Dr Siti Suri Lecture 3 Lab Design And Aseptic Technique

DESIGN, CONCEPTS AND LAYOUT OF CELL CULTURE LABS Concepts Effects of contaminants Facilities in TC labs Equipping the TC labs Essential equipment Beneficial equipment ASEPTIC TECHNIQUE Sources of contaminants Type of contaminants Minimising contamination pipetting sterile handling pouring Objectives of aseptic technique Laminar flow hood and microbiological safety cabinet Types and functions of cabinets Important points regarding the use of the cabinets Organizing the hood for routine work Use of antibiotics

DESIGN CONCEPTS AND LAYOUT OF CELL-CULTURE LAB

CONCEPT

 Tissue culture lab need to maintain asepsis.  Handling of cultured cells in sterile environment.  Provision must be made to ensure that cultures and culture media are maintained free from contaminating micro-organism • The lab should be only for the purpose of TC handling and propagation Should not be used for any other experimental work involving the TC eg. virus infection of TC, cell hybridization

Effects of Contaminant

rapid growing in culture media kill the animal cells release toxin deplete the nutrient medium depress the medium pH FACILITIES NEEDED IN TISSUE CULTURE LAB 1. Sterile handling area 2. Incubation -incubators 3. Preparation area 4. Wash up area 5. Sterilization 6. Storage

Sterile handling area with Minimal movement of people past through the clean area Should have a proper separation between clean area and dirty operations including waste disposal Vinyl flooring with dust proof finishing Air conditioning Other facilities essential to cell culture work Laminar flow cabinets Incubators (CO2) Inverted microscope Instrument and equipment benches-centrifuge, pipettes Media fridges (sterile working solutions only)

Freezers for culture reagents requiring storage at -20°C esp. serum -80°C for cryopreservation of cells before storage in liqiud nitrogen (-196oC) Media and solutions Preparation area General cold storage facility for chemicals and non-sterile reagent General preparation bench In a separate room : Liquid nitrogen freezer (tanks) for frozen cell stocks Storage area for unopened plastic ware Sterilization Autoclave Drying oven Water purification system Wash up area with sinks, soak tanks

EQUIPPING THE TC LABORATORY The specific needs of the tissue culture laboratory like most labs, can be divided into three categories ; Essential – you cannot perform a job without them Beneficial – the work would be done better, more efficiently, quicker, or with less labour Useful – it would make life easier, improve working conditions, reduce fatigue, enable more sophisticated analysis to be made, or generally make your working environment more friendly and attractive.

Essential equipment Incubator – temperature, gas phase and humidity are controlled. Consideration for purchasing incubator : Large enough Have forced air circulation Temperature control ± 0.5°C Safety thermostat Corrosion resistant-stainless steel Easily cleaned Steriliser Antibiotic Autoclave Filtration Chemical sterilisation Fumigation Liquid disinfectants Irradiation Ultraviolet (260nm) – to sterilize air in cell culture cabinet Gamma rays – has a very good penetrating properties, items can be completely sealed and packages

Inverted microscopic Cell freezing unit Water purification Centrifuge

Beneficial equipment

Cell counter-essential for precise quantitative growth kinetics e.g Coulter counter Aspiration pump-a vacuum pump or simple tap siphon Balances, pH meter and osmometer Upright microscope-attach to the camera Dissecting microscope Magnetic stirrer Pipette and micropipette Large column dispensing unit Repetitive dispensing unit Useful additional equipment Low temperature freezer Glassware washing machine Colony counter Controlled rate freezer

ASEPTIC TECHNIQUE Successful cell culture is absolutely dependent on fastidious aseptic technique. Source of contamination - Bacteria - mycoplasma-invisible (need mycoplasma test) - yeast - fungal spores Type of contamination - can be minor and confined to one or two cultures -can spread among and several other cell lines and infect a whole experiment - can be widespread and wipe out the entire stock or even the whole lab

Minimising Contamination by: • cultures are checked through microscopy • culture maintain without antibiotics (where possible) • reagents are checked for sterility before use • bottles of media are not shared with other people or used for different cell lines • the standard of sterile technique and good microbiological practice and techniques are kept high at all times •Sloppy cell culture technique can be very disruptive to the lab. Occasional isolated contamination can happen to anyone, but commonly prove to be no more than a nuisance. CONTAMINATION IS TROUBLESOME, TIME CONSUMING AND EXPENSIVE.

Pipetting Never use pipette more than once. Never enter a culture vessel or reagent bottle with a used pipette Never draw so much fluid into the pipette as to wet the plug Do not allow fluid to drip from the pipette tip Never blow bubbles in media or cell suspensions When using pipette to disperse cells, keep the tip below fluid level and a deep, narrow-mouthed bottle or flask Clean up any spillage immediately with 70% ethanol

Sterile handling Swabbing Swab down working surface before and during work particularly following spillage Swab down after finished Swab bottles, particularly from the cold room, before using for the first time each day Capping Deep screw caps should be used in preference to stoppers Flaming The necks of the bottles and screw caps should be flamed before and after opening a bottle and after closing

If working outside the Class II cabinet, Work close to the flame where there is an up-current due to convection, and do not leave bottles open IT IS NOT ENCOURAGE TO USE FLAMES IN CLASS II CABINETS AS THEY CAUSE TURBULENCE AND DISTURBED THE AIR FLOW Screw caps should be place open-side down on a clean surface and flamed before replacing on the bottle Screw caps may be held in the hand during pipetting, avoiding the need to flame or lay down

Handling bottles and flasks

Pouring The major risk in pouring lies in the generation of a bridge of liquid between the outside of the bottle and the inside, which may infection to enter the bottle. Bottles or flasks that are stored or incubated after pouring are at a significantly higher risk

Thus it is essential that the cell culture specialist adopt the best possible working habits (good microbiological practice and techniques) and continually be on the outlook for problems and their causes

Objectives of aseptic technique To provide a barrier between micro-organism in the environment outside the culture and the pure uncontaminated culture within its flask and dish. Personal hygiene Hand washing Gloves Caps, gowns and face mask Flu-wear face mask Talking is permissible-if working in the Class II microbiological safety cabinet

LAMINAR FLOW HOOD AND MICROBILOGICAL SAFETY CABINET Working in the laminar flow hood Routine handling of cultured cell is recommended to be carried out in a class II microbiological safety cabinet. Functions and limitation of laminar flow Laminar flow hoods are engineered to eliminate particulates, to reduce the chance that a particle will enter an open culture dish or flask. Two basic types of laminar flow hood are used : vertical flow-air flow is straight down horizontal flow – force the air out of the front cabinet, no glass screen

Laminar flow clean air cabinet Horizontal air-flow cabinet

This protects the work against microbial contamination by a laminar flow of sterile air. This type of cabinet is suitable for making media and handling non-infected tissue cultures. It is not suitable for infectious work as personnel are exposed to the exhaust air. 1 Open front and exhaust air 2 Working space 3 Hepa filters and laminar flow 4 Fan

Class I microbiological safety cabinet

. This protects the worker. Air is drawn from the room, with the air-stream away from the worker thus protecting him against infection. The incoming air is not sterile and may contaminate the work. The exhaust air is filtered to protect the environment. 1 Open front with inward airflow 2 Working space 3 Exhaust fan 4 Exhaust hepafilter 5 Sterile exhaust air

This protects both the work and the worker. Incoming and exhaust air is filtered. The open front is protected by an air current, which acts as a curtain. Highly contagious material must be handled in a Class ll cabinet as air and liquid spilling through the front are not absolutely preventable. 1 Open front with air curtain 2 Window 3 Working space 4 Inlet with hepafilter 5 Inlet fan 6 Air inlet with prefilter 7 Air vents in the bottom 8 Exhaust fan 9 Exhaust hepafilter

Class II microbiological safety cabinet (vertical flow)

Important points regarding the use of laminar flow hoods Laminar airflow creates a clean work environment, not a sterile one The laminar flow hood is the most critical work area in the lab, despite the filtered air, it is the most likely site where cultures can become contaminated Horizontal-flow hoods must never be used in work with pathogens or with human and primate cell lines Perform all activities in the hood with the understanding that airflow can carry contaminants to the culture system Hands and arms (or gloves and sleeves) can be a serious source items for of contaminant – laden particulate (eg. Dry skin, talc, and lint).

Organizing the hood for routine work Minimize the clutter Position items for efficiency of movement and to minimize traffic over the centre of the field where handling of cultures will be performed Leave the front and rear vents clear for the best airflow The flasks or dishes of cultured cells or tissue for primary culture should be the last items transferred to the hood Minimize the introduction of dust and particles to the hood by first wiping with 70% ethanol Incubators are a prime source of contamination. Flasks and trays of cultures from the incubator be wiped with 70% ethanol before they are placed in the hood

USE OF ANTIBIOTICS Antibiotics introduced into culture media – to reduce contamination Use of laminar flow hoods and strict/good microbiological practice and techniques makes antibiotic unnecessary Disadvantages of using antibiotics i. They encourage the development of antibiotic resistant organisms ii. They hide the presence of low level cryptic contaminants esp. Mycoplasma that will flare-up once the antibiotic is removed or changes made eg change of culture conditions iii. They have antimetabolite effects that can cross react with mammalian cells vii. They encourage poor aseptic techniques. Fungal and yeast contaminants that cannot be controlled by antibiotics will set in. Use of antibiotics is restricted to primary culture Large scale labor intensive experiments with high consumable costs