Microbiological technqiues

Question 1

How are microorganisms cultured?

Answer 1

• on oslid media, usually prepared using agar and liquid media knwon as broth

Question 2

What does any medium for culturing microorganisms contain?

Answer 2

• energy source
• carbon source
• nitrogen source
• mineral salts
• growth factors
• water

Question 3

How are agar plates prepared?

Answer 3

• agar powder used to make a 1% solution and is booled
• thisis cooled until about 50 degrees
• nutrients added
  
  • unless a ready-made agar plate is used
• whilst molten (45 degrees)
  
  • agar poured into petri dishes

Question 4

Why are aseptic techniques necessary?

Answer 4

• avoid contamination by any other organisms
  
  • for a pure culture
• prevent release of potentially harmful organisms in the environment

Question 5

What are the asepotic techniques toi ensure a pure culture?

Answer 5

• wash hands and/or wear gloves
• make sure all apparatus are sterilised before use
• sterilised culture media
• pour agar into petri dish with minimum risk of contamination (next flashcard)
• hold inoculating (colelcting) loop in bunsen for 30 seconds then allow to cool before using to transfer microorganisms
• do not open plates after they have been inoculated
  *

Question 6

What are the aseptic techniques to ensure the work environment is clean?

Answer 6

• sterilise lab bench
  
  • wipe with disinfectant
• dispose of all glassware by placing in a pressure cooper
• place disposable items, such as syringes and pippete tips into a container marked ‘waste’
• petri dishes should be wrapped in plastic bag, tied and sterilised in an autoclave

Question 7

How is the agar poured with minimum risk of conamination?

Answer 7

• use thumb and index finger to lift lid to 40 degrees
• petri dish rested on bech surface
• bottle neck close to petri dish
• cooled agar
• cloth wrapped around bottle

Question 8

How is the plate inoculated?

Answer 8

• pass the inoculating loop of bunsen flame (roaring)
  
  • sterilised
• remove plug from culture
  
  • flame neck of culture tube
• take sample by placing loiop in bottle
• flame neck again and replace pug
• touch the agar surface gently with the loiop
• sterilise liip again

Question 9

What is direct counting? How is it done? what are the problem?

Answer 9

• samples removed at intervals and examined in a coutnign chamber
• if there are too many to count then they must be diluted with water
• the disadvantage of this method is that it gives total counts, including both dead and alive microorganisms

Question 10

What is viable counting?

Answer 10

• samples diluted and spread onto agar
• each bacterium or yeast grows into a colony
• if the sample has been diluted enough, then the number of colonies can be countred
• this method only counts the cells that were alivew when the samples were taken
  
  • takes about 24 hours for colonies to form

Question 11

What is turbidimetry? What are the cpontrols

Answer 11

• samples placed intoa coloriumeter and readings taken
• method does not distinguish between living and dead
• converteds to poulation number using conversion graphs
• each smaple taken from a liquid culture myust be representative of whole culture to make results valid
• culture vessel shakebn to distribute the bacteria evenly
• sampke renmoved with sterile pipette placed halfway down the vessel
• several sample - at least three - taken to ensure the results are reliable