Subculture involves transferring cells from an existing culture to fresh growth medium in order to prolong the life and expand the number of cells. This is necessary because over time toxic metabolites build up and nutrients are depleted in the original culture. Subculture produces a new culture with lower cell density and fresh nutrients. It allows for long-term maintenance of cell lines and increasing cell numbers for industrial or research purposes. Different techniques are used to subculture adherent versus non-adherent cells.

1. Topic
Subculture and its
importance

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.

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.

18. Cell scrapers and cell lifters