Lab #5

Review for Practical #1 Colony morphology (pg. 19)

Review for Practical #1 Preparing a smear (pg ) Same procedure for all staining methods except Acid fast staining (1% albumin)

Review for Practical #1 Gram staining (pg. 34) 1 min sec 5-10 sec

Review for Practical #1 Bacterial shape and arrangement Gram positive cocci Gram negative bacillus

Review for Practical #1 Endospore staining (pg. 37 – 38) Prepare and heat fix a smear Flood slide with Malachite green for 1 min Steam for 3 minutes Use Bunsen burner like a blowtorch, intermittently Make sure the stain doesn’t dry up Rinse with water for ½ minute Cover smear with 0.5% Safranin stain Rinse and blot dry Observe under the microscope (+) Green = spores (inside cells and outside) Pink = vegetative bacterial cell

Acid fast staining (Pg ): Prepare smear Add 2 loopfuls of 1% albumin (helps hold bacteria onto slide) Mix in bacteria aseptically Air dry and heat fix Flood slide with Carbolfushin Blow torch for 5 minutes, intermittently  DON’T LET STAIN DRY Rinse with water Decolorize with Acid Alcohol until color stops to run Rinse with water Cover smear with Methylene blue for 1 minute Rinse with water Blot dry and observe under microscope Review for Practical #1

Acid fast positive = Red cells Acid fast negative = Blue cells Only Mycobacterium species will be Acid fast positive  resist de-colorization and retain primary stain (Carbolfushin) Review for Practical #1

Hanging drop method to determine motility (pg ) Use a broth culture Need a depression slide, coverslip, and Vaseline Smear Vaseline on palm  scrape onto the four edges of coverslip (Don’t get vaseline mixed w/ culture) Transfer loopful of bacterial culture onto coverslip Place depression slide, concave side down, onto coverslip  DON’T PRESS CAREFULLY invert slide  see the hanging drop Observe under microscope for motility Review for Practical #1

Microscope observation of hanging drop: Start at 10X objective  look for the edge of the drop Center the edge of drop in field of view Switch to 40X objective  FINE focus Bacteria will appear “ghost like” within the drop (no stain!) Motility is distinct movement from point A to point B Review for Practical #1

Streak plate method (pg. 26 – 29): Sample is only take once – only for quadrant 1 Streak from top to bottom – don’t go up and down Flame loop in between each quadrant!! Work aseptically! Review for Practical #1

Capsule staining Some bacteria produce this external structure Capsules composed of polysaccharides Encapsulated bacteria is more virulent Procedure Negative staining (stain the background and the bacteria) Capsule appears as a “halo” around the bacterial cell

Capsule staining