2 Bacterial culture in laboratory Introduction : bacterial growth
3 Bacterial growthMultiplication by binary fission: the bacteria grows and divides into two identical daughter cells.Characterized by a generation time (G) variable depending on the species20 minutes for Escherichia coli, Lactobacillus acidophilus for 100 minutes, 1000 minutes to Mycobacterium tuberculosis
6 Bacterial growth For example, Escherichia coli G= 20 minutes After 1 hour: 3 cycles : n = 23 = 8 bacteriaAfter 2 hours: 6 cycles : n = 26 = 64 bacteriaAfter 3 hours : 9 cycles : n = 29 = 512 bacteriaAfter 4 hours : 12 cycles: n = 212 = 4096 bacteriaAfter 5 hours : 15 cycles: n = 215 = bacteria…After 10 hours : 30 cycles : n = 230 = bacteriaMore than a billion bacteriaafter 10 hours of culture!
7 Bacterial culture in laboratory Culture mediaand 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 mediaBacteriaIncreasing the cell density during the growthClear media
10 1 bacteria 1 visible bacterial colony Culture in solid mediaBacteriaAfter h :1 bacteria 1 visible bacterial colonyAccumulation of cells during the growth
11 Inoculation technique In liquid media :In solid media (usually with Agar Petri dish) :Inoculation with pipetteSpreading with a rakeRidges with a loopInoculation with loopRidges with a swab
12 Bacterial culture in laboratory Aseptic conditions in lab
13 Aseptic conditions in lab Problem 2 :Bacteria can contaminate the technicianProblem 1 :Bacteria from environment can contaminate the preparationBacteria to study…Problem 3 :Technician can contaminate the preparationNeed 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 burnerThis area is use to open tube or agar Petri dish with bacteriaConvection air movement
17 Aseptic conditions in lab Organization of the workspaceBunsen Burner is place in the center of the workspace and all instruments are distributed around (aerial view) :Rack tubesBunsen BurnerBox with instruments (pipettes, inoculation loops…)Trash for contaminated instrumentsAgar Petri dish
18 Aseptic conditions in lab Organization of the workspace
19 Aseptic conditions in lab Consumables materialsMost microbiology labs use sterile disposable equipment :Inoculation needles and loopsVolumetric pipettesSpreadingrakePipettes