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Microbiology Lab Guide: Protocols, Safety, and Techniques

Explore lab procedures from biohazard disposal to cell identification techniques in this comprehensive microbiology lab guide. Learn about aseptic transfers, microscopy tips, and culture transfer techniques. Understand the differences between prokaryotic and eukaryotic cells. Develop skills in identifying common microorganisms and assessing motility.

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Microbiology Lab Guide: Protocols, Safety, and Techniques

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  1. Biology 220 - Microbiology Kathleen Devlin kdevlin@csusb.edu

  2. General Lab Info • Lab Midterm – 100 pts • Lab Final – 100 pts • Lab Assignments – 32 pts • This may include • weekly in-lab assignments • quizzes • Lab Participation – 18 pts • 1 pt each week for signing in on time • 1 pt each week for proper cleanup

  3. Lab Protocols • Be on time • Read your lab prior to lab • Clean up after yourselves

  4. Biohazard Disposal • Put tubes in tube rack • Put plates, pipet tips, microtubes, etc in biohazard bucket • Put dirty slides in “Dirty Slides” bucket • Put broken glass in “Broken Glass” bucket

  5. Lab Safety • No food or drinks • Clean your lab bench with disinfectant before and after lab • Report any broken glass or injuries immediately • Be aware when using bunsen burners

  6. Exercise 1 - Scavenger Hunt • Objective: Find items commonly used in lab

  7. Exercise 2 – The Unseen Organism • Bacteria and some Fungi are too small to be seen, but are everywhere (ubiquitous) • Objective: Demonstrate that microorganisms exist in the air and on surfaces

  8. Types of Cultures/Media • Broth – liquid • Slant – solid, agar containing tube, dried at an angle • Plate – solid, agar containing petri dish • We will use many types of media throughout the quarter, each having different nutrients/properties

  9. Aseptic Transfers • Movement of bacteria from one media culture to another, without introducing contamination • Tools • Loop • Needle • Flame • Sharpie (label everything!!!)

  10. Aseptic Transfer, cont. • Label the new, sterile media (initials and bacteria being transferred) • Label tube glass or bottom of plates • Hold both tubes in hand not holding inoculating loop/needle • Flame inoculating loop/needle until red hot • Cool ~15 seconds • Remove both caps with little finger • Pass tube opening through flame • Take inoculum from tube and quickly place in/on new medium • Flame tube openings • Recap

  11. Exercise 3 - Culture Transfer Techniques • Objective: Transfer a bacterium from a culture to sterile media without introducing contamination • Each group of 4 students will need: • 2 broth tube • 2 slant tube • 2 agar plate • Transfer Micrococcus luteusbroth to each type of media • Transfer Micrococcus luteusslant to each type of media • Place tubes in incubation rack and plates in tub (always incubate plates upside down!!!!)

  12. Microscopy

  13. Microscope Care • Make sure lenses are clean (w/lens paper) • Store with lowest power objective lens in position • Stage should be all the way down • Turn power off

  14. Tips for Using the Microscope • Always start on the lowest power (4x) • Use course focus to bring object into view • Use fine focus to make the image sharp • Move to the next power • Use only the fine focus from here on out • Before you get to the 100x lens, put a drop of oil on the slide • Use the iris diaphram and light intensity to adjust light levels

  15. Exercise 4 – ID of Cells • Objectives: Recognize visual differences between prokaryotic and eukaryotic cells; Identify three basic morphologies of prokaryotic cells. • Eukaryotic – cells of animals, plants, fungi, and protists. Contain membrane bound organelles • Prokaryotic – cells of bacteria. No membrane bound organelles. 3 basic shapes • Cocci = sphere • Bacilli = rod • Spirilla = spiral

  16. Staphylococcus aureus • Gram-positive, cluster forming (like grapes) cocci • Normally found in nose and on skin • Can cause pneumonia, nosocomial infections, food poisoning, TSS

  17. Bacillus cereus • Gram-positive rod • Causes gastrointestinal intoxication • Found in a wide variety of foods

  18. Treponema pallidum • Spirochaete bacterium • Causes syphilis

  19. Yeast • Eukaryotic • Fungus • Used in making beer, wine, and cheese • Can form symbiotic relationship with bacteria • Note: These cells are round, but we do not use the term “cocci” for eukaryotic cells.

  20. Human Blood Smear • Eukaryotic cell • Note: These cells are round, but we do not use the term “cocci” for eukaryotic cells.

  21. Note the size difference between prokaryotic and eukaryotic cells

  22. Exercise 5 - Motility • Objective: Determine if a bacterium is motile • Some bacteria have the ability to move toward or away from a stimulus • TTC will turn red along the path traveled by the bacterium

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