chapter 2

• The Five I’s of Microbiology:

  • Inoculation
  • Incubation
  • Isolation
  • Inspection
  • Identification
• Inoculation:

  • Culture: to grow microorganisms
  • Medium (plural, media): nutrients for the growth of microbes
  • Inoculum: a small sample of microbes
  • Inoculation: the introduction of an inoculum into media to culture microbes
  • Clinical specimens are obtained from body fluids, discharges, anatomical sites, or diseased tissue
• Incubation:

  • Incubator: a temperature-controlled chamber to encourage the multiplication of microbes
  • Temperatures used in laboratory propagation of microorganisms: 20 to 45 °C
  • Atmospheric gases such as oxygen or carbon dioxide may be required for the growth of certain microbes
  • During the incubation period, the microbe multiplies and produces growth that is observable macroscopically
• Various Conditions of Cultures
• Types of Media:

  • Physical state
  • Chemical composition
  • Functional type (purpose)
• Chemical Content of Media:

  • Defined or synthetic: Composition is precisely chemically defined
  • Complex: One or more components is not chemically defined
  • Contains extracts of animals, plants, or yeasts
  • Examples: blood, serum, meat extracts or infusions, milk, yeast extract, soybean digests, and peptone
• Media for Different Purposes:

  • General-purpose media: Grow as broad a spectrum of microbes as possible
  • Enriched media: Contains complex organic substances for the growth of fastidious microbes
• Selective Media:

  • Contains agents that inhibit the growth of certain microbes
  • Important in the primary isolation of a specific type of microorganism
  • Speed up isolation by suppressing unwanted background organisms and favoring the growth of desired ones
• Differential Media:

  • Allow multiple types of organisms to grow but display visible differences in how they grow
  • Variations in colony size or color
  • Media color changes
  • Production of gas bubbles
• Selective and Differential Media:

  • A medium can be both selective and differential
  • Example: MacConkey agar suppresses the growth of some organisms while producing a visual distinction between the ones that do grow
  • Dyes are used as differential agents because many are pH indicators that change color in response to the production of an acid or a base
• Miscellaneous Media:

  • Reducing medium: Contains a substance that absorbs oxygen or slows the penetration of oxygen
  • Important for growing anaerobic bacteria
• Isolation:

  • Based on the concept that if an individual bacterial cell is separated from other cells on a nutrient surface, it will produce a discrete mound of cells called a colony
  • Isolation requires a medium with a firm surface, a Petri dish, and an inoculating loop (streak plate method)
• Inspection and Identification:

  • Microbes can be identified through microscopic appearance, characterization of cellular metabolism, determination of nutrient requirements, products given off during growth, presence of enzymes, and mechanisms for deriving energy, genetic and immunologic characteristics
• Size of Macroscopic versus Microscopic Organisms:

  • The dimensions of macroscopic organisms are given in centimeters (cm) and meters (m)
  • The dimensions of microscopic organisms range from millimeters (mm), to micrometers (μm), to nanometers (nm)
• Microbial Size:

  • Yeast are generally 3 to 4 µm
  • The smallest bacteria measure around 200 nm; largest around 750 µm
  • Most viruses measure between 20 nm and 400 nm; some can be as big as 800 nm or 1500 nm
• Magnification:

  • Real image: formed by the objective
  • Virtual image: formed when the image is projected up through the microscope body to the plane of the eyepiece, the ocular lens forms a second image
  • Total magnification: calculated based on the power of the objective and ocular lenses
• Resolution:

  • Also known as resolving power
  • The capacity of an optical system to distinguish two adjacent objects or points from one another
  • Resolving power of the human eye: 0.2 mm
  • Resolving power of the light microscope using the oil immersion lens: 0.2 μm
• Effect of Wavelength on Resolution
• Oil Immersion Lens Reduces Light Scatter, Increasing Resolution
• Contrast:

  • Refractive index: The degree of bending that light undergoes as it passes from one medium to another
  • Too much light can reduce contrast and burn out the image
  • Increase contrast with special lenses (such as a phase-contrast microscope) and by adding dyes
• Types of Microscopy: Bright-Field:

  • The most widely used type of light microscope
  • Forms its image when light is transmitted through the specimen
  • Can be used for both live, unstained material and preserved, stained material
• Types of Microscopy: Dark-Field:

  • A bright-field microscope can be adapted as a dark-field microscope by adding a special disc called a stop to the condenser
  • The stop blocks a
• Dark-Field Microscopy:

  • Adapted from a bright-field microscope by adding a special disc called a stop to the condenser
  • The stop blocks all light from entering the objective lens, except peripheral light that is reflected off the sides of the specimen itself
  • Results in brightly illuminated specimens surrounded by a dark (black) field
• Phase-Contrast Microscopy:

  • Takes advantage of the fact that cell structures differ in density
  • Transforms subtle changes in light waves passing through the specimen into differences in light intensity
  • Most useful for observing intracellular structures such as bacterial endospores, granules, and organelles
• Fluorescence Microscopy:

  • Furnished with an ultraviolet (UV) radiation source
  • Uses certain dyes and minerals that fluoresce when bombarded by short ultraviolet rays
  • Most useful in diagnosing infections and pinpointing particular cellular structures
• Confocal Microscopy:

  • Overcomes the problem of cells or structures being too thick by using a laser beam of light to scan various depths in the specimen
  • Captures a highly focused view at any level, ranging from the surface to the middle of the cell
  • Most often used on fluorescently stained specimens
• Transmission Electron Microscope (TEM):

  • Method of choice for viewing the detailed structure of cells and viruses
  • Produces its image by transmitting electrons through the specimen
  • Specimen must be sectioned into extremely thin slices and stained or coated with metals for image contrast
• Scanning Electron Microscope (SEM):

  • Provides detailed three-dimensional views of objects
  • Bombards the surface of a metal-coated specimen with electrons while scanning back and forth over it
  • Electron pattern is displayed as an image on a television screen
• Preparing Specimens for the Microscope:

  • Specimens are generally prepared by mounting a sample on a glass slide between the condenser and the objective
  • Preparation depends on the condition of the specimen, aims of the examiner, and type of microscopy available
• Stains:

  • Staining applies colored chemicals (dyes) to specimens
  • Basic dyes have a positive charge, while acidic dyes have a negative charge
  • Different staining techniques include negative versus positive staining, simple versus differential staining
• Differential Stains:

  • Gram Stain:
  • Developed by Hans Christian Gram in 1884
  • Consists of sequential applications of crystal violet, Gram’s iodine, alcohol rinse, and a contrasting counterstain
  • Acid-Fast Stain:
  • Differentiates acid-fast bacteria from non-acid-fast bacteria
  • Used to detect Mycobacterium tuberculosis
  • Endospore Stain:
  • Distinguishes between endospores and vegetative cells
  • Significant in identifying gram-positive, spore-forming members of the genus Bacillus and Clostridium
• Special Stains:

  • Used to emphasize cell parts not revealed by conventional staining methods
  • Capsular staining is used to observe the microbial capsule, while flagellar staining reveals tiny filaments used by bacteria for locomotion