1. Lab #4

2. Review of Lab #3 Oxygen requirements Obligate aerobes (B. subtilis) Obligate anaerobes (C. sporogenes) Facultative anaerobes (E. coli, K. pneumoniae, and S. epidermidis) Microaerophilic Slant – only aerobic and facultative anaerobic Motility tall – only aerobic (top) and facultative anaerobic (throughout) Thioglycolate broth – all organisms will grow but in a different location in the broth

3. Review of Lab #3 Oxygen requirements

4. Review of Lab #3 Catalase test Organisms that utilize oxygen produce enzymes to help protect against toxic oxygen derivatives (H 2 O 2, O 2 -, etc.) Catalase enzyme (Production of O 2 is seen as bubbles) E. coli B. subtilis K. pneumoniae S. epidermidis H 2 O 2 H 2 0 + ½ O 2 Catalase Positive

5. Review of Lab #3 Endospore staining Positive result = spores are present (you see green spores and pink vegetative cells) Negative result = no spores are present (you only see pink vegetative cells) Vegetative cells will be present in both cases! B. cereus – spore positive B. subtilis – spore positive E. coli – spore negative K. pneumoniae – spore negative S. epidermidis – spore negative

6. Culture technique used to obtain isolated colonies  obtain a pure culture Lab #4 – Streak plate method (4D)

7. 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!! Lab #4 – Streak plate method (4D)

8. Each student streak 3 plates – lots of practice! Label plates properly Name Date Organism Incubate the plates INVERTED Why? Lab #4 – Streak plate method

9. Type of differential stain Used to identify members of the genus Mycobacteria Mycobacterium tuberculosis Mycobacterium leprae Mycobacteria cell walls have a high concentration of waxy lipid substances (mycolic acid) Prevent dyes from staining the cells Heat is used to help stain cells Primary stain: Carbolfushin Counterstain: Methylene blue Lab #4 – Acid fast staining (9)

10. Method (Pg. 45-46): 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 Lab #4 – Acid fast staining

11. 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) Lab #4 – Acid fast staining

12. Each student prepare two stains: M. tuberculosis S. epidermidis Record results on pg. 47 Lab #4 – Acid fast staining

13. Lab #4 – Hanging drop method (8A) Method to determine motility (pg. 41 - 42) Use a broth culture Need a depression slide, coverslip, and Vaseline Smear Vaseline on palm  scrape onto the four edges of coverslip (don’t mix vaseline with 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

14. Microscope observation: 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!) Observe to distinguish motility vs. Brownian motion Motility is distinct movement from point A to point B Brownian motion = jiggling in one spot/ gliding in direction Lab #4 – Hanging drop method

15. Each student prepare a hanging drop for: S. epidermidis E. coli Record motile or non-motile on pg. 44 Lab #4 – Hanging drop method