This lecture discusses tissue culture and the necessary equipment and environment. The basic equipment includes an incubator, hood, centrifuge, microscope, etc. The culture environment requires specific chemical factors in the media like essential elements, organic supplements, carbon sources, and plant growth regulators to control cell growth. The lecture outlines the objectives, key equipment, and factors that influence the cellular environment.

1. Lecture 2
By
Dr. Ahmed Metwaly
TISSUE CULTURE

2. Objectives:
Cell Culture Equipments
■ Basic Equipments
■ Expanded Equipment
■ Additional Supplies

3. Objectives:
The culture environment
■ Chemical factors (Plant cell culture media)
■ Essential elements
■ Organic supplements
■ Source of fixed carbon
■ Plant growth regulators

4. Cell Culture Equipments
Basic Equipments
■ Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)
■ Incubator (humid CO2 incubator recommended)
■ Water bath
■ Centrifuge
■ Refrigerator and freezer (–20°C)
■ Cell counter
■ Inverted microscope
■ Liquid nitrogen (N2) freezer
■ Sterilizer (autoclave)

5. Cell culture hood ( laminar-flow
hood or biosafety cabinet)

6. How does laminar hood flow
work?
■ laminar flow is the case occurs when a fluid or air flows in
parallel layers, with no disruption between the layers.
■ Laminar flow hoods are used to exclude contaminants by
passing a sterile air from inside to outside.

7. Incubator
Incubator is a device used to
grow and maintain cell
cultures. The incubator
maintains optimal
temperature, humidity and
other conditions such as
the carbon dioxide (CO2)
and oxygen content of the
atmosphere inside.

8. ■ The purpose of the incubator is to provide the appropriate
environment for cell growth.
■ The incubator should be large enough for your laboratory needs,
have forced air circulation, and should have temperature control .
■ Stainless steel incubators allow easy cleaning and provide
corrosion protection, especially if humid air is required for
incubation.

9. Types of Incubators
Types of Incubators
There are two basic types of incubators, dry incubators and humid CO2
incubators.
■ Dry incubators are more economical, but require the cell cultures to
be incubated in sealed flasks to prevent evaporation. They do not
allow precise control of atmospheric conditions in the incubator.
■ Humid CO2 incubators are more expensive, but allow superior control
of culture conditions. They can be used to incubate cells cultured in
Petri dishes or multi-well plates, which require a controlled
atmosphere of high humidity and increased CO2 tension.

10. Centrifuge
A centrifuge is a piece of equipment
that puts an object in rotation
around a fixed axis (spins it in a
circle), applying a potentially strong
force (outward). The centrifuge
used for sedimentation of insoluble
substances.

11. Water bath freezer (–20°C)
Most cell culture reagents can be stored at –5°C to –20°C;. A
domestic freezer is a cheaper alternative to a laboratory freezer.

12. Cell counter or hemocytometer
■ Automated cell counters sample the culture, quantify,
classify, and describe cell populations using both
electrical and optical techniques.
■ A cell counter is essential for quantitative growth
kinetics.

13. Inverted microscope
■ An inverted microscope is
a microscope with its light
source and condenser on the
top, above the stage pointing
down, while the objectives and
turret are below the stage
pointing up.
■ Inverted microscopes are
useful for observing living cells
at the bottom of a large
container (e.g., a tissue culture
flask) under more natural
conditions than on a glass
slide, as is the case with a
conventional microscope.

14. Liquid nitrogen or cryostorage
container
Liquid nitrogen
is nitrogen in a liquid state
at an extremely low
temperature.
Liquid nitrogen is
a cryogenic fluid that can
cause rapid freezing on
contact with living tissue.

15. Sterilizer (autoclave)
An autoclave is a pressure
chamber used to carry out
sterilization processes under
elevated temperature and
pressure .

16. Expanded Equipment
■ • Aspiration pump
■ • pH meter
• Aspiration pump pH meter

17. ■ Confocal microscope
Confocal microscopy is an optical imaging technique for increasing optical
resolution and contrast of a micrograph. It enables the reconstruction of three-
dimensional structures from the obtained images.

18. ■ Flow cytometer
Flow cytometry is a laser-based, biophysical technology employed in cell
counting, cell sorting, biomarker detection and protein engineering, by
suspending cells in a stream of fluid and passing them by an electronic
detection apparatus.

19. Additional Supplies
■ • Cell culture vessels (e.g., flasks, Petri dishes, roller bottles,
multi-well plates)
■ • Pipettes and pipettors
■ • Syringes and needles
■ • Waste containers

21. The culture environment
■ Chemical factors (Plant cell culture media)
■ Physical factors such as temperature, pH, the gaseous
environment, light (quality and duration).

22. Plant cell culture media
Culture media used for the in vitro cultivation of plant cells are
composed of three basic components:
■ (1) essential elements, or mineral ions, supplied as a complex
mixture of salts;
■ (2) an organic supplement supplying vitamins and/or amino
acids; and
■ (3) a source of fixed carbon; usually supplied as the sugar
sucrose.

23. Essential elements

24. The essential elements are further divided into the following
categories:
■ (1) Macroelements (or macronutrients);
■ (2) Microelements (or micronutrients); and
■ (3) An iron source.

25. ■ Macroelements
those elements required in large amounts for plant growth
and development. Nitrogen, phosphorus, potassium,
magnesium, calcium and sulphur (and carbon, which is
added separately) are usually regarded as
macroelements. These elements usually comprise at
least 0.1% of the dry weight of plants
■ Microelements
These elements are required in trace amounts for plant
growth and development, and have many and diverse
roles. Manganese, iodine, copper, cobalt, boron,
molybdenum, iron and zinc usually comprise the
microelements, although other elements such as nickel
and aluminium are frequently found in some
formulations.

26. ■ Iron Source
Iron is usually added as iron sulphate, although iron citrate
can also be used. Ethylene diamine tetraacetic acid (EDTA)
is usually used in conjunction with the iron sulphate. The
EDTA complexes with the iron so as to allow the slow and
continuous release of iron into the medium.
Uncomplexed iron will precipitate out of the medium as ferric
oxide.

27. ■ Organic supplements
■ Only two vitamins, thiamine (vitamin B1) and myoinositol
(considered a B vitamin) are considered essential for the
culture of plant cells in vitro
■ Amino acids are also commonly included in the organic
supplement. The most frequently used is glycine
(arginine, asparagine, aspartic acid, alanine, glutamic
acid, glutamine and proline are also used). Casein
hydrolysate can be used as a relatively cheap source of a
mix of amino acids.

28. ■ Carbon source
Sucrose is cheap, easily available and relatively stable and
is therefore the most commonly used carbon source.
Other carbohydrates such as glucose, maltose, galactose
and sorbitol) can also be used and in specialised
circumstances may prove superior to sucrose.
■ Gelling agents
Media for plant cell culture in vitro can be used in either
liquid or ‘solid’ forms, depending on the type of culture
being grown. For any culture types that require the plant
cells or tissues to be grown on the surface of the
medium, it must be solidified (more correctly termed
‘gelled’). Agar, produced from seaweed, is the most
common type of gelling agent, and is ideal for routine
applications.

29. Composition of Murashige and Skoog (MS) plant culture medium.

30. ■ Plant growth regulators
Plant growth regulators are the critical media components in
determining the developmental pathway of the plant cells. The
plant growth regulators used most commonly are plant hormones
or their synthetic analogues
■ . There are five main classes of plant growth regulator used in
plant cell culture, namely:
■ (1) auxins;
■ (2) cytokinins;
■ (3) gibberellins;
■ (4) abscisic acid;
■ (5) ethylene.

31. ■ Auxins
Auxins promote both cell division and cell growth The most
important naturally occurring auxin is IAA (indole-3-acetic
acid), but its use in plant cell culture media is limited
because it is unstable to both heat and light.
Occasionally, amino acid conjugates of IAA (such as
indole-acetyl-L-alanine and indole-acetyl-L-glycine), which
are more stable.
2,4-Dichlorophenoxyacetic acid (2,4-D) is a stable
chemical analogues of IAA and is the most commonly
used auxin and is extremely effective in most
circumstances.

32. Commonly used auxins, their abbreviation and chemical name

33. ■ Cytokinins
Cytokinins promote cell division. Naturally occurring
cytokinins are a large group of structurally related
purine derivative compounds. Of the naturally
occurring cytokinins.
These are zeatin and 2iP (2-isopentyl adenine). Their use
is not widespread as they are expensive and relatively
unstable. The synthetic analogues, kinetin and BAP
(benzylaminopurine), are therefore used more
frequently.
Non-purine-based chemicals, such as substituted
phenylureas, are also used as cytokinins in plant cell
culture media.

34. Commonly used cytokinins, their abbreviation and chemical name

35. ■ Gibberellins
There are numerous, naturally occurring, structurally related
compounds termed ‘gibberellins’.
They are involved in regulating cell elongation, and are agronomically
important in determining plant height.
■ Abscisic acid
Abscisic acid (ABA) inhibits cell division and some times stimulate it,
according to plant species. It is most commonly used in plant tissue
culture to promote distinct developmental pathways such as
somatic embryogenesis.

36. The effect of different ratios of auxin to cytokinin on the growth and morphogenesis of callus

37. Summary:
Cell Culture Equipments
■ Basic Equipments
■ Expanded Equipment
■ Additional Supplies

38. Summary:
The culture environment
■ Chemical factors (Plant cell culture media)
■ essential elements
■ Organic supplements
■ Source of fixed carbon
■ Plant growth regulators