Isolation of bacteria by dilution plating

1
Isolation of bacteria by dilution plating

• We will finish this lab next Monday
• We will also begin lab 7 on Monday

2
Pure vs mixed culture

• Pure originate from 1 bacteria strain
• All colonies look the same
• Mixed originate from many bacteria strains
• Colonies have different size/shape

http//smccd.net/accounts/case/biol240/streakplate
.html
3
Streak plate technique

• Spread millions of cells over the surface
• Individual cells deposited at a distance from all
  others
• Divide forming distinct colonies
• Distinct colonies do not touch any other colonies
• Clone of a single bacteria ? pure culture

4

• Disinfect your bench, wash your hands and wear
  gloves
• Label the bottom of a TSA plate
• TSA Tryptic Soy agar (general high nutrient
  media)
• Keep lid closed when the plate is not in use
• You streak the plate on 3 different portion
• You can draw the section that you will streak on
  the bottom of your plate

5
Objective 1 Streak plate
http//www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/la
b_manual/streakplate3.jpg
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Streak plate

• Using a sterile loop take a loopful of your
  bacteria from the broth
• Streak a vertical line
• Then streak gently across section 1
• Zig-zag pattern until a 1/3 of the plate is
  covered
• Do not dig into the agar

http//www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/la
b_manual/streakplate2.jpg
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Streak plate

• Sterilize the loop ? let it cool
• Rotate the plate about 90 degrees and spread the
  bacteria from the first streak into a second area
• Do only one streak (or very few) in the first
  area and once you are in the second area do not
  go back to the first
• Do a zig-zag pattern until the 2nd area is
  covered
• Sterilize again ? do the same for 3rd area

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First section
third section
Sterilize loop, let it cool
Sterilize loop, let it cool
second section
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Sterilized loop

• Make sure that your red hot loop is cool enough
  prior to touch the bacteria
• After you waited a few seconds
• Stab it into the agar in a position away from
  bacteria ? will cool it
• If you stab where bacteria are ? production of
  aerosol

10
Isolated coloniesColony Forming Units CFU
http//faculty.mc3.edu/jearl/ML/ml-9.htm
http//www.bact.wisc.edu/themicrobialworld/Prop.ac
nes_colonies.jpg
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Objective 1Next lab (Monday 18th February)
Gram stain
To confirm you have a pure culture
http//faculty.mc3.edu/jearl/ML/ml-9.htm
http//www.bact.wisc.edu/themicrobialworld/Prop.ac
nes_colonies.jpg
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Objective 2 Spread plate technique and dilutions

• Label four plates for this exercise
• Name, date, dilution
• Pipette 1 ml from the bacteria culture into 99ml
  saline 1100 dilution
• 0.1 ml of this into your first plate 11000
  dilution
• Pipette 1ml of your 100 ml dilution of bacteria
  in saline and put into a 9 ml tube 11000
• 0.1 ml of this into your 2nd plate 110000
  dilution
• Pipette 1ml of your 10 ml dilution of bacteria in
  saline and put into another 9ml tube 110000
• 0.1 ml of this into your 3rd plate 1 100000
  dilution
• Pipette 1ml of your 10 ml dilution of bacteria in
  saline and put into another 9ml tube 1100000
• 0.1 ml of this into your 4th plate 1 1000000
  dilution

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Dilution
33
11000
110000
1100000
11,000000
http//www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/la
b_manual/dilution.jpg
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Pipetting

• Place the end of the pipette into the opening
  pump
• Place the pipette tip into the solution
• Press top button ? aspire
• Read at the bottom of the meniscus
• Press bottom button ? dispense

Bottom of meniscus
Pipette tip
http//www.rlc.dcccd.edu/mathsci/reynolds/micro/la
b_manual/pipet_dilut.html
http//www.lab-services.nl/images/cellmate.gif
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Pipette other considerations

• Always change pipette when going from a more
  concentrated solution to a less concentrated
  solution ? avoid carry over
• Dilution are additive
• 110 dilution is 1 ml into 9 ml, not 1 ml into 10
  (that will be a 111 dilution)
• Mix well (pipette up and down or swirl gently) ?
  prior to take your sample for your dilution

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Spread plate technique

• Quantitative technique that allows the
  determination of the number of bacteria in a
  sample.
• Pipette the required amount of bacteria (from
  your dilution) on the surface of the Petri plate
• Spread the inoculum over the surface of the agar
  medium using a hockey stick
• Incubate the plate inverted at 37oC

http//www.woodrow.org/teachers/bi/1999/projects/g
roup5/gfx/flaming_stick.jpg
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http//www.bact.wisc.edu/Microtextbook/images/book
_3/chapter_10/10-4.jpg
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Counting colonies (1Next lab (Monday 18th
February)

• Count by looking at the bottom of the plate
  (while keeping the Petri plate closed)
• Agar is translucent you should not have to open
  the plate
• If there are a lot of colonies on the plate ?
  helpful to use a marker to mark the colonies
  already counted
• If there are tons of colonies ?TMTC (Too many to
  count)

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Counting bacteria

• We must have between 30 and 300 colonies on the
  plate
• Less than 30 might not be representative
• More than 300 very difficult to count
• Also we might not have isolated colonies

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CFU

• Colonies forming units
• Not the same as bacteria
• 2 bacteria might have been very close and formed
  one colony
• CFU per ml of sample number of colonies /
  (amount plated X dilution)

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CFU calculation example

• You count 46 colonies on your plate
• You put 1 ml of bacterial culture into 99 ml of
  saline and plated 0.1 ml
• Dilution 1/100
• CFU 46
• 1/100 0.1
• 46 100 10 46 000
• Dividing by 1/100 is the same as multiplying by
  100 0.1 1/10 Do not take amount plated in
  consideration twice