🎒 Laboratory Setup and Media Preparation

This lesson builds core elective concepts in BSc Agriculture with practical applications and exam-oriented clarity.

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Laboratory Setup and Media Preparation

Laboratory Design

A tissue culture laboratory must provide an aseptic working environment while supporting all stages from media preparation to plant transfer. A standard laboratory has several functional areas arranged to maintain a workflow from non-sterile to sterile zones.

Essential Sections

• Washing and sterilization room: Glassware cleaning, drying racks, autoclaves, and hot air ovens
• Media preparation room: Balances, pH meters, microwave or heating mantles, and chemical storage
• Transfer area (Inoculation room): Laminar air flow cabinets placed in a dust-free room with UV germicidal lamps
• Culture room (Growth room): Shelves with fluorescent lighting (2000 to 3000 lux), temperature control (25 plus or minus 2 degrees Celsius), and 60 to 70% relative humidity
• Hardening unit: Greenhouse or shade net area for acclimatizing rooted plantlets

Essential Equipment

Equipment	Function
Laminar air flow cabinet	Provides sterile HEPA-filtered air for aseptic manipulation
Autoclave	Sterilizes media, instruments, and water at 121 degrees Celsius, 15 psi, 15 to 20 minutes
Hot air oven	Dry sterilization of glassware at 160 to 180 degrees Celsius for 2 hours
pH meter	Adjusts media pH (typically 5.6 to 5.8) before autoclaving
Analytical balance	Weighs chemicals and growth regulators with precision
Distilled water unit	Produces double-distilled or deionized water for media preparation
Stereomicroscope	Dissects small explants like meristem tips

Nutrient Media

Murashige and Skoog (MS) Medium

The most widely used basal medium, developed in 1962. It contains:

• Macronutrients: Ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, potassium phosphate
• Micronutrients: Iron (as Fe-EDTA), manganese, zinc, boron, copper, cobalt, molybdenum, iodine
• Vitamins: Thiamine (B1), pyridoxine (B6), nicotinic acid, glycine
• Carbon source: Sucrose (20 to 30 g/L) providing energy for heterotrophic growth
• Gelling agent: Agar (6 to 8 g/L) or gellan gum for solid media

Other Common Media

• B5 (Gamborg's): Lower ammonium content; used for legume and cereal cultures
• White's medium: Lower salt concentration; used for root cultures
• WPM (Lloyd and McCown): Woody Plant Medium for tree species
• Nitsch and Nitsch: Used for anther and microspore culture

Plant Growth Regulators

Growth regulators are the most critical component for directing cell differentiation:

• Auxins (2,4-D, IAA, IBA, NAA): Promote callus formation, root initiation, and somatic embryogenesis
• Cytokinins (BAP, kinetin, TDZ, zeatin): Promote shoot multiplication and axillary bud break
• High auxin to cytokinin ratio: Favors root formation
• High cytokinin to auxin ratio: Favors shoot formation
• Balanced ratio: Promotes callus proliferation

Media Preparation Steps

1. Prepare stock solutions of macronutrients, micronutrients, vitamins, and growth regulators
2. Measure appropriate volumes from stock solutions into a flask
3. Add sucrose and dissolve completely
4. Adjust pH to 5.6 to 5.8 using 0.1N NaOH or 0.1N HCl
5. Add agar and heat to dissolve (if solid medium is required)
6. Dispense into culture vessels (bottles, test tubes, or petri dishes)
7. Autoclave at 121 degrees Celsius, 15 psi for 15 to 20 minutes
8. Cool and store in a clean room until use

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Summary Cheat Sheet

Topic	Key takeaway
Main focus	Laboratory Setup and Media Preparation.
Section context	Revise this lesson with the rest of Foundations & Laboratory for stronger conceptual continuity.