ctech topic 1

• 7 main functions


  • Non-Clean Area
  • Storage
  • Sterilization
  • Preparation
  • Wash-Up
  • Clean (Sterile) Area
  • Incubation
  • Observation
• Storage


  • Room temperature
  • 40°C
  • -20°C
  • -196°C
• Sterilization


  • Autoclave
• Preparation


  • Balance
  • Centrifuge
  • Hemocytometer
• Wash-Up


  • Sink Area
  • Water bath
• Clean (Sterile) Area


  • Sterile Handling
  • Incubation
  • Observation
• Sterile Handling


  • Cell Culture Hood
• Incubation


  • 37°C CO2 Incubator
• Observation


  • Inverted Microscope

  
• Cell Culture Hoods (Clean Area)

  • Filter sterilizes air passing over the work area so that the work environment is protected from dust and bacterial/fungal contaminants
  • Provides an aseptic work area while allowing the containment of infectious splashes or aerosols generated by many microbiological procedures
  • Have specific airflow patterns designed to maintain a clean and sterile environment for cell culture work
  • The airflow in these hoods serves to protect both the user and the cell cultures from contamination
• Horizontal Flow Hood


  • Draws in air from above, filters the air, then forces the air out the front of the working area of the hood
  • Minimizes the risk of contaminants settling onto the work surface, as gravity helps to direct particles away from the workspace
  • Provide significant protection to the user and the cell culture
• Vertical Flow Hood


  • Draws in air through the front of the working area into the vent system. 80% of this air is recirculated and the remainder expelled
  • Provides a clean, particle-free workspace for the user and materials placed on the work surface
• Types of Cell Culture Hoods

  • Laminar Flow Hood
  • Class III Biological Safety Cabinet
  • Class II Biological Safety Cabinet
  • Class I Biological Safety Cabinet
• Laminar Flow Hood

  • Do not provide personnel or environmental protection. They only provide a clean, sterile workspace
  • Can have vertical or horizontal airflow, however vertical airflow is used in cell culture
  • Suitable for applications that require aseptic techniques, such as cell culture, tissue culture, or microbiological manipulations
  • Not suitable for working with hazardous or infectious materials unless additional containment measures are in place
• Class I Biological Safety Cabinet

  • Provide significant levels to personnel and environmental protection but not to the product
  • Typically have a vertical airflow
  • Designed with an open front with inward airflow (personnel protection) and HEPA-filtered exhaust air (environmental protection)
  • Suitable for working with low to moderate risk agents or when protection is primarily needed for the operator and the environment
• Class II Biological Safety Cabinet

  • Provide significant levels of protection to personnel, environmental and product
  • Have a vertical or horizontal laminar airflow pattern, depending on the design
  • Designed with an open front with inward airflow (personnel protection), downward HEPA-filtered laminar airflow (product protection) and HEPA-filtered exhaust air (environmental protection)
  • Suitable for handling potentially hazardous materials (e.g., primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents)
• Class III Biological Safety Cabinet

  • Provide the highest level of containment, offering maximum protection to personnel, the environment, and the product
  • Totally enclosed and operated through glove ports or other sealed mechanisms
  • Always gas-tight and have a vertical airflow pattern
  • The cabinet is maintained under negative pressure and supply air is drawn in through HEPA filters. The exhaust air is treated with either double HEPA filtration or single HEPA filtration followed by air incineration and then exhausted outside
  • Suitable for handling human pathogens and other BSL-4 materials
• 37°C CO2 Incubator


  • Distributes heat evenly and maintains temperature at 37°C
  • Provides a 5% CO2 concentration (pH 7.2 -7.4) can be adjusted to 10% if necessary
  • Humidity controlled in the presence of a tray of deionized (DI) water
• Autoclave is a self-locking device used to sterilize instruments by means of steam pressure
• Autoclaving is also called steam sterilization
• The use of pressurized steam to kill agents of infection and denature proteins
• Reduces the risk of contamination by sterilizing apparatus at very high temperatures and pressures
• Sterilization process: 121°C for 15mins
• Proper Maintenance


  • Promoting productivity
  • Reduce contamination
  • Improve safety
• Equipment Maintenance


  • Cell Culture hoods
  • Incubators
  • Autoclave equipment
• Basic Equipment & Lab Requirements

  • Clean Area
  • Sterile handling
  • Incubation
  • Observation
  • Non-Clean Area
  • Preparation
  • Wash-up
  • Sterilization
  • Storage
• Mammalian cell culture


  The technique of growing and maintaining mammalian cells outside the mammal in specially designed containers, which are located in conditions which attempt to mimic the precise environmental conditions that were present in that mammal
• Environmental conditions mimicked in mammalian cell culture

  • Temperature
  • Humidity
  • Nutrition
  • Contamination-free conditions
• Mammalian cell culture


  • It is one of the major tools used in cellular and molecular biology
• Knowledge gained from mammalian cell culture

  • Cell products: secretion, exocytosis, biotechnology, bioreactor design, product formation & harvesting, downstream processing
  • Genetics: genetic analysis, transfection, infection, transformation, immortalization, senescence
  • Environmental interaction: infection, drug action, ligand receptor interactions, cytotoxicity, mutagenesis, cytogenesis
  • Cell-cell interaction: morphogenesis, paracrine control, cell proliferation kinetics, metabolic cooperation, cell adhesion & motility, matrix interaction, invasion
  • Intracellular flux: RNA, hormone receptors, metabolites, calcium, signal transduction, membrane trafficking
  • Intracellular activity: DNA transcription, protein synthesis, energy metabolism, drug metabolism, cell cycle, differentiation, apoptosis