1. SIBiol. The Microbial World Hands-On Session Day One Singapore Institute of Biology Workshop 30th - 31st May 2003

2. SIBiol. Culture Media Preparation Each group is to prepare: –One conical flask (250ml) containing 50ml LB Broth “Bacterial Growth Curve” experiment tomorrow –Two Nutrient Agar plates “Antiseptics and Disinfectants” and “Antibiotics” expts. –Two/Three Nutrient Agar plates one for each member of group for “How Clean Are You?” –Inoculate 5ml of provided LB broth with single colony of Escherichia coli bacteria Incubate 37°C overnight for “Bacterial Growth Curve”

3. SIBiol. Bacteria and Preservatives Dissolve one stock cube in 250ml tap water Divide solution into 4 beakers Add one teaspoon of salt/sugar/vinegar respectively Cover top with cling film and leave overnight “Salt” “Sugar”“Vinegar”“Control” + Salt+ Sugar+ Vinegar Nothing added

4. SIBiol. Sour Milk Compare the two beakers of milk –Appearance –Smell (Do NOT taste, please!!) –pH - Acidic, Neutral or Alkaline? FreshLeft in the open What causes milk to go sour? Why does milk at room temperature go sour faster than milk kept in the fridge? Lactobacillus bacteria http://www.ulb.ac.be/sciences/cudec/ressources/lactobacillus.gif

5. SIBiol. How Clean Are You? Use one of your prepared Nutrient agar plates Do one plate for each person Try different parts of your body or test surfaces Write on underside of agar plate

6. SIBiol. How Clean Are You? Cover plate and seal with parafilm Incubate at 37°C overnight Flip plate over 1.Wash your hands thoroughly 2.Touch “Clean” side of plate with one hand 3.Rub fingers of other hand on body or test surface 4.Touch other hand to “Dirty” side of plate

7. SIBiol. Antiseptics and Disinfectants Use one of your prepared Nutrient agar plates Do one experiment per group Write on underside of agar plate

8. SIBiol. Antiseptics and Disinfectants Ensure soil solution is evenly swabbed over the agar surface Do not use excessive force! Swab agar using cotton bud dipped in soil solution Flip over plate

9. SIBiol. Antiseptics and Disinfectants Cover plate and seal with parafilm Incubate at 37°C overnight Place paper discs soaked in respective solutions onto agar plate A - Clorox B - Dettol C - Alcohol D - Iodine

10. SIBiol. Antibiotics Set up similar agar plate as before –Numbered 1 to 4 instead of A to D Swab with E. coli or environmental microbes Place provided antibiotic discs on agar

11. SIBiol. The Microbial World Hands-On Session Day Two Singapore Institute of Biology Workshop 30th - 31st May 2003

12. SIBiol. Bacterial Growth Curve Procedure –Aseptically pour 5ml of overnight E. coli bacterial culture into 50ml of fresh LB media in 250ml flask –Immediately pipette 3ml of media into cuvette and read absorbance reading at 600nm –Incubate flask at 37°C with vigorous shaking –Take out 3ml of media every 15 minutes and take absorbance reading –Plot data on graph paper

13. SIBiol. Yeast can help fill Balloons! Add one tablespoon full respectively “Yeast”“Sugar”“Yeast and Sugar” Half-fill bottles with water + Yeast+ Sugar+ Yeast + Sugar

14. SIBiol. Growing Mould on Food Growing Mould on Fruits –Examine the specimens (Fruits) –Have a look at fungal colonies under the microscope Mouldy Bread –Examine the specimens (Slices of Bread) –Have a look at fungal hyphae under the microscope Yeast and Food Decomposition –Examine the specimens (Banana) –Have a look at yeast cells under the microscope

15. SIBiol. Follow-up (1) Follow-up to previous day’s experiments: Bacteria and Preservatives –Which beaker is the clearest? –Which beaker is the most cloudy? –What causes the cloudiness? How Clean Are You? –Describe the differences (if any) between the two halves of the plate –Where can you find bacteria/fungi/microorganisms? –What is an effective way to remove bacteria from our body?

16. SIBiol. Follow-up (2) Antiseptics and Disinfectants –Can you tell if there is more than one type of bacteria/fungi growing? –Which chemical disinfectant has the most effect on inhibiting microbial growth? Antibiotics –Can you tell if there is more than one type of bacteria/fungi growing? –Which antibiotic has the most effect on inhibiting microbial growth? –How did you determine this?

17. SIBiol. Clean Up Be Responsible! - Ensure proper disposal and decontamination of microbial samples Microbial samples and culture plates –Flood petri dishes with 10% bleach and seal with tape –Leave aside for 30 minutes, then dispose. Solid and Liquid waste –Collect in proper containers/bags for autoclaving Bench and work areas –Wipe down with 70% alcohol Wash your hands !!