Transcript Microbial World "Hand on Session" powerpoint by Timothy Tan

The Microbial World
Hands-On Session
Day One
Singapore Institute of Biology
Workshop
30th - 31st May 2003
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”
SIBiol.
Bacteria and Preservatives
SIBiol.
+ Salt
+ Sugar
+ Vinegar
Nothing
added
“Salt”
“Sugar”
“Vinegar”
“Control”
• 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
Sour Milk
Fresh
Left in the open
• Compare the two beakers of milk
– Appearance
– Smell (Do NOT taste, please!!)
– pH - Acidic, Neutral or Alkaline?
• What causes milk to go sour?
Lactobacillus bacteria
• Why does milk at room temperature go sour
faster than milk kept in the fridge?
SIBiol.
http://www.ulb.ac.be/sciences/cudec/ressources/lactobacillus.gif
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
SIBiol.
How Clean Are You?
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
• Cover plate and seal with
parafilm
• Incubate at 37°C overnight
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4. Touch other hand to
“Dirty” side of plate
Antiseptics and Disinfectants
Write on underside of
agar plate
• Use one of your prepared Nutrient agar plates
• Do one experiment per group
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Antiseptics and Disinfectants
Flip over plate
Swab agar using
cotton bud dipped in
soil solution
• Ensure soil solution is evenly swabbed over
the agar surface
• Do not use excessive force!
SIBiol.
Antiseptics and Disinfectants
A - Clorox
B - Dettol
Place paper discs
soaked in respective
solutions onto
agar plate
C - Alcohol
D - Iodine
• Cover plate and seal with parafilm
• Incubate at 37°C overnight
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
SIBiol.
The Microbial World
Hands-On Session
Day Two
Singapore Institute of Biology
Workshop
30th - 31st May 2003
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
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Yeast can help fill Balloons!
+ Yeast
+ Sugar
“Yeast”
“Sugar”
+ Yeast
+ Sugar
“Yeast and
Sugar”
• Add one tablespoon full respectively
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Half-fill
bottles with
water
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
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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?
SIBiol.
– 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?
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?
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 !!
SIBiol.