Microbial Control, Antibiotic Sensitivity, and API 20 Enteric Bacterial Analysis

Concepts of Microbial Control and Antibiotic Sensitivity

Control of Microorganisms

  • Involves using physical or chemical agents to stop microorganism growth or to kill them.
  • Applications include:
    • Food preservation
    • Sanitation of buildings
    • Preventing infections in hospitals

Physical Control Methods

  • Heat:
    • Boiling
    • Autoclaving
    • Pasteurization: Process involves heating and cooling milk to denature enzymes and prevent bacterial growth.
      • Enzymes are denatured by heat treatment, losing their function.
  • Cold:
    • Low temperatures slow down metabolic reactions because metabolic reactions require liquid water.
    • Ice crystal formation can disrupt cell membranes.
  • Desiccation:
    • Preserves food by removing water.
    • Examples: legumes, fruits, peas, grains, nuts, yeast.
    • Enzymes need water to function, so desiccation inhibits them.
  • Freeze-drying (Lyophilization):
    • Inhibits enzymes by drying frozen material.
    • Used for foods like freeze-dried coffee.
    • Process:
      • Rapid freezing in alcohol/dry ice or liquid nitrogen to form tiny ice crystals.
      • High vacuum pressure to remove all water.
      • Sealing under vacuum.
  • Filtration:
    • Membrane filters are used for heat-sensitive liquids and gases.
    • Rapid and expensive; filters clog easily.
    • Typically 0.1 mm thick.
  • Osmotic Pressure
  • Radiation

Chemical Control Methods

  • Cleaning agents
  • Alcohol
  • Antibiotics

Temperature Requirements

  • Thermophiles: Thrive in high temperatures (e.g., 40°C).
  • Mesophiles: Thrive in moderate temperatures (e.g., 20°C, normal body temperature of 37°C, room temperature of ~24°C).
  • Psychrophiles: Thrive in low temperatures (e.g., 0°C).
  • Cardinal Temperature: Refers to the temperature range in which an organism can grow.

Relative Susceptibility of Microorganisms

  • Most susceptible:
    • Enveloped viruses
    • Gram-positive bacteria
  • Intermediate susceptibility:
    • Gram-negative bacteria
    • Fungi
    • Active protozoa
  • Most resistant:
    • Non-enveloped viruses
    • Mycobacteria
    • Protozoan cysts
    • Bacterial endospores

Moist Heat

  • Boiling.
  • Autoclaving.

Dry Heat

  • Oven, open flame: Kills by oxidizing cell constituents and denaturing proteins.

Examples of Microbial Control Applications

  • Milk
  • Surgical instruments in hospitals
  • Prepared meals for expeditions (e.g., Aconcagua)
  • Drinking water from streams and lakes
  • Apricots preserved for 6 months
  • Microbial media containing vitamins that cannot be boiled

Antibiotic Sensitivity

Antibiotics

  • Chemicals produced by bacteria/fungi or in the lab that can kill or prevent the growth of other organisms (bacteria).
  • Effective in controlling various infectious bacteria in humans and used extensively in disease control (treatment of bacterial infections).

Antibiotic Sensitivity Testing

  • Essential to determine whether an organism is sensitive to the inhibitory action of an antibiotic.
  • Disc diffusion method:
    • Filter paper discs are saturated with specific amounts of certain antibiotics and placed on a growth of the particular bacterial culture to be tested.
    • If growth is inhibited (large zone of inhibition), the culture is sensitive to the antibiotic.
    • If growth is seen (very small or no zone of inhibition), the culture is resistant to the antibiotic.

Antibiotic Action

  • Bacteriostatic: Stops bacteria from growing.
  • Bactericidal: Kills bacteria.
  • Modes of action:
    • Destruction of cell wall
    • Destruction of cell membrane
    • Inhibition of protein synthesis
    • Inhibition of DNA synthesis
    • Inhibition of intermediary metabolism
  • Most antibiotics are effective against bacteria with a prokaryotic cell structure, differing from eukaryotic cells.

Antibiotic Sensitivity Results

  • Sensitive: A large zone of inhibition where no bacteria grow surrounds the antibiotic disk.
  • Equivocal (Indeterminate): The category ‘intermediate’ is reported, requiring an alternative test.
  • Resistant: A very small (if any at all) zone of inhibition exists, indicating that the antibiotic has little effect on the organism tested.

Diffusion Susceptibility Test: Kirby-Bauer

  • A common method for testing antibiotic resistance.
  • Procedure:
    1. Swab bacteria over the surface of an agar plate uniformly.
    2. Add paper disks with a known dose of antibiotic to the surface.
    3. Incubate; the antibiotic will diffuse into the medium as cells grow.
      • Larger molecular size = slower diffusion.
    4. Examine the plate for clear zones around the disk where growth is inhibited.
    5. Measure the diameter of the clear zones and consult a table to determine if the results are clinically useful.
  • Note:
    • Inhibition does not always equal killing.
    • In vitro results may not equal in vivo results.

Minimum Inhibitory Concentration (MIC) Test

  • MIC = smallest amount of drug that will inhibit growth/reproduction of the pathogen.
  • Serial dilutions of the drug are made.
  • A standard amount of bacteria is added to each tube.
  • After incubation, cloudiness indicates growth.
  • To determine whether antibiotics are bacteriostatic or bactericidal, further testing is needed.

API 20E Bacterial Analysis

API 20E Rapid Test

  • Used to identify Enteric bacteria.

Procedure

  1. Retrieve the test panel from the incubator.
  2. Add reagents (Kovak's reagent, Methyl red, VP reagent I and II) to the appropriate wells.
  3. After reagent incubation, refer to the positive test result panel.
  4. Record the positive results as instructed by your lab instructor.
  5. Log onto the API20 analysis website.
  6. Input the code for your results to determine the genus and species of your bacterial culture.