1. Bacterial culture in laboratory

2. Bacterial culture in laboratory
Introduction : bacterial growth

3. Bacterial growth
Multiplication by binary fission: the bacteria grows and divides into two identical daughter cells.
Characterized by a generation time (G) variable depending on the species
20 minutes for Escherichia coli, Lactobacillus acidophilus for 100 minutes, 1000 minutes to Mycobacterium tuberculosis

4. Bacterial growth

5. Bacterial growth
After n cycles : 2n bactéria

6. Bacterial growth For example, Escherichia coli G= 20 minutes
After 1 hour: 3 cycles : n = 23 = 8 bacteria
After 2 hours: 6 cycles : n = 26 = 64 bacteria
After 3 hours : 9 cycles : n = 29 = 512 bacteria
After 4 hours : 12 cycles: n = 212 = 4096 bacteria
After 5 hours : 15 cycles: n = 215 = bacteria …
After 10 hours : 30 cycles : n = 230 = bacteria
More than a billion bacteria
after 10 hours of culture!

7. Bacterial culture in laboratory
Culture media
and inoculation technique

8. Culture media Solid by adding a gelling agent (agar) to a liquid media

9. Culture in liquid media
After 16h of culture = turbid media
Bacteria
Increasing the cell density during the growth
Clear media

10. 1 bacteria  1 visible bacterial colony
Culture in solid media
Bacteria
After h :
1 bacteria  1 visible bacterial colony
Accumulation of cells during the growth

11. Inoculation technique
In liquid media :
In solid media (usually with Agar Petri dish) :
Inoculation with pipette
Spreading with a rake
Ridges with a loop
Inoculation with loop
Ridges with a swab

12. Bacterial culture in laboratory
Aseptic conditions in lab

13. Aseptic conditions in lab
Problem 2 :
Bacteria can contaminate the technician
Problem 1 :
Bacteria from environment can contaminate the preparation
Bacteria to study…
Problem 3 :
Technician can contaminate the preparation
Need a sterile zone around the preparation !!

14. Aseptic conditions in lab
Equipment to create a sterile area to work…
Microbiological Safety Cabinet :
System which uses an HEPA filter (High Efficiency Particulate Air) to generate a flow of sterile air
 Protection for the preparation and the technician

15. Aseptic conditions in lab
Equipment to create a sterile area to work…
Bunsen Burner :
Gas burner which delivers a flame at 1300°C
 provides a sterile air zone near

16. Aseptic conditions in lab
Working with a Bunsen Burner :
Sterile air area :
Approximately 40 cm diameter around the burner
This area is use to open tube or agar Petri dish with bacteria
Convection air movement

17. Aseptic conditions in lab
Organization of the workspace
Bunsen Burner is place in the center of the workspace and all instruments are distributed around (aerial view) :
Rack tubes
Bunsen Burner
Box with instruments (pipettes, inoculation loops…)
Trash for contaminated instruments
Agar Petri dish

18. Aseptic conditions in lab
Organization of the workspace

19. Aseptic conditions in lab
Consumables materials
Most microbiology labs use sterile disposable equipment :
Inoculation needles and loops
Volumetric pipettes
Spreading
rake
Pipettes