1. Isolation of bacteria by dilution plating

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

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 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?)

5. Objective 1: Streak plate

6. 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

7. 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

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

9. 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 colonies Colony Forming Units: CFU

11. Objective 1: Next lab Gram stain To confirm you have a pure culture

12. 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: 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: dilution

13. Dilution 33
1:1000
1:10000
1:100000
1:1,000000

14. 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

15. 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

16. 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

18. 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)

19. 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

20. 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)

21. 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