plating and counting colonies

• what will a scientist do to estimated a whole population of microorganisms


  they will count a small sample of the culture
• what is meant by serial dilution


  this assumes a single bacterial cell will reproduce asexually to form a single colony of cells
• what techniques is used to proves serial dilution


  total viable count technique
• what is the first step of the total viable count technique to prove serial dilution


  place 9 cm^3 of sterile distilled water into five sterile test tubes using a sterile pipette
• during the total viable count technique what happens after you placed 9cm^3 of sterile water into 5 sterile test tubes


  place 1cm^3 of the original bacterial culture sample into the first tube and gently mix (this bacterial culture has now been diluted 10 times (and is said to be a 10^-1 dilution))
• during the total viable count technique what happens after the original bacterial culture has been placed into the first test tube


  transfer 1cm^3 of the 10^-1 diliution from the first test tube into the second test tube (that contains 9cm^3 of sterile distilled water) and gently mix
• what dilution is the first test tube in the total viable count technique


  10^-1 dilution / 10 times
• what dilution is the second test tube in the total viable count technique


  10^-2 dilution / 100 times
• during the total viable count technique what happens after 1 cm^3 of the first dilution has been added to the second test tube


  now transfer 1cm^3 of each diluted sample onto a sterile nutrient agar plate and use a sterile spreader to spread the sample around
• during the total viable count technique what happens after each of the first 2 test tube samples have been spread onto two agar plates


  repeat this twice more to give a total of 3 plates per dilution
• why is the spreading of each dilution 3 times needed


  to calculate a mean number of colonies
• during the total viable count technique what happens after 3 agar plates have been made of each dilution


  seal each agar plate with tape but not all the way around and incubate each agar plate at 25 degrees for 24-48 hours
• during the total viable count technique what happens after all the agar plates have been incubated for 24-48 hours at 25 degrees

  look for the dilution that shows distinct colonies without merging . count the number of distinct colonies on each plate
• what is meant by the merging within a agar plate

  this is when colonies clump together
• during the total viable count technique what happens after you have counted the number of distinct colonies on each plate


  multiply the number of colonies by the dilution factor to give the number of bacteria in the original 1cm^3 bacterial culture sample
• what inaccuracies may occur when carrying out a serial dilution if the original bacterial culture is under diluted


  if dilution is insufficient then the colonies might merge causing the counting to be inaccurate resulting in a under estimation of cell numbers
• what inaccuracies may occur when carrying out a serial dilution if the original bacterial culture is over diluted


  if dilution is too great then there will be too few colonies on each plate to count to be statistically sound
• what is meant by the term statistically sound

  leading to inaccuracies