Anaphy Lab CHAP 1

• The Microscope is one of the most important instruments in the study of microorganisms.
• Bacterial identification and classification are based in cell forms and structures visible only under high magnification and resolution.
• Antonie van Leeuwenhoek was the first individual to utilized the microscope to discover small organisms he called “animalcules
• Microscopy is defined as the use of a microscope to magnify objects that are too small to be seen by the naked eye.
• Microscope methods may vary. These includes: Bright-field Microscopy, Phase contrast Microscopy, Fluorescent Microscopy, Dark-field Microscopy, Electron Microscopy
• Some dyes or stains are used combined with these microscopy methods can detect etiologic agents in a rapid way.
• This has the widest use and application in the clinical microbiology. Bright Field Microscopy
• Bright light passes through a series of lenses that bend the light in a manner that results in the magnification of the organism present in the specimen.
• Total magnification achieved is the product of lenses used.
• Magnification is the process of enlarging an image of an apparent size.
• These is calculated by multiplying the ocular lenses together with the objective lenses
• Organisms can be magnified >1000x their actual size.
• Resolution is defined in which a magnified object can maintain its detail
• Scanner (4x)
• Scanner + Eyepiece = 40x magnification
• Low Power Objective (10x)
• LPO + Eyepiece = 100x magnification
• High Power Objective (40x)
• HPO + Eyepiece = 400x magnification
• Oil Immersion Objective (100x)
• OIO + Eyepiece = 1000x magnification
• Immersion Oil is used in conjunction with the oil immersion objective
• Must be with same refractive index as glass (1.5)
• Cedarwood Oil (r.i. 1.516) – absorbs blue and ultraviolet light, yellows with age, slightly acidic which can cause damage to the lenses
• Type A and B Synthetic Oils – general purpose immersion oils with low and high viscosity respectively. Almost no interference, easily removable
• Type A - immersion oil with LOW viscosity
• Type B - immersion oils with high viscosity
• To make objects stand out from the background especially those almost transparent microorganisms. Contrast
• Earlier adaptation of contrast is displayed by the use of staining techniques by staining the background making the organism stand out.
• Contrast is Extensively used by Phase contrast microscopy
• In contrast of bright field microscopy, this employs a luminous object against a background with little to no light. Dark Field Microscopy
• Uses a dark filter. Dark Field Microscopy
• Condenser allows light to pass through obliquely (to the side). Dark Field Microscopy
• Greatest utility for detecting certain bacteria because of their thin dimensions such as the spirochetes. Dark Field Microscopy
• Offers another of approach instead of staining by enhancing the contrast of the specimen to the background. Phase Contrast Microscopy
• Uses beams of light passing through the specimen that are partially deflected by the different densities or thicknesses (refractive index). Phase Contrast Microscopy
• The more the refractive index, the more the beam of light is slowed
• Employs the use of dyes or fluorochromes which reacts with excited light (UV lights). Fluorescent Microscopy
• Fluorochrome molecules releases energy in a form of visible fluorescent light called fluorescence.
• Commonly used dyes includes Acridine Orange, Auramine-Rhodamine and Calcofluor White.