Cell Culture: Cell culture is the multifaceted process through which cells are isolated from

animal or plant and their subsequent growth under controlled artificial conditions, generally
outside their natural environment. In this procedure cells are directly isolated from body or
disaggregated by enzymatic or mechanical procedure or they may be derived from cell lines or
cell strains.
There are three major types of cell culture, which include;
 Primary cell culture
 Secondary cell culture, and
 Cell line
Here, we shall focus on primary cell culture.
Primary cell culture: Primary cell culture is the primary step of cell culturing in which the cell
is first isolated from tissue and then proliferated under the appropriate conditions until they
consume all available contents for their growth. Now the cell is ready for subculturing by
transferring them to new growth medium that furnish more opportunity for continued growth.
There are two types of primary cells:
 Adherent cells - Also referred to as anchorage dependent cells, these are the type of cells
that require attachment for growth. Adherent cells are immobile, and obtained from such
organs as kidney.
 Suspension cells - These are the type of cells that do not require attachment in order to
grow. They are therefore also referred to as anchorage independent cells, and include
such cells as lymphocytes found in the blood system.
Cell lines: A cell line is a permanently established cell culture that will proliferate indefinitely in
appropriate fresh medium and space. Cell lines differ from cell strains in that they have
absconded the Hayflick limit and become immortalised.
The Hayflick limit (or Hayflick Phenomenon) is the number of times a normal cell population
will divide before it stops, presumably because the telomeres reach a critical length.
A cell line arises from a primary culture at the time of the first successful subculture. The terms
finite or continuous are used as prefixes if the status of the culture is known.
Cell Strain: By applying cloning, the positive population of cell lines are selected, therefore this
cell lines now becomes a cell strain. A cell strain often acquires additional genetic changes
resulting to the initiation of the parent line.
Cultivation techniques: Batch, Fed-batch and Continuous culture technique
1. Batch culture technique
2. Fed-batch culture technique
3. Continuous culture technique

Batch culture technique

 Batch culture technique is also called as closed system of cultivation.
 A technique used to grow microorganisms or cells. A limited supply of nutrients for
growth is provided; when these are used up, or some other factor becomes limiting, the
culture declines. Cells, or products that the organisms have made, can then be harvested
from the culture.
 In this technique at first nutrient solution is prepared and it is inoculated with inoculum
(culture organism) and then nothing is added in the fermentation tank except aeration.
 In batch culture, neither fresh medium is added nor used up media is removed from the
cultivation vessel. Therefore volume of culture remains same.

Phases
Nutrient consumption and build-up of toxic by-products slow down and stop the growth of
bacteria. One method to avoid this is to use batch culture system. This involves culturing bacteria
in liquid medium within a closed system where no fresh medium is added during the incubation
period and hence the nutrient concentration declines and waste products accumulate during the
growth of bacteria.
The microorganisms reproduce by binary fission and their growth can be expressed as the
logarithm of cell number and can be plotted against incubation time. This result in a curve that
has 4 distinct phases
Lag Phase- when microorganisms are introduced into fresh culture medium no increase in cell
number or mass occurs. This marks the beginning of the Lag Phase which is necessary as the
cells may be old and depleted of ATP, certain essential cofactors and ribosome, the medium may
be different from the one that the microorganism was growing in previously, or the
microorganism may have suffered injuries and may need time to recover
Exponential Phase- during this phase the rate of growth of microorganisms is constant and they
divide and become double in number at regular intervals and at the maximal rate possible given
their genetic potential, nature of the medium and the conditions of growth
Stationary Phase- during this phase the growth curve becomes horizontal as growth of the
population ceases. Bacteria enter this phase at a population level of around cells per ml. The total
number of viable organisms remains constant due to a balance between cell division and cell
death or because the population ceases to divide but still remains metabolically active. Microbial
populations enter the stationary phase due to reasons such as nutrient limitations, oxygen
availability and accumulation of toxic waste products
Death Phase- characteristic feature of this phase is decline in the number of viable cells due to
nutrient deprivation and build-up of toxic wastes. The death of a microbial population is
logarithmic. The extended survival of particularly resistant cells may reduce the death rate. In
this phase the number of cells that die during a given time period is proportional to the number of
cells that existed at the beginning of the time period

Advantages of using batch cultures:

 They allow versatility and can be used for many different reactions.
 They are safe and do not pose a threat of strain mutation.
 They ensure complete conversion of substrate.
 The changing conditions during the use of batch cultures affect the physiology and
growth of bacteria and hence highlight the ability of bacteria to adapt to its environment.
Disadvantages of using batch cultures:
 Use of batch cultures requires highly skilled labor thus increasing labor costs.
 Use of batch cultures is time consuming
 Changing concentrations of products and reactants, varying pH and oxidation-reduction
potential makes interpretation of results difficult.
 Complicated mix of growing, dying and dead cells also makes interpretation difficult.