MCROBIO

• Principles of Microscopy
• Magnification:

  • Shows two adjacent objects as discrete entities
  • Allows objects to be clearer for better understanding
  • Dependent on wavelength of light used
  • Numerical aperture:
  • Function of the diameter of the objective lens in relation to its focal length
  • Doubled by the use of the substage condenser
  • Resolving power = wavelength / (2 * NA)
  • Dependent on refractive index and bending power of light
• Resolution:

  • Ability of lens to show two adjacent objects as discrete entities
  • Greater resolving power with shorter wavelength of light
  • Dependent on numerical aperture
  • Refractive index affects bending power of light
• Illumination:

  • For efficient magnification and resolution
  • Condenser contains two lenses and helps produce maximum numerical aperture
  • Keep condenser close to the state when using oil immersion objective
  • More magnification leads to less working distance and more numerical aperture
• Focusing:

  • Lowest objective to highest magnification
  • Parfocal lenses remain in focus when adjusting magnification
  • Contrast is the difference between the specimen and background, staining is needed
  • Iris diaphragm can adjust illumination and contrast
• Types of Microscope
• Two-dimensional imaging (Light Microscopy):

  • Bright field microscope (Compound Light Microscope):
  • Specimens visualized based on density differences
  • Can observe behavior of living cells
  • Phase contrast microscope:
  • Amplifies differences in refractive index
  • Allows visualization of live samples
  • Dark Field Microscope:
  • Light reaches specimens from slides, not through them
  • Excellent for observing motility
  • Fluorescence Microscope:
  • Visualizes specimens that fluoresce
  • Used in microbial ecology for enumerating bacteria
• Three-dimensional Imaging:

  • Differential interference contrast (DIC) Microscope:
  • Creates 3D appearance of structures
  • Confocal scanning laser microscopy (CSLM):
  • Uses laser source for high contrast and 3D view
  • Electron Microscopy:
  • Uses beam of electrons for higher resolution images
  • Transmission electron microscope (TEM):
  • Operates in a vacuum for high magnification and resolution
  • Scanning electron microscope (SEM):
  • Specimens coated with heavy metal for observation
• Culture Media Preparation
• Culture media composed of nutrients for growth and identification of microorganisms

  • Classified based on physical state, use, and function
  • Solid, semi-solid, and liquid media types
  • Simple, enriched, enrichment, selective, and differential/indicator media
  • Culture media sterilized via filtration if heat-sensitive
• Steps in Preparing Culture Media:

  • Weigh and measure different components
  • Adjust pH
  • Plated media: autoclave, cool, pour, solidify, test for sterility
  • Tubed media: distribute, autoclave, test for sterility
• Aseptic Techniques and Inoculation Methods
• Aseptic techniques prevent contamination and spread of microorganisms

  • Joseph Lister - Father of Modern Surgery
  • Use of sterile materials, disinfectants, biosafety cabinets
  • Inoculating loop or needle used for handling bacteria and fungi
  • Sterilization before and after use
• When working with highly pathogenic bacteria, sterilization chambers should be used
• Bacteria and fungi can be handled using an inoculating loop or needle, which should be sterilized before and after use via flaming
• Instruments should not be shaken while cooling
• The size of an inoculating loop ranges from 0.01 to 0.005 ml of liquid
• Inoculating loops or needles can also be used for picking up colonies
• To avoid spattering and aerosol formation, flame the wire in the inner part of the flame
• Large volumes of liquid can be handled with pasteur pipettes or graduated pipettes, with suction from an aspirator like a rubber bulb
• Washing hands and workstation is important
• Bunsen burners or alcohol lamps produce an airflow via convection, preventing dust from settling from the environment to the surfaces of the workspace
• Inoculating loops/needles and hockey sticks are used for spread plating, with hockey sticks being sterilized by dipping in ethanol and passing through the flame
• Cotton plugs allow air to enter the test tube while protecting it from larger materials
• Methods of Inoculation:

  • Liquid medium: inoculating loop/needle with inoculum dipped into liquid medium, moved slightly, then withdrawn
  • Solid plate medium:
  • Clock streaking leads to individual colony forming
  • Implant streaking directly presses material containing bacteria into the solid medium
  • Direct plate exposure exposes agar plate to the surrounding air for a certain period of time
  • Spread plate involves spreading a small volume of liquid inoculum over the surface of a medium using a hockey stick or sterile bent glass rod
• • Flood plate is done by flooding the surface of a solid medium with a liquid inoculum and withdrawing excess with a sterile pasteur pipette
  • Spread and flood plates result in a lawn plate, where the surface of the agar is covered in a layer of confluent growth
  • Pour plate method involves delivering inoculum into an empty sterile plate using a sterile graduated pipette or streaking out with a loop, then pouring sterile melted nutrient agar medium until the bottom of the plate is covered, swirling the plate to distribute the inoculum
• Plates can be inoculated with sterile cotton swabs, where a sterile swab is used to sample microorganisms at any given site by lightly drawing it on the surface of the agar
• Inoculating a solid tube medium can be done by streaking the surface of slants using an inoculating loop, needle, or swab, or by stabbing the deeper, microaerobic, or anaerobic portion of a butt using a needle
• Types of Staining:

  • Negative staining uses acidic dyes like nigrosine/india ink for capsule staining and crystal violet for staining the background
  • Positive staining uses basic dyes like carbolfuchsin, methylene blue, and crystal violet for staining the bacteria itself
  • Simple staining uses carbolfuchsin, methylene blue, and crystal violet
  • Differential staining uses multiple chemical dyes, including gram staining and spore staining
• Gram positive staining results in a blue/purple color, while gram negative staining results in a pink/red color
• Capsule staining is used to detect sticky substances found when bacteria are overgrowing in particular spaces, such as Bacillus anthracis, which prevents phagocytosis and allows attachment onto tissues
• Spore staining results in green colored spores and red colored vegetative cells
• Physical Factors that Affect Microbial Growth:

  • Temperature affects enzymatic activity, with an increase leading to increased activity and a decrease leading to decreased activity
  • pH levels can be low (acidic), neutral, or high (basic)
  • Osmotic pressure is dependent on water availability in a cell, with halophiles needing some NaCl to thrive
  • Anaerobiosis refers to the amount of oxygen in the environment, with different types of bacteria having varying oxygen requirements
• Desiccation refers to the state of dryness or drying out an organism, with some microorganisms able to survive desiccation depending on their features
• Biochemical Tests can be used to test the presence of enzymes in bacteria, such as carbohydrate fermentation, casein hydrolysis, gelatin hydrolysis, and starch hydrolysis
• Introduction to Fungi:

  • Fungi are eukaryotic, spore-bearing organisms with filamentous, branched somatic structures surrounded by cell walls containing chitin or cellulose
  • Fungi can be yeast (unicellular), mold (multicellular), or dimorphic (mold -> yeast)
  • Basic structures include hyphae (septate or aseptate) and mycelium, a mass/mat of hyphae
• Fungal Reproduction:

  • Reproduces by formation of spores
  • More common to refer to fungi by their asexual designations
  • The teleomorphic phase occurs only under specialized conditions in the laboratory
• Classification of True Fungi based on their method of reproduction:

  1. Zygomycetes (common bread mold)

  2. Ascomycetes (puffballs, common mushrooms)

  3. Basidiomycetes (Dutch elm disease, rye smut)

  4. Deuteromycetes (Fungi imperfecti)
• Identification of Fungi relies heavily on morphology and mode of spore production

  • Two types of asexual spores: Conidia and Sporangiospores
• Culture Media for Fungi:

  • Generally contains a source of Carbon, Nitrogen, and Vitamins
  • Glucose (dextrose) is the most widely utilizable

  1. Potato Dextrose Agar (PDA):

  • General Purposed Medium
  • Isolation of Fungi and Molds
  • Disadvantages: Too rich, encourages mold sporulation and pigment production