3. Sub-culturing
• A subculture is a new cell or microbiological culture made by
transferring some or all cells from a previous culture to fresh growth
medium.
4. • This action is called sub-culturing or passaging the cells.
• Subculture is used to prolong the life .
• Or expand the number of cells or microorganisms in the culture.
5. Role
• Cell lines and microorganisms cannot be held in culture .
• Subculture is therefore used to produce a new culture with a lower
density of cells than the originating culture, fresh nutrients and no
toxic metabolites allowing continued growth of the cells without risk
of cell death.
• Subculture is important for both proliferating (e.g. a microorganism
like E. coli) and non-proliferating (e.g. terminally differentiated white
blood cells) cells.
6.
7. Conti….
• subculture is from a culture of a certain volume into fresh growth
medium of equal volume.
• This allows long-term maintenance of the cell line.
• Subculture into a larger volume of growth medium is used when
wanting to increase the number of cells .
• For example, use in an industrial process or scientific experiment
8. Materials
•
Culture vessels containing your adherent cells
• Tissue-culture treated flasks, plates or dishes
• Complete growth medium, pre-warmed to 37°C
• Disposable, sterile 15-mL tubes
• 37°C incubator with humidified atmosphere of 5% CO2
9. Conti….
• Balanced salt solution such as Dulbecco’s Phosphate Buffered Saline
(DPBS), containing no calcium, magnesium, or phenol red
• Dissociation reagent such as trypsin , without phenol red .
• Reagents and equipment to determine viable and total cell counts
such as Countess II FL Automated Cell Counter, Trypan Blue and
hemacytometer, or Coulter Counter.
10. • In the laboratory, micro-organisms are usually grown or cultured in
liquid medium (broth) or on solid medium (agar plates or slopes).
• Growth of bacteria and yeasts shows as cloudiness or turbidity in the
broth .
• The growth on plates depends upon how the plate has been
inoculated.
• And Types of inoculations on agar plates.
11. • Subculturing is the aseptic transfer of micro-organisms from a culture
to fresh medium.
• The freshly inoculated medium is then incubated at the temperature
appropriate for growing the organism.
12. Procedures:
• There are four sub-culturing procedures they are:
• Solid to solid: the transfer of bacteria or fungi from an agar slope or
plate culture to an agar plate.
• Solid to liquid: the transfer of bacteria or fungi from an agar slope or
plate culture to a broth.
• Liquid to solid: the transfer of bacteria or fungi from a broth culture
to an agar slope or plate.
13. • Liquid to liquid: the transfer of bacteria or fungi from a broth culture
to a broth .
• Containers of culture media to be inoculated must be labelled with
initials, date and name of organism.
• To prevent possible confusion, plates are marked on the underside
while tubes and bottles must be labelled on the side.
• Lids are not labelled.
