ASEPTIC TECHNIQUE

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

(c)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
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
• 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 – F. Services
– B. Size • Electricity
– C. Windows • Gas
– D. Doors • Compressed air and vacuum
– E. Surfacing Materials • Nitrogen
• 1. Floors • Water
• 2. Walls and Ceilings • Waste disposal
(a) Tiles
– G. Furniture
(b) Hard Gloss Paint on • Benches
Smooth Plaster • Seats
(c) Plastic Laminated • Trolleys
Board • Doormat
• 3. Bench Tops • Screens
(a) Stainless Steel – Shack Type
(b) Plastic laminates – 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 those of
the stomach, associated with certain
diseases.