Biology Microbiology Kathleen Devlin

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

Lab Protocols  Be on time  Read your lab prior to lab  Clean up after yourselves

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

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

Exercise 1 - Scavenger Hunt  Objective: Find items commonly used in lab

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

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

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

Aseptic Transfer, cont.  Label the new, sterile media (initials and bacteria being transferred) Label tube glass or bottom of plates 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

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 broth tube 2 slant tube 2 slant tube 2 agar plate 2 agar plate  Transfer Micrococcus luteus broth to each type of media  Transfer Micrococcus luteus slant to each type of media  Place tubes in incubation rack and plates in tub (always incubate plates upside down!!!!)

Microscopy

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

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

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 Cocci = sphere Bacilli = rod Bacilli = rod Spirilla = spiral Spirilla = spiral

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

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

Treponema pallidum  Spirochaete bacterium  Causes syphilis

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.

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

Note the size difference between prokaryotic and eukaryotic cells

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