LBOBI16 LE1

• Essential Knowledge and Lab Skills

  • Pipetting
  • Lab equipment
  • Lab safety
  • Aseptic technique
  • Waste disposal
• Laboratory equipment


  • Analytical balance
  • Centrifuge or ultracentrifuge
  • Vortex mixer
  • Water bath or dry bath
  • Refrigerator/freezer
  • Spectrophotometer
  • Thermal cycler
  • Electrophoresis set-up
  • UV transilluminator
• General guidelines and Precautions

  1. Always read and follow equipment manufacturers' instructions

  2. Before operating, inspect equipment for any visible physical damage and immediately report to the custodian in charge

  3. Check the voltage requirements of an equipment, as well as the output of available electrical outlet, before plugging equipment in

  4. Do not leave equipment unattended when in use
• Analytical Balance


  Weighing instrument
• Analytical Balance


  1. Always close the balance door

  2. Only glass, ceramic, metal or plastic objects and containers should be placed in direct contact with the balance pan

  3. Do not handle objects to be weighed with bare hands

  4. Never weigh chemicals directly in contact with the balance pan. Use containers such as beakers, flasks and weighing bottles. Do not use weighing paper

  5. Be cautious when weighing to avoid spills inside the balance enclosure

  6. If a spill occurs, clean it up immediately
• Centrifuge or Ultracentrifuge


  Separation instrument
• Centrifuge or Ultracentrifuge


  1. Securely cap tubes to be placed inside centrifuge

  2. Load tubes in a "balanced" arrangement

  3. If available, make sure to close the inner lid or rotor cover before running the centrifuge

  4. Wait for the centrifuge to come to a full stop before opening
• Vortex Mixer


  Make sure tubes are capped properly before subjecting to vortex mixing
• Water Bath or Dry Bath

  Heating instrument
• Water Bath or Dry Bath

  1. For water bath, use only distilled or de-ionized water to prevent corrosion

  2. Turn on device at least 30 minutes before intended use to allow the temperature to reach desired level

  3. Regularly drain, clean and replace water in water bath to avoid biofilm formation
• Refrigerator/Freezer


  1. Never store food or beverage in a refrigerator or freezer intended for laboratory use

  2. Avoid keeping the door open for extended periods, as this may cause the temperature inside to increase

  3. When using an ultralow freezer, use the appropriate hand cover or protective equipment

  4. Defrost and clean when deemed necessary
• Spectrophotometer


  Measures the light transmitted through a solution to determine the concentration of a particular substance in that solution
• Thermal Cycler


  Heating device used in in-vitro nucleic acid amplification methods (e.g., PCR)
• Electrophoresis System/Set-up


  Chamber with electrical supply that allows migration of charged solutes or particles in an electric field within the chamber
• UV Transilluminator


  Emits UV light for visualization of fluorescent-stained biological material, almost exclusively used for visualizing ethidium bromide- or SYBR Green-stained DNA
• Biohazard


  An infectious agent, or part thereof, presenting a real or potential risk to the well-being of man, animals and/or plants, directly through infection or indirectly through disruption of the environment
• Biosafety


  Containment principles, technologies, and practices implemented to prevent unintentional/accidental exposure to biohazards or their unintentional/accidental release
• Biosecurity


  Institutional and personal security measures designed to prevent loss, theft, misuse, diversion or intentional release of biohazards
• Biosafety and Biosecurity practices

  • Substitution or elimination
  • Administrative controls
  • Engineering controls – e.g., biosafety cabinets
  • Personal protective equipment
• Biosafety Levels


  Levels of containment for working with biohazards
• BSC Class I


  • Inward air moving into the cabinet contains aerosols generated during microbiological manipulations, it then passes through a filtration system that traps all airborne particles and contaminants, finally, clean, decontaminated air is exhausted from the cabinet
• BSC Class II


  • Inflow air flows through the front inlet grill, near the operator, none of the unfiltered inflow air enters the work zone of the cabinet, so the product inside the work zone is not contaminated by the outside air
• BSC Class III


  • Provides an absolute level of safety, which cannot be attained with Class I and Class II cabinets, usually of welded metal construction and are designed to be gas tight, used for BSL-4
• Proper BSC use


  1. Location: remote from traffic & potentially disturbing air currents

  2. Cabinets should be turned on at least 5 min before beginning work & after completion of work to allow cabinet to "purge"

  3. Materials to be placed inside cabinet should be surface-decontaminated w/ 70% alcohol

  4. Ensure front intake grill is not blocked w/ paper, equipment or others

  5. Materials placed as far back in the cabinet, towards the rear edge of the work surface, w/o blocking rear grill

  6. Place all necessary items into the cabinet before beginning manipulations

  7. Arms should be moved in & out of BSC slowly

  8. Aerosol-generating equipment (e.g. mixers, centrifuges, etc.) should be placed towards the rear of cabinet

  9. Observe "clean to dirty work flow/set-up"

  10. Open flames should be avoided in the BSC, as they disrupt air flow, dangerous when volatile, flammable structures are also used
• Example of Class II Biosafety Cabinet
• Personal Protective Equipment must be donned & doffed in proper order
• Micro-Pipette Principle


  Air displacement pipette – operate by piston-driven air displacement
• Piston


  • Disk enclosed in an air-tight cylinder, its movement expels or displaces a certain volume of air, which creates a vacuum that allows an equivalent volume of liquid to be aspirated upon the return of the piston to its original position
• Micro-Pipette parts


  • Plunger button & plunger
  • Tip ejector button connected to the tip ejector
  • Volume indicator or volumeter display
  • Piston
  • Shaft "tip holder"
  • Disposable pipette tips
• Micro-Pipette Volume Capacity


  • P2 (0.1-2 μL)
  • P10 (0.5-10 μL)
  • P20 (2-20 μL)
  • P100 (10-100 μL)
  • P200 (20-200 μL)
  • P1000 (100-1000 μL)
• Pipette use - Setting the volume

  1. Set volume of liquid to be aspirated & dispensed by turning the plunger button or thumbwheel

  2. Numbers in volume indicator correspond to the desired volume in microliters

  3. Exception: P1000 – numbers that appear in the volume indicator, indicate an actual volume in microliters that is 10x

  4. Red number appearing in the volume indicator of smaller volume capacity pipettes, P-2, P-10, and P-20 – indicate the tenths decimal place of the set volume

  5. Avoid using the lowest capacity of a pipette!! (max is okay)

  6. After using pipette, return settings to max capacity
• Pipette use - Aspirating a Liquid


  1. Push plunger up to 1st stop

  2. Immerse the disposable tip of the micro-pipette into the desired liquid, while still holding down the plunger button

  3. After immersing tip into liquid, releasing plunger creates a partial vacuum, liquid is aspirated

  4. Place tip into the target vessel

  5. Push to "first stop" – cause the air to dispense the liquid

  6. To empty tip completely, push plunger to reach "second stop"
• Pipette use guidelines: proper care & use

  1. Never adjust/set pipette outside volume range as it will instantly & permanently damage

  2. Never drop pipettes on bench/floor

  3. Clean the pipette before and after use with approved disinfectant such as 70% ethanol and by wiping with soft cloth or paper material
• Correct immersion depth
• Slow and careful Plunger handling
• Aseptic Technique


  Absence of pathogens
• Aseptic Technique practices


  • Hand wash
  • Use of sterile gloves, masks, gowns
  • Sterilization of surgical instruments
  • Use of disinfectants including antiseptics
• Sterilization


  Complete destruction of all living organisms including cells, viruses, spores, "devoid of life"
• Disinfection


  Destruction/removal of pathogens from objects by physical/chemical methods
• Physical methods of Sterilization & Disinfection

  • Heat
  • Incineration
  • Autoclave
  • Filtration
  • Radiation
  • Ultrasonic waves/Sonicator