CELL CULTURE

REAGENTS
LITHU.RADHAKRISHNAN
D3 BIOTECH
 INTRODUCTION
Animal cell culture basically involves the in vitro maintenance and
propagation of animal cells in a suitable nutrient media .
Thus , culturing is a process of growing of cells artificially .
Cell culture has become an indispensible technology in various branches of
life sciences .
It was in 1907 , ROUS HARRISON first developed a frog tissue culture
technique . He probably chose frog for two reasons being cooled blooded
animal , no incubation is required and tissue regeneration is fast in frog .
Biological industries reagents and supplements are specially designed for cell
culture allowing for the growth and propogation of a wide spectrum of cells
types under controlled culture .
In vitro cell culture systems provide the researcher with the appropriate means
for effectively studying cell growth and differentiation , in order to understand
the cellular response to specific environmental stimuli .
 Cell culture reagents
Cell culture is the processing by which cells are grown under
controlled conditions , generally outside of their natural
environment .
In practice , the term cell culture now refers to the culturing of
cells developed from multicellular eukaryotes , especially animal
cells , in contrast with other types of culture that also grow cells ,
such as plant tissue culture , fungal culture and microbiological
culture ( of microbes ) .
Cell culture reagents are used for culturing of animal cells . There
are different types of reagents used in animal cell culture .
 Antibiotics
Antibiotics or antibacterials are a type of antimicrobial used in treatment and
prevention of bacterial infection . They may either kill or inhibit the growth of
bacteria .
Several antibiotics are also effective against fungi and protozoans , and some are toxic
to humans and animals , even when given in therapeutic dosage .
Antibiotics are effective against viruses such as the common cold or influenza , & may
be harmful when taken inappropriately .
In 1928 ALEXANDER FLEMING identified PENICILLIN the first chemical
compound with antibiotic properties .
Sometimes the term antibiotic is used to refer to any substances used against microbes ,
synonymous to antimicrobial , leading to the widespread to but incorrect belief that
antibiotics can be used against viruses .
Two types of mechanisms of antibiotics are : Bacteriostatic and Bactericidal .
Bacteriostatic include Tetracycline , Spectinomycin , Sulphonamides , Macrolides ,
Chloramphenical , Trimethprim .
 Bactericidal include Penicillins , Cephalosporins , Fluoroquinolones ,
Glycopeptides , Monobactams , Carbapenems .
Antibiotics modes of action include :
i. Inhibitors of DNA synthesis
ii. Inhibitors of bacterial protein synthesis
iii. Inhibitors of bacterial cell wall synthesis
 BIOLOGICAL BUFFERS
Many biochemical process are markedly impaired by even small changes in the
concentration of free H+ ions .
Different inorganic substances were originally used as buffers ( Eg : phosphate , borate ,
bicarbonate) and later weak organic acids were also used .
Many of these buffer substances , however , have the disadvantage that they are not inert
and have lasting effects on the system under investigation ( Eg : inhibition of enzymes ,
interactions with enzyme substrates ) .
Requirements of biological buffers :
i. Solubility
ii. Permeability
iii. Ionic strength
iv. Dependence of the pKa value
v. Complex formation
vi. Inert substances
vii. UV absorption
 TRIS buffer is probably the most frequently used buffer substance in biological experiments .

The reasons for this are that TRIS is comparatively inexpensive , very freely
soluble in water , is inert in many enzymatic systems and has a high buffer
capacity .
TE buffer 10mM , Tris-Hcl (ph 7.4 , 7.5 , or 8 ) , 1mM EDTA (ph 8.0 ) . This
buffer has become the standard buffer for the storage of nucleic acids . It is
used at different ph values .
BALANCED SALT SOLUTION
The balanced salt solutions (BSS) are primarily composed of inorganic salts .
It is a solution made to a physiological ph and isotonic salt concentration .
Sometimes , sodium sodium bicarbonate , glucose and HEPES ( N-2 hydroxy
ethyl piperazine N-2-ethane sulphonic acid ) buffer may also be added to BSS
.
BSS are used for washing tissues and cells and are usually combined with
other agents to treat the tissues and cells .
They provide the cells with water and inorganic ions , while maintaining a
physiological ph and osmotic pressure .
Sometimes glucose is added as an energy source and phenol red as a ph
indicator .
BSS is also useful for a short period (upto 4hrs ) incubation of cells .
 The important functions of balanced salt solutions are :
1. supply essential inorganic ions
2. provide the requisite ph
3. maintain the desired osmolality
4. supply energy from glucose
List of balanced salt solution
Alsevers solution
Earles balanced salt solution (EBSS)
Geys balanced salt solution (GBSS)
Hanks balanced salt solution (HBSS)
Phosphate buffered saline (PBS)
Ringers balanced salt solution (RBSS)
Pucks balanced salt solution
Simms balanced salt solution (SBSS)
TRIS buffered saline (TBS)
Tyrodes balanced salt solution (TBSS)
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