Laboratory Techniques in Biotechnology

Laboratory Techniques

Chemical Use Guideline

• Always use a product with a label to reference
• Do not mix chemicals without specific authorization
• Always use personal protective equipment
• When pouring chemicals, pour concentrates into the water and not vice versa
• Never pour chemicals into an empty, unlabeled container
• Do not store flammable chemicals near a source of heat
• Pesticides, fungicides, etc. must be stored in a safe and elevated position

Types of Chemicals

• Toxic chemicals: poisonous, examples include hydrogen sulfide and cyanide
• Corrosives: irritants, examples include chlorine and HCl acid
• Flammables: chemicals that burn readily, examples include gasoline, benzene, and ethyl ether
• Reactive: require stability and careful handling, examples include nitroglycerine

Basic Tips of Safe Chemical Handling

• Read the label
• Dress properly
• Follow directions
• Know emergency procedures
• Be careful
• Report any suspected problems
• Keep your work area neat, clean, and organized
• Store everything properly

Basic Rules of Chemical Safety

• Be aware, be alert, and be alive
• Don't buy or store chemicals you do not need
• Store chemicals in their original container
• Always wear appropriate clothes and work in a safe environment
• Always dispose of chemicals safely

Safety Rules of Chemistry Laboratory

• Protect your eyes and wear appropriate protective clothing
• Do not apply cosmetics, eat, or drink in the laboratory
• Pour from large containers to smaller ones (always add acid to water)
• Work with volatile chemicals under a fume hood
• Do not smell any chemicals directly and do not pipette solutions by mouth
• Know the safety of equipment

Basic Laboratory Procedures

• Weighing: requires careful weighing of all components
• Measuring liquids: use calibrated glassware, pipettes, and a hand-operated device
• Cleaning glassware: follow proper method of cleaning, including soaking and rinsing

Sterilization

• Sterilizing glassware and instruments
• Sterilizing nutrient media
• Sterilizing plant materials
• Sterile culture techniques

Acids and Bases

• Strong acids: corrosive, examples include hydrochloric (HCl), nitric (HNO3), sulfuric (H2SO4), and hydrobromic (HBr)
• Weak acids: often organic, examples include formic acid (HCOOH), acetic acid (CH3COOH), salicylic acid (C6H4(OH)COOH), and citric acid (C5H7O5COOH)
• Bases: ionic compounds containing metal ions and hydroxide ions, examples include sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH)2), magnesium hydroxide (Mg(OH)2), and ammonium hydroxide (NH4OH)

Salts

• Ionic compounds composed of metallic ions and nonmetallic ions
• Salts dissociate in water
• Salt solutions are generally electrolytes

pH

• pH scale measures the hydrogen ion concentration
• pH 7 indicates a neutral solution, acids are less than 7, bases are greater than 7

Safety Equipments in Laboratory

• Eye wash fountain
• Safety shower
• Fire extinguisher
• Emergency exits

Personal Safety while Handling Pesticides

• Avoid contact with pesticides
• Wear all designated safety equipment
• Be careful of drips and spills
• Keep hands away from eyes and mouth
• Wash your hands before smoking, eating, or bathroom breaks
• Use designated safety equipment according to the WPS statement on the label and regional requirements

Instruments in Meteorology, Microbiology, and Biochemistry/Chemistry

• List of instruments includes:
  • Meteorology: sunshine recorder, anemometer, wind vane, pyranometer, Stevenson screen, hygrometer, ordinary rain gauge, self-recording rain gauge, thermometer, and barometer
  • Microbiology: microscope, balance/scale, centrifuge, laminar airflow, spectrophotometer, refrigerator, freezer, autoclave, hot air oven, incubator, pH meter, and water bath
  • Biochemistry/Chemistry: spectrophotometer, balance/scale, centrifuge, stirrer, pH meter, refrigerator, freezer, flame photometer, hot air oven, water bath, EC meter, and atomic absorption spectrophotometer

Cleaning and Handling Glassware

• Conventional method of washing glassware: soaking in chromic acid-sulfuric acid bath, followed by rinsing and drying
• Alternative methods: washing with hot water and commercial detergents, rinsing with distilled and double-distilled water, and drying
• Cleaning and handling of glassware: inspect, dry, and store in a closed cabinet

GLASSWARE

• Beaker: used for stirring, mixing, and heating liquids, available in various sizes
• Burette: used to dispense known amounts of liquid reagent, measures from the top
• Condenser: used to cool hot vapors or liquids, maintains a thermal gradient
• Erlenmeyer/Conical flask: used for titration, has a flat, conical body, and a cylindrical neck
• Volumetric flask: used to measure a precise amount of liquid, has a flat bottom and long neck
• Measuring/Graduated cylinder: used to accurately measure the volume of an object, often more accurate than flasks and beakers### Laboratory Equipment and Techniques

Pipettes

• Come in various designs for different purposes, with varying levels of accuracy and precision
• Types of pipettes:
  • Volumetric pipettes (bulb pipettes): used to measure a specific volume of solution with high precision
  • Graduated pipettes: used to measure and dispense small volumes of liquid
  • Micropipettes: used to measure and dispense very small volumes of liquid (less than 1ml)

Glassware Cleaning

• Importance of cleaning glassware: essential in chemistry, necessary for accurate results
• Two levels of cleaning:
  • Quantitative cleaning: required for high-precision applications, such as analytical or physical chemistry
  • Normal cleaning: used for general laboratory purposes
• General cleaning tips:
  • Disassemble apparatus immediately after use
  • Triple rinse with solvent, followed by tap water and DI water rinse
  • Air dry on a drying rack
• Specialized cleaning methods:
  • Acidic peroxide solution: used to clean glassware contaminated with organic residues
  • Chromic acid: used to clean glassware, but not recommended due to environmental and health risks
  • Concentrated hydrofluoric acid: used to clean glassware, but not recommended for calibrated volumetrics due to risk of damage

Microscope

• Definition: an instrument used to see objects that are too small for the naked eye
• Types of microscopes:
  • Optical microscope: uses light to image the sample
  • Electron microscope: uses an electron beam to image the sample
• Rules for using a microscope:
  • Carry the microscope properly
  • Always begin focusing with the 4X objective
  • Use the coarse focus only with the 4X objective in place
  • Use immersion oil only with the 100X objective in place

Analytical Balance

• Definition: a class of balance designed to measure small masses in the sub-milligram range
• Features:
  • Measuring pan inside a transparent enclosure with doors
  • Used to measure accurate masses of chemicals in laboratory experiments

Laminar Flow Cabinet

• Definition: a carefully enclosed bench designed to prevent contamination of sensitive materials
• Features:
  • Air drawn through a HEPA filter and blown in a smooth, laminar flow towards the user
  • Stainless steel construction with no gaps or joints
  • Optional UV-Germicidal lamp for sterilization

Incubator

• Definition: a device used to grow and maintain microbiological cultures or cell cultures
• Features:
  • Maintains optimal temperature, humidity, and other conditions
  • Used in cell biology, microbiology, and molecular biology experiments

Spectrophotometer

• Definition: an instrument used to measure the amount of light absorbed by a sample
• Features:
  • Uses Beer-Lambert's Law
  • Used for analysis of proteins, carbohydrates, and other substances

Centrifuge

• Definition: a device used to separate particles or fluids based on density
• Features:
  • Small table-top centrifuge with maximum revolutions per minute of 6000
  • Used for sediment examination, separation of serum from clotted blood, and concentration of materials

Magnetic Stirrer

• Definition: a device used to stir liquids using a rotating magnetic field
• Features:
  • Used to mix liquids quickly and efficiently

Water Bath

• Definition: a water container with an electrically operated heating device
• Features:
  • Used to incubate samples at a constant temperature over a long period of time
  • Used to warm reagents, melt substrates, or incubate cell cultures
  • Precautions: regular monitoring of water level, disinfectants can be added, and raised to 90°C for decontamination

Oil Bath

• Definition: a type of heated bath used to heat reaction mixtures
• Features:
  • Uses silicone oil or other heat-stable oils
  • Used in conjunction with a hot plate or heating mantle

Sand Bath

• Definition: a container filled with heated sand used to provide even heating
• Features:
  • Used in conjunction with a hot plate or heating mantle
  • Used for chemical reactions and solution preparation

Fume Hood

• Definition: a ventilated cabinet used to safely handle and discharge environmentally harmful gases
• Features:
  • Used for chemical reactions and solution preparation
  • Provides a safe working environment for laboratory personnel### Earthing and Electrical Safety
• Earthing is a method of protecting against electric shocks by providing a path for fault current to flow to earth.
• Key function of equipment earthing is to prevent the buildup of static electricity, reducing the risk of electrical discharge in hazardous environments.
• Current refers to flowing electricity.
• Earth refers to a connection to the ground.
• Electrical installation refers to a fixed wiring system.

Handling of Chemicals and Safety

• There are four types of chemicals:
  • Toxic agents: poisonous to humans and can act rapidly.
  • Corrosives: irritants that can damage body tissues.
  • Flammables: burn readily and may explode or burn if sparks or flames are present.
  • Reactives: require stability and careful handling, can explode or react violently if not handled properly.
• Good handling practices:
  • Obtain the minimum amount of chemicals needed.
  • Ensure all containers are clearly labelled.
  • Toxic materials must be locked away.
  • Corrosive substances must be stored securely at a low level.
  • Flammable materials must be stored in specially designed cupboards.
• Safety precautions:
  • Always wear personal protective equipment (PPE).
  • Never mouth-pipette.
  • Always dilute concentrated acids by adding acid to water.
  • Never carry winchesters by the neck.
  • Always leave benches and balances clean and tidy.
  • Don't mix chemicals without authorization.

pH Meter and Its Function

• A pH meter measures the electro-chemical potential between a known liquid and an unknown liquid.
• The pH meter consists of a glass electrode and a reference electrode.
• The glass electrode measures the electro-chemical potential of hydrogen ions.
• To complete the electrical circuit, a reference electrode is needed.
• The pH meter measures the electrical potential difference between the unknown liquid and the solution inside the glass electrode.
• Calibration of the pH meter is done using standard solutions with known pH values.

Preparation of Media and Sterilization

• Media/microbe culture: a nutrient blend used to support microbial growth.
• Culture: part of a specimen grown in culture media.
• Culture media: a medium (liquid or solid) that contains nutrients to grow bacteria in vitro.
• Types of culture media:
  • Simple (basal, ordinary)
  • Enriched culture media
  • Selective media
  • Differential media (indicators)
• Properties of media:
  • Supports the growth of bacteria.
  • Nutritive (contains the required amount of nutrients).
  • Suitable pH (neutral to slightly alkaline).
  • Suitable temperature and atmosphere.

Organic Farming

• Organic farming: a system that avoids the use of synthetic inputs and relies on crop rotations, crop residues, animal manures, and biological systems of nutrient mobilization and plant protection.
• Principles of organic farming: care, health, ecology, and fairness.
• Components of organic farming:
  • Manure
  • Green manuring
  • Vermicompost
  • Bio-fertilizers
  • Crop rotation
  • Animal husbandry
  • Biological management

Seed Viability

• Seed viability: a measure of how many seeds are alive and can develop into plants.
• Why test seed viability: to ensure that seeds stored in a gene bank will grow and produce plants.
• When to determine viability: at the start of storage and at regular intervals during storage.

Tissue Culture of Crop Plants

• Tissue culture: the aseptic culture of plant protoplasts, cells, tissues, or organs under conditions that lead to cell multiplication or regeneration of organs or whole plants.
• Techniques:
  • Micro propagation
  • Preparation of plant tissue for tissue culture
  • Factors affecting plant tissue culture: growth media, environmental factors, explants source, and genetics.

Preparation and Standardization of Reagent Solutions

• Volumetric (titrimetric) analysis: involves determining the volume of a solution of accurately known concentration that is required to react quantitatively with the solution of the substance being determined.
• Standardization of reagent solutions: essential for accurate analytical results.
• Types of reagent solutions:
  • Acid-base titration
  • Redox titration
  • Complex formation titration
  • Precipitation titration

Buffer Solutions

• Buffer solution: an aqueous solution that resists a change in pH when a small or moderate amount of strong acid or base is added to it.
• Biological buffers: should meet specific criteria, including a pKa between 6.0 and 8.0, high water solubility, and minimal toxicity towards cells.

This quiz covers basic laboratory techniques and safety guidelines in biotechnology, including chemical handling and personal protective equipment usage.