Lab Procedures and Staining Techniques

Lab Procedures and Guidelines

Test Tube Racks

  • One rack per person: Each student is allowed one test tube rack.
  • Distribution of cultures: Confirm if anyone still needs a culture (all should have one).

Labeling Procedures

  • Initials on plates: It is essential to label your plates with the initials of the culture provided.
    • Example initials: p c a f s a s e e f

Safety Protocols

  • Goggle usage: Always wear goggles before starting inoculation.
  • Use of gloves: Ensure gloves are worn to maintain sterility.

Plate Arrangement in Bin

  • Place blood agar plates in the front and other types in the back of the bin.

Inoculation Guidelines

  • Microscopic Observation: All observations should be done at 100x magnification since the specimens are all bacteria.
  • Assistance for Lab 6 or 7: Feel free to ask for help regarding Lab 6 or Lab 7 operations; assistance is available.

Acid Fast Staining Overview

  • Red Dye Introduction: The acid fast stain begins with the application of a red dye called carbofusion.
  • Heating required: To penetrate the mycolic acids, a steaming process is necessary for the stain to adhere effectively.
  • Staining outcomes:
    • Acid-fast bacteria: Retain the red color, indicating they contain mycolic acids.
    • Non-acid-fast bacteria: When treated with the decolorizer (acid alcohol), they lose their color and turn blue.

Examples of Cultures

  • Mycobacterium: Should appear red under the microscope.
  • E. Coli: As a non-acid-fast organism, it should appear blue.
  • Bacillus shape: Both Mycobacterium and E. Coli are described as bacillus-shaped, which refers to their rod-like appearance.
    • E. Coli (blue) on the left.
    • Mycobacterium (red) on the right.

Observational Challenges

  • Cord Factor: The phenomenon of clumping observed in Mycobacterium is due to the presence of something called the cord factor.

Preparations and Maintenance of Slides

  • Initial preparations: Finish Lab 7 or Lab 6 before proceeding to acid-fast staining.
  • Using prepared slides: Be cautious with oil; remove it using lens paper rather than alcohol.
    • Importance: Using lens paper preserves the quality of prepared slides, which should not be discarded.
  • Visual examination: When stained, individual dots should be discernible with clarity.

Microscopic Techniques

  • Use a higher condenser setting for better visibility of stained specimens as it enhances resolution.
  • For unstained specimens, the condenser can be lowered to adjust visibility.

Cleanup Procedure

  • Students are instructed to begin the process of cleaning up after microscope usage, ensuring the workspace is tidy for the next session.