ASEPTIC TECHNIQUE Dr J Domenech Topics Sources of

ASEPTIC TECHNIQUE Dr. J. Domenech Topics: • • • Sources of Contamination Design of an asepsis Laboratory Cleaning methods of asepsis laboratory Clothing Asepsis room ventilation

What is the Aim of Aseptic Technique? • To prevent the access of micro organisms during the preparation and testing of pharmaceutical products. When is Aseptic Technique unnecessary? If the last two stages in the processing of a sterile product are – • Packing in a container and sealing to prevent contamination after sterilization, followed by • Sterilization by one of the three official heat sterilization processes – – Dry heat – Autoclaving or – Heating with a bactericide

A terminal heat treatment is not possible for several classes of product: (a) Thermolabile Soluble Substances, Stable in Solution. – can be filtered through a bacteria proof filter – aseptic technique is required to prevent contamination of the filtrate during collection and while it is being packed and sealed in the final containers. – Eg. Thiamine Hydrochloride Injection. (b) Thermolabile Soluble Substances, Unstable in Solution. – They must be dissolved aseptically in a sterile solvent just before use, – Eg. Chorionic Gonadotrophin Injection.

A terminal heat treatment is not possible for several classes of product (Contd. ): (c) Thermolabile Suspensions, Stable in the Vehicle, – Eg. Propyliodone and Propyliodone Oily Injections. (d) Thermolabile Powders that require Dilution with Other Powders, – Eg. antibiotic dusting powders. (e) Thermolabile Powders that require Incorporation in a Semi solid base, – Eg. the eye ointments of the British Pharmacopoeia. • Filtration is impossible in the last three cases and, therefore, • The medicament must be mixed with the appropriate sterile vehicle aseptically.

SOURCES OF CONTAMINATION Satisfactory rules for good aseptic technique can only be devised if the possible sources of contamination are fully appreciated. 1. The Atmosphere – The atmosphere has no flora of its own cannot support the growth of micro organisms. – a shaft of light in a darkened room demonstrates heavy contamination with particles. – micro organisms are associated with many of these.

TYPES OF CONTAMINATION (a)Dust Outdoors: • dust particles in outside air come from the soil may carry soil bacteria. • these are saprophytes and • include cocci (mainly species of Sarcina and Micrococcus) and • sporing rods (particularly Bacillus spp. ) • pathogenic anaerobic sporing rods (e. g. Clostridium tetani and Clostridium welchii) quite common.

Indoors • the dust stirred up by cleaning operations sometimes contains resistant pathogens. • Eg. Staphylococcus aureus, Haemolytic streptococci, Mycobacterium tuberculosis and intestinal bacteria.

(b) Droplets • droplets are expelled from the respiratory tract by coughing and sneezing • contain organisms from the nose, mouth, throat and lungs. • healthy carriers often distribute Staphylococcus aureus and Beta haemolytic streptococci • transfer of the common cold, Influenza, the virus diseases of childhood and tuberculosis by droplet infection (c) Droplet Nuclei • The smaller droplets evaporate quickly • they contain saliva or mucous • the residue consists of tiny protcin flakes carrying any organisms previously in suspension. (d ) Free Micro organisms • naked yeasts and mould spores are often abundant.

People are the greatest source of contamination Sneezing produces 100, 000 – 200, 000 aerosol droplets which can then attach to dust particles

2. The Breath • in normal breathing few organisms pass into the atmosphere • coughing, sneezing and spitting can cause contamination • Haemolytic streptococci and Staphylococcus aureus present in the noses and throats • Staphylococcus aureus is found just inside the nostrils

3. The Hands • major means of transmitting infection. • there are not less than 10 000 organisms per cm 2 of normal skin • Organisms fall into 2 groups: • The Resident Flora: bacteria can live and multiply on the surface of the skin or in the hair follicle and the ducts of sebaceous glands • Eg. Mostly non-pathogen; Staphylococcus occasionally aureus. • The Transient Flora: composed of organisms collected from the environment or from other parts of the body.

4. Clothing • Atmospheric dust becomes entangled in the fibres of fabrics is dislodged by body movements • Can raise the level of contamination around a person who is working carelessly. • A special danger is the load of contaminated particles shed from a handkerchief that has dried after previous use.

5. The Hair • Hair is constantly exposed to atmospheric dust • Atmospheric dust becomes entangled in the hair • Dust may liberate during ?

6. The Working Surface • organisms sediment on the surface 7. Equipment • In aseptic technique no source of contamination is more serious than unsterile equipment.

THE DESIGN OF AN ASEPSIS LABORATORY A. Site • site the asepsis laboratory as far as possible from the rooms to which non pharmaceutical staff have access. • should be away from stairs, lifts and corridors • should contain few or no storage facilities, • the sterile equipment and products store must be adjacent or near by. • access to the lab should be through one or 'more rooms with washing and changing facilities.

B. Size • the maximum number of people using the asepsis room at any one time. • a large, fairly high room is more pleasant to work in • the overall level of micro organisms in its atmosphere is less affected by – local air disturbances or – contamination produced by individual workers. • cleaning the upper walls and ceiling, often neglected

• a small room is more economical – the capital and maintenance costs of the equipment are reduced • for controlling the microbial content • temperature and • humidity of the atmosphere – Cleaning is easier if the ceiling is low. . • the room must be much bigger for – preparing heat sterilised injections, including infusion fluids. • The clean atmosphere greatly assists the production of particle free solutions.

C. Windows • not pleasant to work day after day in a windowless room • efficiency is likely to suffer • bright sunshine is the best detector of dust • Large windows of clear glass are most acceptable to staff BUT they must not open • ventilation should be provided by an air filtration system. • The heat losses that occur from extensive areas of glass can be reduced by double glazing • Shading from the sun in summer can be given by a venetian blind.

D. Doors Air lock with double door • If possible, the laboratory should be entered through an air lock with double doors about 1 m apart • this process prevents a sudden inrush of air when the door is opened • The method of using the lock is to confirm first that the door to the laboratory is shut • for this, a small window is needed in the outer one. • Then enter in the air lock • after the outer door has been closed again • the laboratory door can be opened.

Sliding doors • Less air disturbances • Create dust traps • not be opened easily without using the hands

Swing doors • generally fit better, particularly if their openings are surrounded with insulation strip. • They can be made to push open and, therefore, can be foot operated; • but footplates are necessary to protect the wood.

E. Surfacing Materials The floors, walls and bench tops of an asepsis room must be • (a) Easily cleaned frequent washing will be necessary to prevent accumulation of dirt. • (b) Smooth cleaning is easier if there are no cracks and pores in which dust and micro organisms can lodge. • (c) Impervious, e. g. to cleaning agents and spilt liquids. • (d) Resistant to chemicals. They should not be softened or swollen by solvents, stained permanently by dyes or damaged by strong acids or alkalis.

1. FLOORS: The most suitable are (a) Terrazzo • a mixture of cement and crushed marble. • both can be coloured. • It is spread in plastic form on the site or is obtainable as tiles.

Terrazzo Floor • It stands up to energetic cleaning and, if desired, the floor can be gently sloped to carry the water away. • It is expensive, cold, , tiring to stand on, noisy, slippery when wet, and badly marked by rubber heels. • It is attacked by acids and stained by dyes but can be given a protective surface to increase its resistance.

(b) Linoleum • Heavy grade linoleum has many good features. • It is in expensive, reasonably warm, comfortable, quiet, obtainable in many colours and easily cleaned. • Sheet and tile forms are available • The polished surface is slippery when wet

Linoleum

• (C) Plastics • The non slip or matt finish grades of PVC are suitable. • Obtainable as sheets or tiles • The polished surface is very slippery • Oils and organic solvents attack it • Dyes are absorbed

2. WALLS AND CEILINGS The possible surfaces are (a) Tiles: • Good quality modern tiles seem more satisfactory • Ceramic surfaces are cold • not stand up to hard knocks. (b) Hard Gloss Paint on Smooth Plaster • Gloss paint is inexpensive and quite satisfactory • must be renewed as soon as cracking or peeling begins. • Plaster walls are easily damaged (c) Plastic Laminated Board • This material has been used for covering the walls and ceilings of asepsis rooms in industry. • The cost is high.

2. WALLS AND CEILINGS (c) Plastic Laminated Board • This material has been used for covering the walls and ceilings of asepsis rooms in industry. • The cost is high.

3. BENCH TOPS • The most popular surfaces for asepsis work are (a) Stainless Steel • This is virtually indestructible. • solution of iodine: one that noticeably attacks it. • The attaching screws should be under the bench

3. Bench Tops (b) Plastic Laminates • The major advantages – bright colouring – lower cost. • less noisy and not cold • Although their heat resistance is good, the radiation from an autoclave can raise and distort the laminate • Resistant to reagents, except strong solutions of phenols • although dyes cause staining this is easily removed if treatment is not delayed.

F. Services An asepsis laboratory will require many, if not all, of the following services (a) Ventilation: This may include removal of micro organisms, control of humidity and temperature, and provision of fresh air. (b) Electricity: for lighting and sometimes for a hot plate, ultraviolet lamp, aerosol producer or vacuum pump. (c) Gas: for the Bunsen burner. (d) Compressed air and or vacuum: for clarification and bacterial filtration.

1. ELECTRICITY • Strip lighting is the most pleasant to work in. • Dust collecting surfaces within the room can be avoided by fitting the tubes above flush glass or plastic panels in a false ceiling • Switches and sockets should be flush fitting and have finger plates of plastic • Most of the controls can be outside the room • A red indicator window above the entrance

2. GAS • Gas cocks may be on the wall or at the back of the bench but the controls must be easy to reach. 3. COMPRESSED AIR AND VACUUM • Some types of rotary Pump can separately provide both these services. • Pumps are noisy and are best housed outside the laboratory

4. NITROGEN • A cylinder can be kept near to the vacuum pump 5. WATER • It is difficult to justify a water supply in the laboratory itself. • Hand washing facilities are undesirable. • Water baths can be filled in the washing room • water baths are sometimes needed to melt or softer semi solid bases or to heat anaerobic culture media

Others Services • In a hospital two or three sinks may be enough but • in industry much larger facilities are essential. • Stands of the drinking fountain type are economical • Soap is conveniently supplied, in liquid form, from dispensers on the wall or on the tops of fountains. • Electric hand driers are often very large, are usually slow and cause considerable air disturbance; • paper towels, sterile if preferred, are an acceptable alternative. • in some industrial suites showers are provided

6. WASTE DISPOSAL • Wrapping paper, bags, plugs, pieces of twine, elastic bands, tops of ampoules etc. collect during aseptic technique and must be cleared from the working surface immediately. • A foot operated waste bin is popular • a metal ring can be clipped under or out from the bench and a plastic or stainless bucket or large bowl can be slipped into the ring • this can be close enough to the hands to make effective use

G. Furniture 1. BENCHES • conventional benching may be replaced by tables or wall mounted work shelves to reduce dust collection and facilitate its detection and removal • When storage space is considered essential, cup boards are preferable to drawers • The cupboards should be dustproof • To assist cleaning, as many surfaces as cost per mits should be faced with plastic laminate

2. SEATS • These must be adjustable and comfortable. • user's face is well above the front opening of the screen breath is kept away from the materials underneath. • Comfort is best assured by chairs rather than stools 3. TROLLEYS • Trolleys with removable trays, preferably of stainless steel, are better • They are easy to clean, • can be taken away for replenishment and, • if necessary, the trays can be steam sterilised.

4. DOORMAT • The soles of outdoor shoes are heavily contaminated • unless special footwear is worn in the laboratory, it is useful to have, in the air lock, a mat part immersed in a detergent disinfectant solution. • Mats are obtainable, in which the upper part has a honeycomb structure for holding liquid disinfectant • Johnson and Johnson, Slough, developed a disposable adhesive faced pad that fits into an aluminium frame • The resinous facing traps dirt but does not adhere to shoes or the wheels of trolleys.

5. SCREENS • Aseptic technique is carried out under a screen. • (a) Shack Types • Originally these consisted of a wooden case with a sloping front of glass • Plastic screens are transparent, – giving excellent visibility of the contents, and – light in weight, which makes them easy to move about. – The sloping front should raise for the introduction of large items of equipment such as a balance. – If clean air is supplied to the room, there is no need to enclose the screen front and work through arm holes or rubber sleeves

(b) Fume cupboard Types • Tall apparatus, such as burettes, has to project through a hole in the screen top when used with the shack type. • Fume cupboard types are high enough for this equipment to fit entirely inside.

Summary of the Lecture • What is aseptic technique? • Terminal heat treatment is not possible in several classes of product. • Sources of contamination • The Atmosphere – What are the Types of contamination • • • Dust Droplets nuclei Free microorganism The Breath The Hands Clothing The Hair The working surfaces Equipment

Summary of the Lecture (Contd. ) The Design of an Asepsis Laboratory – – – A. Site B. Size C. Windows D. Doors E. Surfacing Materials • 1. Floors • 2. Walls and Ceilings (a) Tiles (b) Hard Gloss Paint on Smooth Plaster (c) Plastic Laminated Board • 3. Bench Tops (a) Stainless Steel (b) Plastic laminates – F. Services • Electricity • Gas • Compressed air and vacuum • Nitrogen • Water • Waste disposal – G. Furniture • Benches • Seats • Trolleys • Doormat • Screens – Shack Type – Fume cupboard Types

• Saprophytes: Organisms living on dead or decaying organic matter that help natural decomposition of organic matter in water. • Cocci: Spherical shaped Bacteria • Sarcina A genus of bacteria found in various organic fluids, especially in those of the stomach, associated with certain diseases.