SAFETY IN THE LABORATORY

Design, organization and Layout of the Laboratory

The design of a laboratory is determined by hazards e.g. (biological, radiological and chemical)
and the type of work to be performed.

The facility (laboratory) design is dictated by the placement of the equipment and systems
required to perform laboratory procedures,

Biosafety and Biosecurity must be considered when selecting the facility design and its
features.

Design (features) of a safely organized laboratory.

1. Facility space. (Laboratory space)

2. Storage
3 Surfaces and finishes (walls, doors, windows)
4. Furniture
5. Facilities and systems.
6. Laboratory equipment

1. Facility space. (Laboratory space)

a) Laboratory floor space,
 The laboratory must have enough floor space for the intended laboratory activities.
 The floor space should be adequate for the laboratory activities to be conducted
safely.
 The space must be sufficient to accommodate, hand-washing basins, benches, sinks
and worktops as well as equipment such as refrigerators and freezers.

b) Corridors and doors

 Corridors, doors and laboratories must be of sufficient width to allow easy delivery,
removal and replacement of laboratory equipment.
 These corridors and exits must be kept clear at all times to allow emergency exit; they
must not be used as storage locations.

c) Floor space for other facilities.

 Floor space must be allocated for additional facilities/rooms for personnel use, such
as toilets, bathrooms, eating/drinking areas and office facilities.
 Spaces for personnel to leave and store personal items, outer garments (coats).

2. Storage
a) Consumables and reagents
 Sufficient floor space, shelving must be available to house consumables and reagents

Prepared by josomuny@gmail.com 1
 safely and securely in the long and short term.

b) Chemicals
 Specialized storage cabinets need to be available for hazardous reagents and
chemicals, such as those with flammable, oxidizing or corrosive properties.
d) Specimens
 Specimen storage, space to accommodate large amounts of refrigerator or freezer
within the facility/laboratory should be available.
e) Waste
 Enough floor space to enable safe and secure storage of waste before it is
decontaminated or transported for disposal.
 Space must also be provided to facilitate waste movement, i.e. for the trolleys used
3 Surfaces and finishes
a) Walls and floors

 Walls and floors must be smooth and of continuous surfaces.

 Walls floors must be easy to clean, and impermeable and resistant to the chemicals
and disinfectants used in the laboratory. For example, vinyl or linoleum are suitable
materials for floors.

 Walls must be solid and properly finished according to function. For example, wall
protection may be required to prevent damage by trolleys, or splash
b) Windows
 Windows should normally be sealed but may be openable when the laboratory is
designed to be naturally ventilated.

 If openable, they must be designed to prevent insects entering the laboratory, and
they should be lockable.
c) Doors
 Doors to the laboratory must be lockable and must have a vision panel to see into the
laboratory.

 Doors should be appropriately labeled with the international biohazard symbols

4. Furniture.
 Furniture must be easy to clean,

 Furniture must not include any fabric surfaces which may absorb and hold
contaminants.

 Bench tops must be impervious to water and resistant to heat and the chemicals and
disinfectants that may be used in the laboratory, for example, acids, alkalis and
organic solvents.

 Bench tops should have curved edges wherever possible for easy cleaning.

Prepared by josomuny@gmail.com 2
5. Facilities and systems.
a) Hand washing
 Must have hand-washing facilities in each room of the laboratory where procedures,
including waste handling, are performed,
 These facilities should be located as close as possible to the exit door.
 Running water must be available, preferably operated by a hands-free mechanism
(elbow, wrist, knee or foot) and soap (in dispensers)
b) Electrical supplies
 Electrical supplies must be of sufficient capacity and reliability for safe and effective
operation of all electrical and electronic devices.
 These supplies include cabling, fuses and outlets, which must be earthed to prevent
shocks in case of malfunction

c) Lighting
 Lighting must be adequate for all activities.
 The direction of light sources must be designed so that personnel can avoid working
in their own shadow.

d) Environmental controls
 Environmental controls, should be in place, to ensure comfort cooling and/or heating
systems (to provide a comfortable temperature) and air conditioning (to control of
the condition of the air), to control temperature and/or humidity for a comfortable
working environment for personnel to perform their tasks safely and with optimal
efficiency.

e) Safety systems
 Should have Installations of safety systems for fire, including fire alarms, and for
laboratory gases, where applicable, must be considered.

6. Laboratory equipment
The laboratory must have adequate floor space for safe use of the equipment. The space is
required for effective equipment cleaning, decontamination and maintenance.

Prepared by josomuny@gmail.com 3
2. SAFETY
Safety is a state of being free from danger or hazards.

A hazard is a source or a situation / anything that has a potential to cause harm. e.g.
electricity, chemicals. Spillages, broken glass ware etc.

A risk is the possibility that harm (death, injury, or illness) might occur when people are
exposed to a hazard.
Or is a likelihood of undesirable event happening, that involves a specific hazard or threat and
has consequences. It’s a factor of likelihood and consequences

A threat. Is a person with intent to harm.

Types of laboratory hazards.

1. Physical – It refers to the stereotypical workplace hazards for example, lighting issues,
objects obstructing walkways, unsafe machinery, slippery floors, noise, heat and steam burns
from autoclaves.

2. Chemical – This includes any form of liquid, vapour, dust, fumes or gases that could be
spilt, leaked or misused.

3. Ergonomic – conditions that pose a risk of injury to the musculoskeletal system of the
worker, such as;
 Repetitive and forceful movements.
 Vibrations
 Extreme temperatures
 Awkward postures that a raise from improper work methods and improperly designed
work station tools and equipment.
However, this category can also include hazards associated with lack of training (e.g. manual
handling) or unsafe working conditions leading to injury.
4. Radiation – This may be more prevalent in a clinical setting and covers x-rays, gamma

Prepared by josomuny@gmail.com 4
rays, UV and microwaves.

5. Psychological – This applies heavily across all sectors in many forms. Examples of
psychological risk include stress, working shifts, problems dealing with the public, internal
harassment and lack of empowerment.

6. Biological –Biological substances that pose a threat to a health of a living organism,

primarily humans, this includes bacteria, viruses, toxins, parasites and moulds of fungi.
biological risks involve viruses, this can happen through bites, cuts, or contamination through
contact with an infected person.

7. Fire hazards, common causes of fire hazards in the laboratory.

 Electrical circuit overloading
 Poor electrical maintenance, eg poor and perished insulation on cables.
 Equipment unnecessarily left switched on.
 Equipment not designed for the laboratory environment
 Open flames.
8. Noise the effect of excessive noise is insidious over time. E.g. some type of laboratory
equipment such as lesser systems, as well as facilities where animals are housed can
produced significant noise expose to workers

The following are hazards and accidents associated with medical laboratory work: biohazards,
chemical and physical hazards.

Factors contributing to laboratory accidents/injuries.

Although inexperience may cause some accidents, others may be a result of ignoring known
risks, haste, carelessness, fatigue and mental preoccupation.

 Fires, fires can result if flammable liquids are kept/ placed next to the section that
handle procedures involving heating.

 Electrical short circuits and shocks, as a result of over loading sockets or due to poor
earthing.

 Leakages and spillages, from gas cylinders and chemicals respectively.

 Unplanned storage, stocking materials in passages as it can lead to blockage of

escape routes during emergencies.

Slippery lab floors due to poor design.

 Use of defective material handling equipment, such as trolleys.

Careless handling of containers, e.g. carrying solvent bottles from the neck alone.

Prepared by josomuny@gmail.com 5
Accidents that can be encountered in the laboratory.

1. Chemical burns
Corrosive substances have the potential to break degrade, burn the skin.
2. Heat Burns
Heat from Bunsen burners and other heating devices commonly used in the lab.
3. Eye Injuries
Contact of hazardous chemicals with the eyes as a result of splashes when working in the
laboratory.
4. Cuts From Glassware glass ware can break and cut the skin, if handled roughly, such as
applying to much force when thin smears.
5. Inhaling Dangerous Gases
Many dangerous chemicals emit hazardous vapours and gases that are hazardous to human
health.
6. Spills And Breaks
In the lab, glass beakers may be dropped and break. Liquids may be spilled. Generally, these
accidents are caused by rushing, being negligent and not properly following procedures.
7. FIRES
When working with hot surfaces and flammable materials, fires become a common danger.

Infections – Are acquired through .

 Inhalation,
 Ingestion
 Entry Through The Skin
 And mucous membranes

Specimen borne pathogens.

All specimen handled in the laboratory are potentially infectious and must be handled with
care.

Specimen borne pathogens get into the body in various ways.

a) Aerosols are droplets of infectious material that can be generated through the
following processes –
 Rapid closing and opening of snap closed containers,
 Rapid dispensing of infectious fluid matter,
 Break of specimen containers with infectious fluid,
 Spillage of infectious fluid,
 Breakage of specimen containers with infectious fluid in them during centrifugation,
Heating of contaminated wire loops,
 Rapid rinsing of Pasteur pipettes,
 Contaminated dust.
All these may have pathogens that get into the body to cause infection by being inhaled. They
can also contaminate surfaces and get ingested

b) Ingestion – Pathogens are ingested in contaminated fingers, foods and drinks

Prepared by josomuny@gmail.com 6
 contaminated during storage in laboratory refrigerators, mouth pipetting of
specimens and cultures, contaminated butts of cigarettes, licking labels or oral
contact with contaminated pens/pencils.

c) Entry through the skin e.g. punctures to the skin, cuts, scratches, insect bites, sores,
and other skin lesions. Important infections maybe contracted through inoculations
with needles and broken glass
d) Other routes are through the mucous membranes of the eye, mouth and respiratory
tract. Organisms from contaminated work benches and equipment may accidentally
contaminate hands and these pathogens can be transferred to the mucous
membranes

2 Chemical hazards

Harmful effects of chemicals.

Harmful chemicals include the following:

 Toxic,
 Corrosive,
 Irritant,
 Flammable

Categories of chemical hazards.

a) Corrosive chemicals destroy tissues. They can cause immediate death, organ or tissue
damage, drowsiness, allergies and hypersensitivity. Examples are sulphuric acid, glacial
acetic acid, caustic soda and alkalis,

b) Toxic chemicals are poisonous. They cause death and ill health if swallowed, inhaled or
allowed to come into contact with the skin. Examples of toxic chemicals are potassium
cyanide, formaldehyde solution, sodium nitroprusside, chloroform, methanol, barium chloride,
c) Explosives chemicals cause blasts and destruction to life and property. Examples are
picric acid and sodium azide
d) Flammable chemicals catch fire very easily causing burns and destruction to property
Carcinogens cause cancer by ingestion, inhalation or contact with the skin Examples are
benzidine, O- toluidine, O - dianiside and selenite
e) Mutagens cause genetic changes in cells
f) Teratogens cause birth defects

Ways through which chemicals can enter into the body:

 Inhalation of powders or fumes
 Absorption on the skin or mucous membranes
 Ingestion
 Injection through the skin by a foreign body
All hazardous chemicals should have product labels.

Biosafety.
Biosafety are practices and control measures that reduce the risk of unintentional exposure
or release of biological materials.

Prepared by josomuny@gmail.com 7
Avoid generating aerosols from infectious material,
 By not using snap closing containers,
 Rapid dispensing of infectious fluid matter,
 Avoid breaking of specimen containers with infectious fluid,
 Avoid spillage of infectious fluid,
 Avoid breaking specimen containers with infectious fluid in them during
centrifugation, avoid heating of contaminated wire loops in the hottest part of the
flame,
 Do not rapidly rinse Pasteur pipettes,
 Avoid accumulating dust.

Ingestion of pathogens can be avoided by observing the following.

 By not eating, smoking cigarettes and drinking in the laboratory,
 Avoid storing foods and drinks in the laboratory refrigerators, do not mouth pipette
specimens and cultures, avoid licking labels or oral contact with contaminated
pens/pencils.
 Take care to avoid damage to the skin and entry through the mucous membranes of
the eye, mouth and respiratory tract
 Use personal protective equipment always e.g. gowns, aprons, eye goggles, gloves,
boots. Protect the respiratory tract by use of fume hoods, gas masks, and automatic
dispensers.
 Use fume hoods and cabinets properly, ensure that they function properly, always
test for their function.

Handling of sharps
 Do not recap needles or other sharps
 Do not bend, shear or break off needles
 Dispose of sharps into puncture proof containers
 Do not overfill sharps containers
 Seal sharps containers prior to disposal

Chemical safety
To avoid any injury which could occur if they are accidentally knocked off from a high shelf.
 Store corrosive chemicals at lower levels
 Use fire proof metal boxes at ground level preferably in an outside cool locked store
to store flammable chemical. If a metal box is unavailable use a container with well
lined tin foil.
 Transport Winchester bottles of flammable chemicals in carrier with handles.
 Separate chemicals that react with each other.
 Store oxidising chemicals separate from organic and reducing agents.
 Limit the volumes of chemicals to store
 Keep poisons such as potassium cyanide and deadly drugs under key and lock
 Keep the chemicals at correct temperatures
 Allow only restricted quantities of flammable chemicals to the laboratory and clearly
label them keep away from fire. Have fire extinguishers available in the room.

Prepared by josomuny@gmail.com 8
 Regularly conduct fire drills
 All hazardous chemicals should have labels of the product namely chemical and
common name, warnings and physical hazards, names and addresses of
manufacturers signs and symbols of hazards

Safety laboratory precautions/practices

 All laboratory staff should be made aware of the hazardous nature of the laboratory
environment and procedures required to follow work safely.
 Induct all new staff to laboratory safety practices
 Collect all specimens in leak proof containers
 Transport specimens in secondary containers for example trays, boxes and plastic
bags to avoid leakage of spillage into the environment. Always use disposable gloves
when handling specimens for transport. On receipt examine packages for leakage
and spillage. Decontaminate secondary containers by autoclaving or chemically.
 Use pipetting aids for pipetting all chemicals and fluid specimens
 Eyes must be washed out well in the event of a chemical or biological splash. In the
event of extensive splashing of chemicals or biological fluid,

 Basic protective clothing consists of a white laboratory coat, must be worn by all staff
when working in the laboratory. The laboratory coat must be buttoned up when
working in the laboratory.

 Laboratory coats must be removed before exiting the laboratory,

 Always wash hands before leaving the laboratory, and after handling hazardous
chemicals and biological agents.

 No eating, drinking, smoking, gum-chewing or application of cosmetics is allowed in

the laboratories.

 No-smoking in the Laboratory room..

 Fingers should not be inserted into the mouth or eyes while working with chemicals
or biological agents.
 Handle and examine potentially infectious materials in safety hoods and safety
cabinets
 Procedures with a high potential of producing aerosols (grinding, blending, vigorous
shaking or mixing of infectious fluids) must be conducted in a biological safety
cabinet
 Soak all contaminated clothes in 1% hypochlorite overnight. Leave protective

Prepared by josomuny@gmail.com 9
 clothing in the laboratory and never take home or wear in a room where refreshment
are taken
 Label chemicals and reagents with signs of flammable, toxic, corrosive, harmful
irritant, oxidising, explosive.
 Store and secure cylinders to the walls, bench or floor holders in the upright position
so that they can not fall. Protective caps must be in place for compressed gas
cylinders except when in use. Store extra cylinders away from the laboratory in a
secure position preferably in a locked ventilated fireproof space. Switch of the gas
cylinders from the mains take in valves and allow to burn and later switch off the
reduction valves.

 Broken glass must be cleaned up immediately and disposed of in specially designated

container..

 Inspect all glassware carefully for cracks and chipped rims before use.

Explain the good laboratory practices (GLP)

GLP is a quality system concerned with the organisation process and conditions under which
non clinical health and environment safety studies are planned, performed, monitored,
recorded, archived and reported.

Or Is a set of principles intended to assure the quality and the integrity of non clinical
laboratory studies.

The purpose of GLP is to;

 Ensure quality test data
 To obtain comparable data between countries.
 Ensure sound laboratory management
 Ensure robust conductance of laboratory testing
 Ensure accurate reporting of test findings
 Ensure safe archival of laboratory data
Elements of GLP
 Quality assurance program
 Standard operating procedures.
 Storage and retention of records
 Performance of the study.
 Apparatus, materials and reagents.
 Reporting of study results

Safety equipments used in the laboratory

Prepared by josomuny@gmail.com 10
 -
Biosafety cabinets
Definition; Biosafety cabinets (BSCs) are enclosed work places with a ventilated hood that is
designed to contain pathogenic microorganisms during microbiological processes.

A biological safety cabinet (BSC) is a primary engineering control used to protect personnel
against biohazardous or infectious agents.
 Are only used for certain risk group organism and for processes that might result in
aerosol formation
 This cabinets are provided with HEPA( High Efficieny Particulate Air) filters that
decontaminate the air moving out of the cabinet.
 Also helps to maintain quality control of the material being worked with as it filters
both the inflow and exhaust air, it is sometimes referred to as a laminar flow or tissue
culture hood.
 These filtered cabinets are primarily designed to protect against exposure to
particulates or aerosols.
 All procedures should be performed in a manner that reduces the generation of
aerosolized material and prevents spills.
 Operations such as centrifugation, vortexing, sonication, and opening containers of
infectious materials whose internal pressure may be different from the ambient
pressure are known aerosol-generating procedures. These procedures should be
conducted inside the BSC or additional measures must be available to mitigate the
safety concern.
 Aportion of the air in most BSCs is re-circulated back into the lab through its exhaust
HEPA filter. (High Efficiency Particulate Air) this purifies the air of potentially
infectious aerosols,
Biosafety cabinets are classified into 3 three classes, each with specific performance
characteristics and applications.
Class 1 and II Biosafety cabinets are used for Biosafety levels 1 and II when used correctly
this will provide an effective containment system.
Class three (3) are most suitable for work with hazardous agents and require that require
Biosafety level 3 0r 4.

Biosafety levels.

Prepared by josomuny@gmail.com 11
Biosafety level 1, describes labs that handle materials with a minimal risk, these affords basic
protection for operator and the environment.
Biosafety level II laboratory.
This level is for labs handling materials that may present a moderate risk to personnel and
environment.
Biosafety level III laboratory. More advanced than BS level II in terms of design,
infrastructure and operational procedures and practices.
Biosafety level IV laboratory.
This lab is applicable for work with dangerous and exotic agents that pose a high individual
risk of life threatening disease which may be transmitted through via the aerosol routes.

Protective gears, sometimes referred as protective equipment, (PPE) is any equipment (for
example, gloves) worn by a person to protect that individual from exposure to one or more
hazards.
a) Laboratory coats,
Laboratory coats are designed to prevent personal clothing from becoming splashed or
contaminated by biological and/or chemical materials.
b) Footwear,
Footwear must be worn in the laboratory. Footwear should cover the top of the foot, and
should be well fitting and comfortable to allow personnel to perform their tasks without their
feet hurting.
c) Gloves,Appropriate gloves must be worn for all procedures, Disposable gloves can be
used but they should not be disinfected or reused
d) Eye protection. Safety glasses, safety goggles, face shields (visors) or other
protective devices must be worn whenever it is necessary to protect the eyes and
face from splashes
e) Aprons
Additional hazard-specific splash protection may be required for certain procedures, such as,
removing specimens from liquid nitrogen
f) Coveralls
Coveralls cover the whole body and are generally worn on top of scrubs or personal clothes.
Depending on the quality, they may be disposable or reused if properly decontaminated.

g) Respiratory protection
Respiratory protective equipment is a form of PPE designed to protect the wearer from
inhaling particles that contain biological agents that may be present in the air or generated
during certain laboratory procedures.

Prepared by josomuny@gmail.com 12
Fire extinguisher,

Fire extinguishers and their classes

There are four classes of fire extinguishers – A, B, C and D – and each class can put out a
different type of fire.

Class A extinguishers will put out fires in ordinary combustibles such as wood and paper.
Class B extinguishers are for use on flammable liquids like grease, gasoline and oil
Class C extinguishers are suitable for use only on electrically energized fires
Class D extinguishers are designed for use on flammable metals
Multipurpose extinguishers can be used on different types of fires and will be labeled with
more than one class, like A-B, B-C or A-B-C.

Fire extinguishers are fairly easy to use in the case of a fire. Most of the types operate using
the P.A.S.S. technique:

P. Pull the pin on the fire extinguisher in order to break the tamper seal.
A. Aim the fire extinguisher low, with the nozzle pointed at the base of the fire.
S. Squeeze the handle of the fire extinguisher to release the extinguishing agent.
S. Sweep the nozzle from side to side while pointed at the base of the fire until it is
extinguished.
If the fire re-ignites, repeat the last 3 steps.

Common causes of fire in the laboratory

The causes of lab fire accidents can be classified into five categories, that is,
 Chemical reaction fires, as a result of spills from flammable liquids.
 Static electricity fires, due to short circuits as a result of over loading sockets and
poorly earthed laboratories
 Equipment failure fires,
 Fire due to operational errors (man-made fire),
 Fires caused by nature disaster.

Explain how fire outbreak can be prevented in the laboratory

 Plan work. The majority of lab fires have resulted from mental or procedural errors or
carelessness.
 Minimize materials e.g flammable liquids. At work area and use only the minimum
quantities necessary to work in progress. Not only does this minimize fire risk, it
reduces costs and waste.
 Observe proper housekeeping. Keep work areas uncluttered, and clean frequently.
Put unneeded materials back in storage promptly. Keep aisles, doors, and access to
emergency equipment unobstructed at all times.
 Observe proper safety practices.

Prepared by josomuny@gmail.com 13
  Store solvents properly.
 Keep barriers in place (shields, hood doors, lab doors).
 Wear proper clothing and personal protective equipment.
 Avoid working alone.

Disinfection and Sterilization

Disinfection describes the process that eliminates many or all pathogenic microorganisms,
except bacterial spore.

Sterilization is a process that destroys/eliminates all form of microbial life including spores
from the article.

Decontamination, reduces the microbial contamination of materials/surfaces.

Sterilization by autoclaving.

Sterilization at temperatures below 1000 C

1. Pasteurization below 1000 C.
• Used for serum, body fluids, milk, cream and certain beverages, e.g. wine and beer.
• Mycobacterium tuberculosis (milk) is destroyed within 15’ at 600 C; Rickettsial
organisms, e.g. Coxiella burnettii (Q fever) also present in milk is destroyed at 720 C
within 20’’.
• The test is best carried in water-bath or controlled hot air oven.
Types of pasteurization:
• Holder’s method: milk is heated for 30 minutes at 630 C.
• Flash method: milk is heated for 20 seconds at 720 C.
Note: Sera or body fluids are sterilized for 1 hour at 560 C; and vaccine at 600 C for 1 hour for
several successive days.

2. Steaming at 1000 C:
• Boiling water produces ‘’free steam’’ of 1000 C at the atmospheric pressure (760 mm
Hg).
• Used for sterilizing culture media (N.broth or N.agar) and heat labile culture media (
e.g. DCA, TCBS and Selenite F broth) , and dissolving agar.
• Steam is produced by Koch & Arnold steamer , which is inexpensive and simple to
operate; and uses gas, steam or electricity as source of heat.
Diagram?
It consists of a vertical metal cylinder with removable conical lid, having an opening for the
escape of steam while preventing the ingress of steam. Contains water which is boiled by a
heater. Perforated tray carries the article to be sterilized.

Two methods of sterilization using this steamer:

i. Single exposure at 1000 C x90 minutes:
The steaming period of 90 minutes includes the time required for tubes or bottles of
media to be heated for room temperature to 1000 C.

Prepared by josomuny@gmail.com 14
 • Heating up period: 20’ x 10-100 ml fluid
(bottle).
30’ x 600 ml fluid (bottle),
45’ x 2-5 L fluid (flask).

ii. Tyndallization/Fractional/Intermittent sterilization:

• Involves an intermittent exposure at 1000 C for 20-40’ or 30’ on three successive days,
and having incubation in between at room temperature.
• Principle: The 1st exposure kills vegetative organisms. The 2nd and 3rd exposures kill
the vegetative forms which arise from spores that survived 1st and 2nd exposures
respectively.
• The technique:
o Good for sugar containing media;
o Not good for media containing thermophilic, anaerobic and other bacteria
whose spores do not germinate in such media and under conditions of
storage between the heating .

Sterilization at temperatures above 1000 C

Autoclave - is the most reliable method of sterilization for culture media and lab
supplies.
Materials to be sterilized should be water-containing, permeable or wettable, and not
liable to damage by this process.
Principle: It provides moist heat at temperatures higher than 1000 C, the boiling of
water under normal atmospheric pressure. In a closed vessel, under increased pressure, the
temperature at which water boils and that of the steam it forms are above 1000 C.

The condensed steam sterilizes articles in three ways:

1. Wets the microorganisms on the articles and so provides the essential condition for
their killing by moist heat.
2. Liberates latent heat which heats up the articles to the sterilizing temperature.
3. Causes a great contraction in the volume of the steam and so promotes the ingress
of fresh steam.
Thus the combined effects of the 3 factors ensure the destruction of
bacterial endospores as well as vegetative cells by coagulating and
denaturing microbial proteins and enzymes.

There are two main types of autoclaves:

1. The simple non-jacketed laboratory autoclave, e.g. Portable autoclave.
2. The steam jacketed autoclave with automatic air and condensate discharge.

Description: It consists of an upright tank, heated below by electricity or gas; having a lid with
a gasket to make it hermetically fitted. The lid carries a pressure gauge, air outlet valve and
safety valve (Pressure stat). At the bottom is water
Procedure:

Prepared by josomuny@gmail.com 15
 1. Put enough water at the bottom of the autoclave chamber.
2. Put the articles on a trivet or perforated tray.
3. Screw down the lid to close the chamber hermetically. Open the air outlet valve and
turn on the source of heat.
4. As water boils, allow steam and air to escape through the air outlet valve for about 5
minutes.
5. Close the air outlet valve and allow pressure to rise to required level.
6. Allow the autoclaving to continue at the chosen pressure and temperature (1210 C)
for 15 minutes.
7. Turn off the heater and let pressure drop to zero.
8. When the autoclave is cool, open the lid and remove the content.

Autoclave controls and sterilization indicators/controls

• Automatic process control: Monitors the whole sterilization cycle [heating up,
holding, cooling and drying stages], according to pre-set duration, temperature and
pressure at each stage.
• Recording thermometer: It makes a graphic timed record of the temperature changes
in the chamber discharge channel thus helps an operator to avoid errors in timing
holding period in the absence of No.1.
• Thermocouple measurement of Load Temperature: It discovers the heating up time
required for a given type load. It is inserted deeply inside the test article, and its wires
are carried out under the chamber door or through a leak proof port to a
potentiometer, which indicates the temperature inside the load during the course of
autoclaving.
• Chemical indicators: These chemicals are put inside the load, e.g. a) Browne’s tubes
[Type 1 (Black spot), 1150 C; type 2 (Yellow spot), 1210 C; type 3 (Green spot), 1600 C;
and type 4 (Blue spot), 1800 C]. These tubes contain red solution which turns from red
to green or other appropriate colour when the above given temperatures and time are
achieved.
b) Adhesive tape (Bowie – Dick) tests for steam penetration and used in
conjunction with other tests, and gives valuable information.
• Spore indicators: A dried bacterial spores filter is put within the load and autoclaved,
then tested for viability, e.g. Geobacillus stearothermophilus (a thermophile) grows
within 550-600 C temperature. Its spores are killed at 1210 C within 12 minutes.
The recovery medium used is thioglycollate broth or cooked meat medium;
incubation for 7 days; C+ve (un-autoclaved spores), C-ve (un-inoculated medium), and
examined for growth.

Disinfectants

• Definition: Disinfection is the selective elimination of certain undesirable organisms

in order to prevent their transmission; its achieved by action on their structure or
metabolism, irrespective of their functional state.
• Principle: Disinfectants kill vegetative bacteria, fungi, viruses and occasionally
spores by the destruction of proteins, lipids or nucleic acids in the cells or their
cytoplasmic membranes.

Types of disinfectants

Prepared by josomuny@gmail.com 16
 1. SOAPS/DETERGENTS
• Soaps and detergents do not disinfect.
Use/application
• Decrease the surface tension between microorganisms and surfaces and thereby
cleanse the surface.
• Soaps emulsify the oil film on the body surface carrying the oils, debris and
microorganisms away in a degerming action.
Mode of action. (MOA)
• The cationic detergents are quaternary ammonium compounds. They solubize the
cell membranes of microorganisms, e.g. benzalkonium chloride and acetyl pyridinium
chloride.

2. ALCOHOLS

Alcohols are the base ingredient for many other disinfectants.

• Excellent pathogen destroyer; but must be left in contact with the items to be
disinfected for long periods to be effective,
• Contact time of about 20’ is considered proper for disinfection with ethyl alcohol,
• Active against bacteria and fungi, but not bacterial spores,e.g. 70% Ethanol.
MOA
Antimicrobial action of alcohol is denaturation of proteins.

3. HALOGENS
a) Iodine
• Iodine solutions frequently used as antiseptics for cleaning wounds and skin.

MOA
• Iodine combines with microbial proteins and inhibits their functions.

b. Chlorine
MOA
• Combines with microbial protein.
• Used as sodium hypochlorite (bleach).
Use.
• Chloramines, a combination of chlorine and ammonia is used to sanitize glassware
and eating utensils.
• Chlorine used as a gas to maintain a low microbial count in drinking water.

4. ALDEHYDES
MOA
• E.g. Formaldehydes and glutaraldehydes inactivate microbial protein by cross-linking
the functional groups in the proteins.
• Both are volatile and toxic by skin contact and inhalation.
Use
• Formaldehyde gas (commonly used as formalin); 37% solution, employed as for
embalming purpose.
• Glutaraldehyde, used as a liquid to sterilize hospital equipment, but requires several
hours to destroy bacterial spores.

Prepared by josomuny@gmail.com 17
 5. PHENOLS.
Use
• Phenol is the first chemical to be used in disinfection ( Used by Joseph Lister ).
• Phenolics a derivative of phenol, are useful molecules that act as antiseptics.
MOA
• Phenolics damage cell membrane and inactivate enzymes of microorganisms, while
denaturing the proteins (e.g.cresols-lysol; bisphenols-hexachlorophenol)

6. QUATERNARY AMMONIUM COMPOUNDS

• "Quats" are a large class of disinfectants which add organic compounds to ammonia.
Use
• Many quats also function as a detergent, and help remove organic debris from
objects.
• The presence of organic debris, however, may deactivate the disinfectant in the quat
compound.
• Quats are effective against many types of bacteria, some viruses, and chlamydia; but
not spores, mycobacteria or fungi, Pseudomonas, and hydrophilic viruses (e.g.
Polyoma ).

• MOA inactivation of energy-producing enzymes, denaturation of essential cell

proteins.

7. ETHYLENE OXIDE
• Used to sterilize objects sensitive to temperatures greater than 600 C or radiation;
such as plastics, optics and electrics.
• Performed at 300 C and 600 C , with relative humidity above 30%; gas concentration
between 200 and 800 mg/l for 3 hours.
MOA
• Kills bacteria, viruses, fungi and bacterial spores
• Denatures microbial proteins.

8. OXIDIZING AGENTS
• Examples, H2O2 kills microorganisms by releasing large amounts of oxygen which
contributes to the alteration of microbial enzymes.
• H2O2 is effective on inanimate objects, but on skin surface, it is quickly broken down
by enzyme catalase, liberating oxygen.
 Benzoyl peroxide – applied on the skin to treat acne due to anaerobic bacteria,
release oxygen that inhibits anaerobic growth.
 Ozone is used to disinfect water and air ; oxidizes the cellular components of
contaminating microbes.

9. HEAVY METALS
• A number of heavy metals have antimicrobial activity.
• E.g. silver in silver nitrate (eye-drop for newborns) active against N.gonorrhoae; zinc
in zinc chloride in mouth wash and zinc oxide in antifungal agent in paints; copper in
copper sulfate, retards growth of algae.
• Heavy metals act by combining with sufhydryl groups on cellular proteins.

Physical methods of sterilization – autocalving, hot air oven, filtration and ionizing

Prepared by josomuny@gmail.com 18
radiation

Hot air oven sterilization

• A suitable method for sterilization of loads that cannot be reliably penetrated by
steam and can tolerate high temperatures required, e.g. 160-1800 C.
• Used for materials in sealed containers, powders, oils, and greases, e.g. petroleum
jelly; glassware ( test tubes, glass petri dishes, flasks, pipettes, and all glass syringes),
metallic instruments ( forceps, scalpels and scissors).
• Wrap articles in aluminum foil or Kraft paper and plugged with stoppers or cotton
wool when using this technique.
Description: Consists of a fan, to provide forced air circulation throughout the chamber;
Temperature indicator; a control thermostat and timer; open mesh shelving; and adequate
insulation. There should be a temperature chart recorder and a port for thermocouples.
• Safety features include an over-heat cut out, operating at and above 2000 C; door inter
-locks to ensure that heating starts when the door is properly shut, and opens when
temperature has dropped below 600 C.

Preparation of Load
1. Cap test tubes with slip-on aluminum caps and place vertically in metal racks.
2. Plug pipettes to a depth of 2cm with non-absorbent cotton wool and place in metal
canisters or wrap in Kraft paper.
3. Wash all-glass syringes and grease its piston with silicone and pack in aluminum
case with metal or foil cap or double wrap in aluminum foil.
4. Wash and pack syringe needles separately in a small test tube with constriction to
prevent needle tip touching the bottom.
5. Use screw-capped bottles with metallic caps and liners which are of Teflon,
polypropylene or silicone rubber with high temperature resistance.
6. Use preferably dry glassware or exposed at 1000 C in the oven.
7. Sterilize powders, fats and oils when sealed in glass or metallic containers; packed in
small quantities not exceeding 10g in weight or 1 cm in depth.
8. Position articles or loads spaciously to allow free circulation of hot air between and
around them. Close the oven door and switch on the heat source
Sterilizing cycle

Sterilization hold time’ starts when:

1. The chamber has reached the chosen temperature, and
2. A further period has been allowed for all parts of the load to be raised to that
temperature.
Commonly used time and temperature relations for sterilization hold time are:
• 20/30 minutes at 1800 C,
• 40 minutes at 1700 C or
• 60 minutes at 1600 C.
Cooling: It is facilitated by fitting a thermostatically controlled cooling fan, circulating cool
outside air drawn in through HEPA filter, or an external cooling coil.
• NB: Do not open the chamber until the chamber and the load have cooled to below 600
C. Glassware is liable to crack if cold air is admitted suddenly while it is still very hot.
Testing the efficacy of Hot air oven:

Prepared by josomuny@gmail.com 19
 1. Compare temperature record chart produced during each run with the master
temperature record.
0 0
2. Include Browne’s tube type 3/Green spot, (160 C) or type 4/Blue spot (180 C) and
observe for colour change.
3. Use the spores of Bacillus subtilis var niger (NCTC 10073 or ATCC 9372). The spores
should have a D – value of 5-6 minutes at 1600 C and a Z – value of about 25 minutes
[ Z = thermal resistance of the spore to the process; D = rate of kill at a given
temperature be about 90%/minute].
4. Make reference to half yearly and yearly thermocouple tests.
5. Use sulfur metal that melts at 1550 C.

Common Terms:
Heat generation time: - the time taken for heat to penetrate (completely) each item in the
load. It is longer than the sterilizing time.

Holding time: - the time necessary to ensure that all organisms (vegetative or spore forms)
are killed at the temperature being used. Materials with low (5.0) PH or High (9.0) PH are
more readily sterilized than those of intermediate PH.

Safety time: extra time is added on as a safety factor, which is usually half holding time and
the total period constitutes the sterilizing time or cycle.

STERILIZATION BY RADIATION
Definition:
Radiation is a form of energy transmitted through space.
Electromagnetic radiation has properties of continuous wave phenomenon and
discontinuous particle phenomenon.
These particles are packets of or quanta of energy known as photons.

Interactions of electromagnetic radiations with matters

• Gamma rays and X-rays (with high energy), pull electrons from molecules and ionize
them. When they pass through cells, free H2 and hydroxyl radicals and some
peroxides are created, and these cause intracellular damages.
• Ultra violet (UV) rays (less energetic), do not ionize but excite electrons and raise
them to higher energy levels; therefore creating new chemical species, which
interferes with the chemical reactions of the organisms.
• Apart from electromagnetic radiations, acoustic (sound waves) radiation and sub-
atomic particles also have their effects against organisms.
• Gamma radiation: Emitted by radioactive isotopes such as 60Co. It has a high
penetrating power and microbicidal effect. Used for sterilizing materials of
considerable thickness or volume, e.g. packed food and plastic syringes.
• X-rays: It has high penetrating power; and lethal to microorganisms and higher forms
of life. Uses as the above but can produce mutant organisms.
• UV radiation: It has low penetration power; sunlight also has UV components, hence
can kill bacteria. The UV wave of sunlight that reaches the earth is of 290mµ, majority
of the shorter wavelengths are filtered out by the atmosphere.

Prepared by josomuny@gmail.com 20
 • Mercury vapour lamps emit UV rays within the region of 250-260 nm, and are
bactericidal and to lesser extent sporicidal. The energy level is low and therefore,
poor penetration power.
• UV rays should be shielded from the eyes and skin, and should not be put on while the
cabinet is being used. NB: Control- Bacillus pumilus.

STERILIZATION BY FILTRATION

Used for sterilizing biological fluids: enzymes, vitamins, antibiotics or sugar solutions (
thermolabile and easily by radiations, hence the need for filtration).
Types of filters:
i. Earthenware filters, e.g. Berkefeld and Chamberland.
ii. Asbestos and asbestos-paper discs, e.g. Seitz filters.
iii. Sintered glass filters.
iv. Cellulose membrane filters.
Berkefeld filters: Are made from kieselguhr, a fossil diatomaceous earth (Germany and other
parts of the world). The filters are relatively coarse and have large pores.
Grades of porosity:
i. V (viel) – Coarsest.
ii. W (wening) – Finest.
iii. N (normal) – Intermediate.
Berkefeld V is normally used and does not allow small organisms such as Serratia
marcensens to pass through. NB: Mandler filter manufactured in USA is similar to Berkeld.

Chamberland filter: made from unglazed porcelain and produced in various grades of
porosity:
i. Finest grade allows the passage of only minutest virus.
ii. L1 is most porous, allows most organisms and it is used as clarifying filter.
iii. L1a, L2 and L3 are comparable to Berkefeld V, N and W filters respectively.
The filters are made in the form of a hollow candle and fluid is forced by suction or pressure
from inside to the outside or vice versa. When clogged with organic matters they are heated
to redness and allow to cool slowly.
Sintered glass filter: It is made from finely grounded glass and fussed sufficiently to make
small particles to adhere.
• Supplied in form of a disk fussed into a glass funnel.
• Special grade is manufactured by supporting a specially fine filter on a stronger
coarse one.
• After use it is washed with running water in the reverse direction and cleaned with
strong sulphuric acid.
Assessment.
• What is safety?
• Why is safety important?
• What is a biohazard?
• What is the universal precaution you must take when dealing with specimens?
• What are the rules related with the handling of sharps and wastes?
• How do you prepare 10% hypochlorite?
• What do you do when there is a spill?
• What do you do when an accident occurs?

Prepared by josomuny@gmail.com 21
4.4 Containment Cabinets:
A biosafety cabinet (BSC); also called a biological safety cabinet or microbiological safety
cabinet, is an enclosed, ventilated laboratory workspace for safely working with materials
contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety
level. However, adequate training and proper technique are required to ensure the user
obtains optimum performance from the biosafety cabinet.

Prepared by josomuny@gmail.com 22