar formation.
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