2. Subculture
• In biology, a subculture is a
new cell or microbiological culture made by
transferring some or all cells from a previous
culture to fresh growth medium. This action is
called sub culturing or passaging the cells.
• Subculture is used to prolong the life and/or
expand the number of cells or microorganisms
in the culture.
3. Importance/Role
• microorganisms cannot be held in culture for
long time due to the gradual rise
in toxic metabolites, use of nutrients and
increase in cell number due to growth.
• 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.
4. • Subculture is important for both proliferating
(e.g. a microorganism like E. coli) and non-
proliferating (e.g. white blood cells) cells.
• Typically, 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 (culture).
5. • Subculture into a larger volume of growth
medium is used when wanting to increase the
number of cells for, for example, use in an
industrial process or scientific experiment.
6. Culture medium
• A growth medium or culture medium is a
liquid or gel designed to support the growth
of microorganisms or cells, or small plants
7. Components of Culture medium
• Nutrients: proteins/peptides/amino-acids.
• Energy: carbohydrates.
• Essential metals and minerals: calcium, magnesium,
iron, etc.
• Buffering agents: phosphates, acetates etc.
• Indicators for pH change: phenol red, bromo-cresol
purple etc.
• Selective agents: chemicals, antimicrobial agents.
• Gelling agent: usually agar.
• Vitamins
8. Passage number
• It is often important to record the approximate
number of divisions cells have had in culture
by recording the number of passages or
subcultures.
• In the case of plant tissue cells somaclonal
variation may arise over long periods in
culture. Similarly in mammalian cell
lines chromosomal aberrations have a
tendency to increase over time.
9. • For microorganisms (contaminations) there is
a tendency to adapt to culture conditions,
which is rarely quite like the microorganisms
in natural environment, which can alter their
biology
10. Protocols for passaging or sub
culturing
• The protocol for sub culturing cells depends
heavily on the properties of the cells involved.
• Non-adherent cells
• Adherent cells
11. Non-adherent cells
• Many cell types, in
particular many
microorganisms, grow in
solution and not attached
to a surface.
• These cell types can be sub
cultured by simply taking a
small volume of the parent
culture and diluting it in
fresh growth medium.
12. • Cell density in these cultures is normally
measured in cells per milliliter for large
eukaryotic cells.
• The cells will often have a preferred range of
densities for optimal growth and subculture
will normally try to keep the cells in this range.
13. Adherent cells
• Adherent cells, for example many mammalian
cell lines, grow attached to a surface such as
the bottom of the culture flask. These cell
types have to be detached from the surface
before they can be sub cultured.
14.
15. • For subculture cells may be detached by one of
several methods including
• Trypsin EDTA (Ethylenediaminetetraacetic
Acid) treatment to break down the proteins
responsible for surface adherence,
16. or
• mechanical methods like repeated washing or
use of a cell scraper.
• The detached cells are then re suspended in
fresh growth medium and allowed to settle
back onto their growth surface.
