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Bacterial culture in laboratory

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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

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