Lab 8- Control Microbial Growth/ Antiseptics/ Water Testing

UV damages DNA and is maximally absorbed at…

210-300nm

UV causes…

• Thymine dimerization
  – Covalent bond between two adjacent thymine molecules on the same DNA
  strand
  – Removed with either the excision repair system or the light repair system

3 organisms used for UV experiment

• Bacillus megaterium, Staphylococcus epidermidis, and Escherichia coli
  – Exposure times for each organism
  • 15 sec, 1 min, 3 min, 5min, 8 min, and 8 min+ lid (control)

“lawn”

is when the organism grows in an even layer across the whole
surface of the plate

Part of the plate will be exposed for the assigned time while the
remaining part will be covered as a control…

– This allows us to directly compare exposed vs. unexposed plate growth

Antiseptic-

used to inhibit or kill microorganisms on a person’s
skin

Disinfectant-

harsher, used to inhibit or kill microorganisms on
inanimate surfaces

• Bacteriostatic-

prevent or inhibit group but do not kill bacteria
•

Bactericidal -

capable of destroying bacteria
•

Germicidal -

capable of kill or inactivate microbes

Procedure for Antiseptics and Disinfectants

Per table will use 2 organisms, work as a pair
• Divide Room:
– Groups 1&4: Staphylococcus epidermidis and Escherichia coli
– Groups 2&3: Klebsiella oxytoca and Bacillus megaterium
• Swab the surface of your plate with assigned organism in three directions
• Write the names of chemicals on the bottom of the plate
• Apply untreated discs onto the swabbed agar evenly spaced apart
• Make sure to tap down the discs to remove any bubbles and adhere them to the
surface
• Then add 4uL of compound to the respective filter paper
• Next week we will measure the diameter of the zone of inhibition (ZOI), the area of
no growth around disc
• No standard chart instead just presence or absence of clearing

Coliforms-

non-endospore forming, gram negative, rods, facultative
anaerobes, that ferment lactose to produce acid and gas within 24hrs
at 37 ⁰C

E.coli, a coliform… 

is a good indicator of fecal contamination
– Rarely found in the soil or water
– Instead inhabits the digestive tract of warm blood animals
– Less fastidious (survives longer in environment) than other potentially
pathogenic enteric organisms

Presence of coliforms suggests…

other more harmful organisms may
also be present without testing for each individually

Presumptive Test

• Statistical method to determine the most probable number(MPN) of coliforms in a
  sample
  - Uses the production of gas from lactose fermentation to determine a positive result
  - Tubes are scored +/- for gas and compared to a chart of values
  - Modified today to only use 3 replicate tubes for sample volume
  - 10mL gets Double Strength Lactose Broth
  - 1mL and 0.1mL use Single Strength Lactose Broth

Confirmed Test

• Performed to confirm if the organism is gram negative and produces acid from fermentation of lactose

• Usually streak positive sample from Presumptive test tube onto an

• Eosin Methylene Blue plate

Wet Mount

Culture is alive-allowing you to see motility of organism
• Wet mount procedure:
– Draw target circle
– Add several loops of sample
– Gently place coverslip on top of liquid in target circle
• Observe results at 10X and 40x objective. Pictures only taken at
400X total magnification