Bacterialculture in laboratory

Bacterialculture in laboratory

Introduction : bacterial growth

Bacterial growthBacterial 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

Bacterial growthBacterial growth

Bacterial growthBacterial growth

After n cycles : 2n bactéria

Bacterial growthBacterial growthFor 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 = 32768 bacteria … After 10 hours : 30 cycles : n = 230 = 1073741824 bacteria

More than a billion bacteriaafter 10 hours of culture!

Bacterialculture in laboratory

Culture mediaand inoculation technique

Culture mediaCulture media Solid Solid by adding a

gelling agent (agar) to a liquid media

LiquidLiquid

Culture in liquid mediaCulture in liquid media

Clear media

After 16h of culture =

turbid media

Increasing the cell density during the growth

Bacteria

Culture in solid mediaCulture in solid media

Accumulation of cells during the growth

After 16 - 24h : 1 bacteria 1 visible bacterial

colonyBacteria

Inoculation techniqueInoculation technique

In liquid media : In solid media (usually with Agar Petri dish) :

Spreading with a rake

Ridges with a loop

Ridges with a swab

Inoculation with pipette

Inoculation with loop

Bacterialculture in laboratory

Aseptic conditions in lab

Aseptic conditions in labAseptic conditions in lab

Bacteria to study…

Problem 1 :Bacteria from environment can contaminate the preparation

Problem 2 :Bacteria can contaminate the technician

Problem 3 :Technician can contaminate the preparationNeed a sterile zone around the

preparation !!

Aseptic conditions in labAseptic conditions in labEquipment to create a sterile area to work…

System which uses an HEPA filter (High Efficiency Particulate Air) to generate a flow of sterile air Protection for the preparation and the technician

Microbiological Safety Cabinet :

Aseptic conditions in labAseptic conditions in labEquipment to create a sterile area to work…

Gas burner which delivers a flame at 1300°C

provides a sterile air zone near

Bunsen Burner :

Aseptic conditions in labAseptic conditions in labWorking with a Bunsen Burner :

Convection air movement

Sterile air area :

Approximately 40 cm diameter around the burner

This area is use to open tube or agar Petri dish with bacteria

Aseptic conditions in labAseptic conditions in labOrganization of the workspace

Bunsen Burner is place in the center of the workspace and all instruments are distributed around (aerial view) :

Box with instruments (pipettes,

inoculation loops…)

Trash for contaminated instruments

Rack tubes

Agar Petri dish

Bunsen Burner

Aseptic conditions in labAseptic conditions in labOrganization of the workspace

Aseptic conditions in labAseptic conditions in labConsumables materials

Most microbiology labs use sterile disposable equipment :

Inoculation needles and loops

Pipettes

Volumetric pipettes

Spreadingrake