The Microbial World

1
The Microbial World

• Hands-On Session
• Day One

Singapore Institute of Biology Workshop 30th -
31st May 2003
2
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 37C overnight for Bacterial Growth
  Curve

3
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

4
Sour Milk

• Compare the two beakers of milk
• Appearance
• Smell (Do NOT taste, please!!)
• pH - Acidic, Neutral or Alkaline?

• What causes milk to go sour?
• Why does milk at room temperature go sour faster
  than milk kept in the fridge?

5
How Clean Are You?
Write on underside of agar plate

• Use one of your prepared Nutrient agar plates
• Do one plate for each person
• Try different parts of your body or test surfaces

6
How Clean Are You?
Flip plate over

• Cover plate and seal with parafilm
• Incubate at 37C overnight

7
Antiseptics and Disinfectants

• Use one of your prepared Nutrient agar plates
• Do one experiment per group

8
Antiseptics and Disinfectants

• Ensure soil solution is evenly swabbed over the
  agar surface
• Do not use excessive force!

9
Antiseptics and Disinfectants
Place paper discs soaked in respective solutions
onto agar plate
A - Clorox B - Dettol C - Alcohol D - Iodine
Initials Date
A
B
C
D

• Cover plate and seal with parafilm
• Incubate at 37C overnight

10
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
The Microbial World

• Hands-On Session
• Day Two

Singapore Institute of Biology Workshop 30th -
31st May 2003
12
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 37C with vigorous shaking
• Take out 3ml of media every 15 minutes and take
  absorbance reading
• Plot data on graph paper

13
Yeast can help fill Balloons!

• Add one tablespoon full respectively

14
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
Follow-up (1)

• Follow-up to previous days 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
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
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 !!