1. Lab #3

2. Review of Lab #2 Gram staining  record results on pg. 35 Gram positive – purple Gram negative – pink Bacillus subtilis – Escherichia coli – Klebsiella pneumoniae – Staphylococcus epidermidis –

3. Review of Lab #2 Motility tall (pg. 43 – 44) Semisolid media (1/3 the normal amount of agar) Motile bacteria are able to move around in the media Observe motility for: E. coli – B. subtilis – K. pneumoniae – S. epidermidis – What bacterial structure provides motility? Flagella

4. Review of Lab #2 Aseptic technique Observe growth on slant, broth (pg. 20), and motility tall (pg. 43 - 44) Slant Bacterial growth

5. Review of Lab #2 Aseptic technique Observe growth on slant, broth (pg. 20), and motility tall (pg. 43 - 44)

6. Lab #3 – Oxygen requirements Oxygen requirement varies for microbes 4 major groups Obligate Aerobes Grow only in presence of oxygen Use O 2 as final electron acceptor in electron transport chain Obligate Anaerobes: Grow only in the absence of oxygen – O 2 is toxic to them Use other molecules (nitrates, sulfates, etc) as final electron acceptor Microaerophilic Require oxygen to be lower than atmospheric level for growth Facultative anaerobes Use oxygen when available but can grow in its absence

7. Removal of oxygen in lab Reducing agent Thioglycollate broth (inoculated in lab #2) Reduces 0 2 to H 2 0 Produces a range of oxygen levels Mechanical removal Vacuum to remove O2 and replace with other gas Chemical Reaction Reaction of O 2 and other chemicals results in loss of O 2

8. Oxygen requirements (thioglycollate broth)

10. Oxygen requirements (pg. 109-111) Use the slant, motility tall, and the thioglycolate broth to determine the oxygen requirements

11. Oxygen requirements (pg. 109-111) Use the slant, motility tall, and the thioglycolate broth to determine the oxygen requirements of the following organisms: E. coli – B. subtilis – K. pneumoniae – S. epidermidis – C. sporogenes – Record results on pg. 110 and 111

12. 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 H 2 O 2 H 2 0 + ½ O 2

13. Lab #3 – Catalase test Test for enzyme catalase (pg. 85-86): Aseptically transfer a visible amount of bacteria into the depression well of a concave slide Spread bacteria Add a drop of 3% or 10% H 2 O 2 to the bacteria on the slide Observe for bubbles (positive result) NOTE: Do not touch H 2 O 2 with loop

14. Catalase test result Test all four organisms: E. coli B. subtilis K. pneumoniae S. epidermidis Record results on pg. 86

15. Lab #3 – Endospore staining Type of structural staining Endospores Round or oval in shape Resistant structures produced for survival of some bacteria under stressful conditions Resistant to heat, drying, UV radiation, acids/bases, disinfectants and even Gram staining Usually found in Bacillus and Clostridium species

16. Endospore cycle

17. Endospore staining procedure Procedure on 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 Do this step 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

18. Endospore staining results 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 – B. subtilis – E. coli – K. pneumoniae – S. epidermidis – Record results on pg. 39