Practical Manual of Medical Microbiology CP Prince
Chapter Notes

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Laboratory Safety1

Laboratory safety is a vital component of functioning of any laboratory. Safety procedures and precautions to be followed in the Microbiology laboratory should be designed to:
  • Restrict microorganisms present in specimens or cultures to the vessels in which they are contained.
  • Prevent environmental microorganisms (normally present on hand, hair, clothing, laboratory benches or in the air) from entering specimens or cultures and interfering with the results of the studies.
Laboratory Biosafety Levels
Four Biosafety levels have been recommended based on the type of microbes you are working with.
Biosafety Level -1 (BSL-1): Adherence to standard microbiological practices. No special requirement as regards containment equipment.
Biosafety Level-2 (BSL-2): In addition to the use of standard microbiological practice, laboratory coats, decontamination of infectious wastes, limited access, protective gloves and display of biohazard sign and partial containment equipment are the requirements for this level.
Most peripheral and intermediate laboratories need BSL-1 or BSL-2 laboratory facilities.2
BSL-3: In addition to BSL-2, it has special laboratory clothing, controlled access to laboratory and partial containment equipment.
BSL-4: BSL-3 plus entrance through change room where laboratory clothing is put on, shower on exit, all wastes are decontaminated before exit from the facility. It requires maximum containment equipment.
Laboratory facilities in BSL-2 (Figs 1.1 and 1.2)
  • Laboratory should be designed in such a way that it can be easily cleaned.
  • Laboratory contains a sink for washing.
  • Laboratory tops are impervious to water but resistant to acids, alkalis and organic solvents.
  • An autoclave to decontaminate infectious material is available.
  • Illumination is adequate for all laboratory activities.
  • Storage space is adequate.
  • Preventive measures against laboratory infections
These are aimed to protect workers, patients and cultures. Following steps are suggested:
  • Perform adequate sterilization before washing or disposing waste.
  • Provide receptacle for contaminated glassware.
  • Provide safety hood.
  • Ensure that tissues are handled and disposed of properly.
  • Promote regular hand washing and cleaning of bench tops.
  • Ensure use of gloves.
  • Provide mechanical pipetting devices.
  • Protect patients from laboratory personnel with skin or upper respiratory tract infections.
  • Provide special disposal containers for needles and lancets.
Pipetting and suctioning have been identified as the significant and consistent causes of occupational infections. Various important precautions that must be taken while pipetting are:
  • Develop pipetting techniques that reduce the potential for creating aerosols.3
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    Figure 1.1: Hazard warning symbols
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    Figure 1.2: Biological safety cabinets
  • Plug pipettes with cotton.
  • Avoid rapid mixing of liquids by alternate suction and expulsion.
  • Do not forcibly expel material from a pipette.
  • Do not bubble air through liquids with a pipette.
  • Prefer pipettes that do not require expulsion of last drop of liquid.
  • Drop material having pathogenic organisms as close as possible to the fluid or agar level.
  • Place contaminated pipettes in a container having suitable disinfectant for complete immersion.
A variety of pipettes are available. Selection should depend upon the ease of operation and the type of work to be performed.
Hypodermic Syringes and Needles
Accidents involving the use of syringes and needles while drawing blood from patients or performing experiments on laboratory animals are among the most common causes of occupational infections in laboratories and health care facilities. They account for almost 25% of the laboratory-acquired infections that occur by accidents. The practices which are recommended for hypodermic needle and syringes are:
  • Avoid quick and unnecessary movements of the hand holding the syringe.
  • Examine glass syringes for chips and cracks, and examine needles for barbs and plugs.
  • Use needle locking (Luer Lock type) syringes only and be sure that needle is locked securely.
  • Wear surgical or other gloves.
  • Fill syringes carefully to minimize air bubbles and frothing.
  • Expel excess air, liquid and bubbles vertically into a cotton pledget moistened with suitable disinfectant.
  • Do not use syringe to forcefully expel infectious fluid into an open vial for mixing. Mixing with a syringe is appropriate only if the tip of the needle is held below the surface of the fluid in the tube.
  • Do not bend, shear, recap or remove the needle from syringe by hand.
  • Place used needle-syringe units directly into a puncture-resistant container and decontaminate before disassembly, reuse or disposal.
Opening Containers
The opening of vials, flasks, petri dishes, culture tubes, embryonated eggs, and other containers of potentially infectious materials poses potential but subtle risks of creating droplets, aerosols or contamination of the skin or the immediate work area. The most common opening activity in most health care laboratories is the removal of stoppers from containers of clinical materials. It is imperative that specimens should be received and opened only by personnel who are knowledgeable about occupational infection risks. Various precautions that can be taken in this regard are:
  • Open containers with clinical specimens in well-lighted and designated areas only.
  • Wear a laboratory coat and suitable gloves.
  • If possible, use a plastic-backed absorbent paper towel to:
    • Facilitate clean-up
    • Reduce generation of aerosols
  • Specimens which are leaking or broken may be opened only in safety cabinets.
Tubes containing bacterial cultures should be handled with care. Vigorous shaking of liquid cultures creates a heavy aerosol. When a sealed ampoule containing a lyophilized or liquid culture is opened, an aerosol may be created. Ampoules should be opened in a safety cabinet (Fig. 1.3).
Laboratory Access
  • As far as possible children and pregnant women visitors should not enter the microbiological laboratories.
  • Appropriate signs should be located at points of access to laboratory areas directing all visitors to a receptionist or receiving office for access procedures.
  • The universal biohazard symbol shall be displayed at specific laboratories in which manipulations of organisms with moderate and heavy risk are being carried out.6
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    Figure 1.3: Biological safety cabinet
    Only authorized visitors shall enter the laboratory showing universal biohazard sign (Fig. 1.4). Doors displaying biohazard symbol shall not be propped open, but shall remain closed when in use.
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Figure 1.4: Universal biohazard sign
  • All employees and visitors in microbiological laboratories shall wear laboratory clothing and laboratory shoes or shoe covers.
  • Disposable gloves shall be worn wherever radiological, chemical, carcinogenic materials or virus preparations of moderate to high risk are handled.
  • Laboratory clothing including shoes shall not be worn outside the work area.
Accidents in Laboratory
In the microbiological laboratory, infections pose the most frequent risk. The important pathogens are:
Hepatitis B virus,
Shigella spp.
Salmonella spp.
including S typhi
Brucella spp.
Bacillus anthracis
Leptospires Yersinia pestis
Mycobacteria spp.
Accidents and Spills
The order of priorities is as follows:
  • Protection of personnel
  • Confinement of contamination
  • Decontamination of personnel
  • Decontamination of area involved
Decontamination of skin: The area is washed thoroughly with soap and water. Detergents or abrasive materials must not be used and care must be taken not to damage the skin.
Decontamination of cuts\eyes: These are irrigated with water taking care to prevent the spread of contamination from one area to another.
Decontamination of clothing: Contaminated garments should be removed immediately and placed in a container. They should not be removed from the spill location until contamination has been monitored.8
Decontamination of work surfaces
  • Flood the total spillage area including the broken container with disinfectant.
  • Leave undisturbed for 10 minutes.
  • Mop with cotton wool or absorbent paper.
  • Wear disposable gloves, apron and goggles.
  • If a dustpan and brush or forceps have been used these too require disinfection.
  • For blood or viruses, hypochlorites (10 gm/L) are used.
  • Do not use hypochorite solution in centrifuges.
  • Use activated gluteraldehyde (20 gm/L) on surfaces for viral decontamination.
  • Place all potentially contaminated materials in a separate container and retain until monitored.
  • Restrict the entry to such an area until contamination monitoring has been carried out.
Management of Laboratory Accidents
An adequately equipped first-aid box should be kept in the laboratory in a place that is known and accessible to all members of staff. The box must be clearly marked and preferably be made of metal or plastic to prevent from damage by pests. A medical officer should be consulted regarding the contents of the box. A first-aid chart giving the immediate treatment of cuts, burns, poisoning, shock and collapse, should be prepared and displayed in the laboratory.
General Laboratory Directions for Safety
The salient general laboratory directions which must be obeyed by all are:
  • Long hair should be bound back neatly away from shoulders.
  • Do not wear any jewellery to laboratory sessions.
  • Keep fingers, pencils, bacteriological loops, etc. out of your mouth.
  • Do not smoke in the laboratory.
  • Do not lick labels with tongue (use tap water).
  • Do not drink from laboratory glassware.
  • Do not wander about the laboratory; uncontrolled activities cause:
    • Accidents
    • Distract others
    • Promote contamination
  • Do not place contaminated pipettes on the bench top.
  • Do not discard contaminated cultures, glassware, pipettes, tubes or slides in wastepaper basket or garbage can.
  • Avoid dispersal of infectious materials.
  • Operate centrifuges, homogenizer and shakers safely.
  • Immunize the laboratory workers against vaccine-preventable diseases such as hepatitis B, meningococcal meningitis, rabies, etc.