Microbiology Lab Basics

• Proper hand washing and table disinfection with soap and disinfectant solution is vital before and after lab work.

Scientific Names

• Must be written properly.
• Proper form requires the genus (capitalized) followed by the species (lower case), for example Escherichia coli.
• Genus and species should be underlined or italicized.
• Genus can be abbreviated, for example E. coli or E. coli.
• Incorrect citation of scientific names results in a deduction of points.

Growing/Culturing Bacteria

• Requires nutrients, temperature, and oxygen.
• "To culture" is defined as "to grow microbes."
• A culture medium provides nutrients. These nutrients include carbon, nitrogen, energy, minerals, and growth factors, and are provided in the form of protein and/or sugars.
• Optimal temperature is 37°C or 98.6° F in most cases.
• Incubators help maintain humidity, heat, CO2, and oxygen ratios.
• Bacteria are classified according to oxygen requirements:
  • Strict/obligate aerobes require oxygen to survive.
  • Facultative anaerobes can live with or without oxygen.
  • Strict/obligate anaerobes cannot tolerate the presence of oxygen.
  • Microaerophiles grow in high levels of CO2 and lower levels of O2.

Culture Media

• Two general classifications: chemically-defined and complex.
• Chemically-defined media's exact composition is known.
• Complex media's exact chemical composition varies, with a protein source in the form of meat extract/peptones.
• Culture media has 4 general forms:
  • Broth (liquid)
  • Agar deep (semisolid)
  • Agar slant (solid)
  • Agar plate (solid)
• Nutrient media includes peptones, meat extract, sodium chloride, and distilled water.
• Agar is extracted from marine red algae, and few microbes can degrade it. It solidifies media and withstands high temperatures (100 °C).

Transfer of Bacteria

• To inoculate is to transfer a small amount of a microorganism from one culture medium to a fresh medium.
• Proper tools include a loop and needle.

Culture Media Types

• Broth (liquid): No agar added, used to culture large numbers of bacteria in a small space, and inoculated with an inoculating loop.
• Agar slant (solid) 1.5-1.7% agar: Solid surface for bacteria to grow, used for long-term storage of bacterial cultures, and inoculated with an inoculating loop.
• Agar deep (semisolid): Contains 0.5-0.7% agar, used to determine an organism's motility and oxygen requirement, and inoculated with an inoculating needle.

Aseptic Technique

• Series of steps taken to prevent contaminating ourselves, surroundings, media, and cultures.
• Steps include:
  • Washing hands and sterilizing the table, properly flaming loop/needle, never placing test tube caps on the table, properly flaming the opening of the tube, and cooling down the loop/needle before transferring.
• Sterilize loops and needles with flame before and after each use.
• Cool instruments after sterilizing, to avoid killing the specimen.
• Your TA will demonstrate proper aseptic technique and how to inoculate each of the following types of media
  • Broth
  • Slant
  • Deep

Incubation

• After inoculating bacteria, store cultures in the incubator for 24-48 hours at 37°C to grow the microbes.
• Incubation involves placing the microbe in an environment that provides the correct optimal temperature.
• Screw caps must be left a little loose unless otherwise instructed; just in case the organism used to inoculate the medium is a strict aerobe.

Labeling

• Labels are important.
• Labels should include:
  • Your name, lab section, date, organism's name, and media name.
• Use masking tape and waterproof ink pens/Sharpie to write the label.
• Do not use pencils for labeling.

Supplies & Procedures

• Supplies include: -Loops, needles, spark lighters, wax pencils, sharpies, student test tube racks
• Test tubes must be disposed of; remove masking tape and place in the "Discard Area" in an ORANGE test tube rack
• Do not invert or shake capped tubes because caps are not tight therefore spills will occur
• Always use a green test tube rack, located in the back of the lab.
• Always hold test tubes by the glass, never the caps.
• Caps are meant to be loose to allow bacteria access to Oxygen
• Label glassware using masking tape and a Sharpie or Pen with:
  • Name, Date, Lab Section, Organism, and Media Name
• Alert TA immediately if spills occur, do not attempt to clean yourself!!

Today's schedule

• Procedure First Period (pg. 42)
• Inoculate each culture into three forms of media (1 broth, 1 slant, and 1 agar deep per organism).
• Caps should be left a little loose before incubation.
• Once finished, incubate all media properly.
• Stock bacterial cultures should remain in green/blue full test tube racks.
• Disposal of damaged media tubes in discard area aka ORANGE RACKS.
• Inform TA to get a replacement culture if a culture is killed.

Learn about proper aseptic techniques, including hand washing and table disinfection, for microbiology labs. Understand scientific name conventions, bacterial culture requirements like nutrients and temperature and classification by oxygen needs.