LBOBI16

• 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 and plunger
  • Tip ejector button connected to the tip ejector
  • Volume indicator or volumeter display
  • Piston
  • Shaft
• Plunger button


  When pressed down causes the piston to move within the airtight cylinder
• Plunger button


  Also serves as volume adjustment knob and thumbwheel – for setting the desired volume of liquid
• Disposable pipette tips


  Prevent cross-contamination or carry over between different samples and reagents
• Micro-Pipette Volume Capacity


  • P1000 (Blue, 1000 – 200 μl)
  • P200 (Yellow, 200 – 20 μl)
  • P20 (Yellow, 20 – 2 μl)
  • P10 (White, 10 – 0.5 μl)
  • P2 (White, 2 – 0.1 μl)
• Pipette Use Step 1: Setting the Volume

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

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

  3. Exception: 1000 microliter capacity pipette – numbers that appear in the volume indicator, indicate an actual volume in microliters that is ten times what is indicated

  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 in microliters
• Pipette Use Step 2: Aspirating a Liquid

  1. The piston moves to the appropriate position when the volume is set

  2. This limits the depth to which you the plunger can be pushed (first stop)

  3. Push the plunger up to the first stop

  4. Then immerse the disposable tip of the micro-pipette into the desired liquid, while still holding down the plunger button

  5. After immersing the tip of the micro-pipette into a liquid, releasing the plunger creates a partial vacuum, and the specified volume of liquid is aspirated into the tip
• Pipette Use Step 3: Dispensing a Liquid


  1. Place the tip of the micropipette into the target vessel

  2. Pressing the plunger until the "first stop" again will cause the air to dispense the liquid

  3. To empty the tip completely, you must push the plunger even further to reach what is known as the "second stop"
• Pipette Use Guidelines: Proper Care and Use

  • Never adjust or set a pipette outside of its allowed volume range as it will instantly and permanently damage the pipette
  • Never drop pipettes on the bench or floor
  • 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


  Depends on the volume of liquid being aspirated
• Slow and Careful Plunger Handling

  • The plunger must be pressed and released slowly and carefully
  • Pressing and releasing the plunger too fast can affect air displacement leading to inaccurate volumes
• Correct Liquid Discharge/Dispense


  • Discharge liquid by touching the wall of the target vessel
  • For volumes below 10 μL, it is necessary to discharge directly into the bottom of the target vessel or into the liquid already present in the target vessel
• Vertical Pipette Posture


  • Keep pipettes upright at all times, especially during actual use to prevent the liquid in the tip to get into the pipette
  • Holding angle of the pipette affects accuracy. Keep the pipette upright especially when aspirating liquid to ensure accurate volume
• Common Cell Biology Lab Equipment

  • Analytical balance
  • Centrifuge or ultracentrifuge
  • Vortex mixer
  • Water bath or dry bath
  • Refrigerator/freezer
  • Spectrophotometer
  • Thermal cycler
  • Electrophoresis set-up (Polyacrylamide, Agarose)
  • UV transilluminator
• General Guidelines and Precautions

  • Always read and follow equipment manufacturers' instructions
  • Before operating, inspect equipment for any visible physical damage and immediately report to the custodian in charge
  • Check the voltage requirements of an equipment, as well as the output of available electrical outlet, before plugging equipment in. Make sure that they are compatible
  • Do not leave equipment unattended when in use
• Analytical Balance


  • Weighing instrument
  • Always close the balance door
  • Only glass, ceramic, metal or plastic objects and containers should be placed in direct contact with the balance pan
  • Do not handle objects to be weighed with bare hands
  • Never weigh chemicals directly in contact with the balance pan. Use containers such as beakers, flasks and weighing bottles. Do not use weighing paper
  • Be cautious when weighing to avoid spills inside the balance enclosure
  • If a spill occurs, clean it up immediately
• Centrifuge or Ultracentrifuge


  • Separation instrument
  • Securely cap tubes to be placed inside centrifuge
  • Load tubes in a "balanced" arrangement
  • If available, make sure to close the inner lid or rotor cover before running the centrifuge
  • 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
  • For water bath, use only distilled or de-ionized water to prevent corrosion
  • Turn on device at least 30 minutes before intended use to allow the temperature to reach desired level
  • Regularly drain, clean and replace water in water bath to avoid biofilm formation
• Refrigerator/Freezer


  • Never store food or beverage in a refrigerator or freezer intended for laboratory use
  • Avoid keeping the refrigerator/freezer door open for extended periods, as this may cause the temperature inside to increase
  • When using an ultralow freezer, use the appropriate hand cover or protective equipment
  • 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
  • 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 Practice

  • Substitution or elimination
  • Administrative controls
  • Engineering controls – e.g., biosafety cabinets
  • Personal protective equipment
• Biosafety Cabinets (BSC)


  Containment devices equipped with HEPA filter(s), designed to provide personnel or both personnel and product protection from 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
• Proper BSC Use


  • Location – remote from traffic and potentially disturbing air currents
  • Cabinets should be turned on at least 5 min before beginning work and after completion of work to allow the cabinet to "purge"
  • Materials to be placed inside the cabinet should be surface decontaminated with 70% alcohol
  • Ensure the front intake grill of BSC is not blocked with paper, equipment or other items
  • All materials should be placed as far back in the cabinet, towards the rear edge of the work surface, without blocking the rear grill
  • Place all necessary items into the cabinet before beginning manipulations
  • Arms should be moved in and out of the BSC slowly, and manipulations within the BSC should be delayed for about 1 min after placing hands and arms inside to allow the cabinet to adjust and to "air sweep" the surface of the hands and arms
  • Aerosol-generating equipment (e.g. mixers, centrifuges, etc.) should be placed towards the rear of the cabinet
  • Observe "clean to dirty work flow/set-up"
  • Open flames should be avoided inside the BSC, as they disrupt the air flow patterns and can be dangerous when volatile, flammable substances are also used
• Personal Protective Equipment


  • Dress, eye, mask, gloves
• Asepsis


  Absence of pathogens
• Sterilization


  • Complete destruction of all living organisms including cells, viruses, and spores
  • When something is sterile, it is devoid of life
• Disinfection


  • Destruction or removal of pathogens from objects by physical or chemical methods
  • Pasteurization: method of disinfecting liquids
  • Disinfectants: strong chemicals used to disinfect inanimate objects but cannot be used on living tissue
  • Antiseptics: solutions to disinfect skin and other living tissues
• Physical Methods of Sterilization and Disinfection

  • Heat (Incineration, Autoclave)
  • Filtration (Water, Air)
  • Radiation (Ionizing, Non-ionizing)