single cell culture

1. Single Cell Culture
Mrs. Praveen Garg
VITS College, satna

2. Introduction
 Single cell culture is a method of growing isolated single
cell aseptically on a nutrient medium under controlled condition.
 The single cells can be isolated from a variety of tissue and organ of
intact plant or from callus tissue or from cell suspension by using
either mechanical method or chemical method.
 The cell aggregates or clumps are removed from the suspension by
using mesh only and then the isolated single cells in the filtrate can be
cultured either in liquid or in solid media.
 The basic principle of single cell culture is the isolation of large
number of living cells and cultures them on a suitable nutrient
medium for their requisite growth and development.

3. Methods of Isolation
 In 1902, Haberlandt was the first person who made pioneering
attempts to isolate and culture a single cell from a flowering plant.
There are two methods of isolation.
• Mechanical isolation: It means chopping of the tissue material
by fine scalpel which releases few intact single cells or by glass
homogenizer the tissue may be crushed where the homogenate
containing only few intact cells can be cultured.
• Chemical isolation: In this method, the macerozyme or pectinase
can be used to dissolve the middle lamella and releasing single cells
from intact tissue.

4. Development of Plant cells from a single cell culture in Microchamber

5.  The single cells are isolated from the established friable callus tissue
and cell suspension culture.
 Mechanically, single cells are carefully isolated from cell suspension
or friable callus with a needle or fine glass capillary.
 Alternatively, the friable tissue is transferred to liquid medium and
the medium is continuously agitated by a shaker.
 Agitation of liquid medium breaks and dispenses the single cells and
cell clumps in the medium. As a result, it makes a cell suspension.
 The cell suspension is first filtered to remove cell clumps and the
filtrate is then centrifuged to collect the single cells from the pellete.

6. There are various methods of single cell culture.
 Paper raft nurse culture technique
 Micro-chamber technique
 Micro-droplet technique
 Nurse culture technique
 Cell plating technique.

7. Paper Raft Nurse Culture Technique
 The isolated single cells are placed aseptically on nutrient medium
soaked filter paper and placed on a actively growing callus tissue.
After small aggregate formation those are transferred to fresh media.
 With the help of paraffin oil and cover glass a micro-chamber is
formed on a glass slide and droplet containing single cells in
medium is placed inside this micro-chamber and incubated for
division.
Micro-Chamber Technique

8. Micro-Droplet Technique
 In this technique the single cells are cultured in special kind of
apparatus named Cuprak dishes which have two kinds of chambers,
small outer chamber filled with water and large inner chamber
carrying numerous wells each filled with micro-droplet of medium
containing single cells.
Nurse Culture Technique
 In this technique the growth or division of single cell is induced by
nurse callus. It has been observed that the single cells plated near
the callus tissue divide quickly to give rise to callus tissue. This
happening must be due to some leaching effect on the division of
single cells by the growing callus mass.

9. Cell Plating Technique
• The basic technique of plating is to first count the cell number
without maceration stage, this will enable a known number of cell
units to be established per unit volume of plating media.
• Both the cell suspension and nutrient medium containing agar are
prepared in double concentration separately.
• The equal volumes of suspension and the agar medium cooled at
35°C are mixed and then dispersed rapidly into petriplate in such a
manner that cells are evenly distributed in a thin layer.
• The dishes are then sealed and incubated for cell division, which will
give rise to callus.

11.  The plates may be observed under an inverted microscope and single
cells develop into callus, this method ensures the isolation of pure
single-cell clones.
 Usually, plating at cell densities of 103— 105 cells/ml or more yields
a high plating efficiency.
Observation

12. Factors Affecting Single Cell Culture
 The composition of the medium for the growth of single cell culture
is generally more complex than callus and cell suspension culture.
 Induction of division of single cells using paper raft technique
indicates that isolated cells get the exact essential nutrient from the
callus mass.
 It has been suggested that the callus mass leaches out the essential
nutrient through plasma membrane of the cells.
 In case of petri dish plating technique the initial plating cell density
is very critical.

13. Importance of Single Cell Culture
 Single cell culture technique is very important for the fundamental and
mutation studies and it has a wide industrial application.
 Single cell culture could be used successfully to obtain single cell clones.
 Plants could be regenerated from the callus tissue derived from the
single cell clones.
 Single cell culture are very important in relation to crop improvement
programmes.
 Isolated single cells can be handled as a microbial system for the
treatment of mutagens and for mutant selection.
 Many plants synthesize various important natural compounds in the
form of alkaloids, steroids etc. Some of these natural compounds are
highly medicinally important.
 Single cell culture is an ideal system for the study of biotransformation.

14. THANK YOU