Isolation of bacteria by dilution plating

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

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

Disinfect your bench, wash your hands and wear gloves Label the bottom of a NA plate NA = nutrient 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 (why not top?)

Objective 1: Streak plate http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/streakplate3.jpg

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/lab_manual/streakplate2.jpg

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

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

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

Isolated colonies Colony Forming Units: CFU http://faculty.mc3.edu/jearl/ML/ml-9.htm http://www.bact.wisc.edu/themicrobialworld/Prop.acnes_colonies.jpg

Objective 1: Next lab 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.acnes_colonies.jpg

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 1:100 dilution 0.1 ml of this into your first plate 1:1000 dilution Pipette 1ml of your 100 ml dilution of bacteria in saline and put into a 9 ml tube 1:1000 0.1 ml of this into your 2nd plate 1:10000 dilution Pipette 1ml of your 10 ml dilution of bacteria in saline and put into another 9ml tube 1:10000 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 1:100000 0.1 ml of this into your 4th plate 1: 1000000 dilution

Dilution 33 1:1000 1:10000 1:100000 1:1,000000 http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/dilution.jpg

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/lab_manual/pipet_dilut.html http://www.lab-services.nl/images/cellmate.gif

Pipette other considerations Always change pipette when going from a more concentrated solution to a less concentrated solution  avoid carry over Dilution are additive 1:10 dilution is 1 ml into 9 ml, not 1 ml into 10 (that will be a 1:11 dilution) Mix well (pipette up and down or swirl gently)  prior to take your sample for your dilution

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/group5/gfx/flaming_stick.jpg

http://www.bact.wisc.edu/Microtextbook/images/book_3/chapter_10/10-4.jpg

Counting colonies (1:Next 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)

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

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)

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