LABORATORY SAFETY IN MICROBIOLOGY

Polle Krystle Macaranas, RMT ARSRL

INTRODUCTION
Microorganisms are normally present in the environment and many of which are carried on and in the human body.

The Microbiology laboratory poses the hazard of exposure to infectious agents. Laboratory personnel should observe safety precautions to avoid laboratoryacquired infections.
Laboratory acquired infections are often attributed to carelessness and poor technique.

4 Main Sections in Microbiology Safety Procedures

1. Good Laboratory Practice
1.1 Personal Safety Procedures 1.2 Labeling 1.3 Biosafety Guidelines

2. Aseptic Technique

2.1 Streak Plate Method 2.2 Broth Transfer Method

4 Main Sections in Microbiology Safety Procedures

3. Decontamination
3.1 Decontamination by Chemical Disinfectants 3.2 Decontamination by Autoclaving 3.3 Decontamination by Incineration

4. Transport of Biohazardous Materials

4.1 Safe handling of Sharps 4.2 Disposal Containers

1. Good Laboratory Practice

Objectives To perform safely the various activities in the laboratory To prevent contamination of the laboratory as well as the lab. personnel with the organism being handled

1. Good Laboratory Practice

1.1 Personal Safety Precautions
a.) No food or drink of any kind should be consumed in the laboratory. Smoking and applying of cosmetics are not allowed.
b.) Observe proper hand washing use a germicidal soap, if unavailable use 70% isopropyl alcohol always wash hands after handling viable materials, after removing gloves and before leaving the laboratory

1. Good Laboratory Practice

c.) Mouth pipeting is not allowed. use rubber bulbs and mechanical pipeting devices

1. Good Laboratory Practice

d.) Wear protective clothing such as laboratory coat/gown, gloves, face mask, laboratory goggles.

e.) Cuts, scratches, sores, and other lesions on the hands and exposed parts of the body should be covered with adhesive plasters.

1. Good Laboratory Practice

f.) Vortexing and centrifugation should be done in closed
containers to minimize creation of splashes and aerosols

g.) Decontaminate bench tops and other surfaces before and after leaving the laboratory or whenever there are spills (1% dilution of sodium hypochlorite/bleach, 70% isopropyl alcohol) h.) Laboratory work area should be kept clean and free of unnecessary clutter

1. Good Laboratory Practice

1.2 Labeling Label properly all specimens, bottles, reagents and other containers to avoid accidents and confusion

1. Good Laboratory Practice

1.3 Biosafety Guidelines

Biosafety levels are determined by the WHO, NIH and CDC WHO recommends that the health authorities of each country should make lists of organisms and viruses in each Risk Group as appropriate to local circumstances so that suitable precautions may be applied.

Classification of Microorganisms by RISK GROUP

RISK GROUP Description
NO or LOW individual and community risk Unlikely to cause human or animal disease
MODERATE individual risk, LOW community risk Can cause disease but unlikely to be a serious hazard Effective treatment and preventative measures are available Risk of spread of infection is limited

Organisms involved
Food spoilage bacteria Common molds yeasts

Risk Group I
Risk Group II

Staphylococci Streptococci
Enterobacteria (except

S. typhi) Clostridia Vibrios

Classification of Microorganisms by RISK GROUP

RISK GROUP Description
HIGH individual risk, LOW community risk Usually causes serious human or animal disease Effective treatment and preventative measures are available Does not ordinarily spread HIGH individual and community risk Usually causes serious human or animal disease Effective treatment and preventative measures are not usually available Can be readily transmitted

Organisms involved
M. Tuberculosis Brucella Salmonella typhi

Risk Group III

Risk Group IV

Small pox virus Ebola virus

Classification of LABORATORIES

WHO recommends 3 kinds or levels of Laboratory

1. BASIC LABORATORY (Biosafety levels 1 and 2) 2. CONTAINMENT LABORATORY (Biosafety level 3)

3. MAXIMUM CONTAINMENT LABORATORY (Biosafety level 4)

3 KINDS of LABORATORY
1. BASIC LABORATORY (Biosafety Level 1)

Simple; Adequate for work with organisms in Risk Group I and II Varies between rooms used at the Primary Health Care Level and the hospital diagnostic laboratory
Clean, comfortable, with enough space Adequate sanitary facilities, especially for handwashing

Typical Biosafety Level 1 Laboratory

3 KINDS of LABORATORY
BASIC LABORATORY (Biosafety Level 2) same as Biosafety Level 1 Laboratory must be equipped with an autoclave and a Biological Safety Cabinet Doors are posted with a biohazard sign a separate bin for infectious and non infectious waste

Typical Biosafety Level 2 Laboratory

3 KINDS of LABORATORY
2. CONTAINMENT LABORATORY (Biosafety Level 3)

More sophisticated; Used for the work with organisms in Risk Group III
A separate room with controlled access by authorized staff only Requires a microbiological safety cabinet Only highly skilled and experienced staff are allowed to work in this laboratory

Typical Biosafety Level 3 Laboratory

3 KINDS of LABORATORY
3. MAXIMUM CONTAINMENT LABORATORY (Biosafety Level 4) Intended for work with viruses in Risk Group IV The most strict safety precautions are necessary Separate buildings with strictly controlled access through air locks and exit through decontaminant showers Laboratory staff is specially trained for the work

MICROBIOLOGICAL SAFETY CABINETS

BIOLOGICAL SAFETY CABINETS (BSCs) A ventilated cabinet that protect laboratory workers from aerosols through sterilization by either HEAT, UV light, or passage of air through a high-efficiency particulate air (HEPA) filter that removes particles. Cabinets are classified as Class I, II, or III based on performance characteristics with regard to biological containment

MICROBIOLOGICAL SAFETY CABINETS

Class I and II Biological Safety Cabinets provide effective partial containment for procedures with Risk Group III organisms (i.e M. tuberculosis, Salmonella typhi, Brucella) Most microbiology laboratories use Class II BSC Class IIa fixed opening Class IIb variable sash opening

Class III cabinets are used for Risk Group IV viruses (i.e. smallpox, Ebola virus)

MICROBIOLOGICAL SAFETY CABINETS

Class I Biological Safety Cabinet
-Open-fronted, negative pressure ventilated cabinet -The operator sits at the cabinet, looks through the glass screen, and works with the hands inside. -Unsterilized room air enters and circulates within the cabinet and the exhaust air from the cabinet is filtered by a HEPA filter

MICROBIOLOGICAL SAFETY CABINETS

Class II Biological Safety Cabinet
-More complicated, sometimes called the laminar flow cabinet -70 % of the air is recirculated through filters so that working area is bathed in clean air -The air flow carries along any aerosols produced in the course of the work and then removed by the filters. -30 % of the air is exhausted to the atmosphere

MICROBIOLOGICAL SAFETY CABINETS

Class III Biological Safety Cabinet
-Provides the highest level of safety -Entirely enclosed; all air entering and leaving the cabinet is sterilized with HEPA filter. -Supply air is drawn through a HEPA filter while exhaust air is filtered through two HEPA filters -All infectious materials are handled with rubber gloves that are sealed to the cabinet

Biological Safety Cabinetry Protocol

Surfaces in the BSC should be wiped down with a germicide before and after each use. A clean cloth soaked with a 70 to 85% solution of ethanol may be used. Gloves and a gown should be worn during cleaning.

Biological Safety Cabinetry Protocol

The cloth used, as well as the gloves and gown, should be autoclaved before disposal. It is important that the UV lamp is cleaned with alcohol on a weekly or biweekly basis to ensure that the lamp intensity is maintained. UV lamps should not be turned on while an operator is working in the cabinet.

2. ASEPTIC TECHNIQUE

A method that prevents the introduction of unwanted organisms into an environment. In microbial cultures, it is used to prevent introducing additional organisms (contaminants) into the culture.

2. ASEPTIC TECHNIQUE
Purposes: To prevent microbial contamination of laboratory worker which may result in infection To prevent contamination of the environment they are working in (e.g. fomites) To prevent contamination of the specimen especially when a pure culture is desired

2. ASEPTIC TECHNIQUE
General Guidelines Always use the proper aseptic technique when transferring cultures from one medium to another.
Heat the entire piece of metal of the inoculation instrument in the flame: it should be RED HOT. Be sure to COOL your inoculation instrument before picking the inoculum. Use an inoculating needle for agar deeps and an inoculating loop for the agar plate and the broths. Label all test tubes and petri plates

2. ASEPTIC TECHNIQUE
General Guidelines Place test tubes in racks when working at your table Keep test tube caps and petri dish covers on media to reduce contamination ALWAYS check agar plates carefully to make sure that there are no mold or bacterial contaminants on the plate: if so, discard the plate. Do the same with any tubed media.

2. ASEPTIC TECHNIQUE
To practice ASEPTIC TECHNIQUES one must: Know how to prepare and clean a work area Know how to handle bacteria in different culture containers Know how to transfer organisms from one place to another without contamination

2. ASEPTIC TECHNIQUE
2.1 STREAK PLATE METHOD

The most common way of separating bacterial cells on the agar surface to obtain isolated colonies To isolate individual organisms into pure culture

2. ASEPTIC TECHNIQUE

2. ASEPTIC TECHNIQUE
2.2 BROTH TRANSFER METHOD the sterile technique of inoculating a bacterial organism into a liquid broth

Used for bacterial isolation and identification

3. DECONTAMINATION

A process of removing or neutralizing hazardous clinical and radioactive wastes by chemical or mechanical means TYPES OF DECONTAMINATION Decontamination by Chemical Disinfectants Decontamination by AUTOCLAVING Decontamination by INCINERATION

A. Decontamination by Chemical Disinfectants

chemical agents used to kill microorganisms that may cause a disease Phenolic Compounds

Alcohols
Chlorine Aldehydes

A. Decontamination by Chemical Disinfectants

PHENOLIC COMPOUNDS

most widely used today e.g. LYSOL

ALCOHOLS most popular 70% isopropyl alcohol CHLORINE Sodium hypochlorite (household bleach) 1: 10 or 1: 100 dilution with water

A. Decontamination by Chemical Disinfectants

ALDEHYDES

4 - 10 % formaldehyde
2% glutaraldehyde (e.g. Cidex) excellent laboratory disinfectant

B. Decontamination by AUTOCLAVING

one of the most dependable methods of sterilizing laboratory equipment and decontaminating biohazardous waste

For BSL-2/3 laboratory

operated by a properly trained personnel

C. Decontamination by INCINERATION
A treatment technology involving destruction of waste by controlled burning at high temperatures; to remove the water and reduce the remaining residues to a safe, non-burnable ash that can be disposed of safely on land, in some waters, or in underground locations.

used for biomedical waste

waste should be initially treated at the containment barrier then packaged before removing off-site

4. TRANSPORT of BIOHAZARDOUS MATERIALS

Safe handling of Sharps (Needles, syringes, slides, pipets, capillary tubes, scalpels ,etc.) Dispose in designated containers Used disposable needles must not be bent, sheared, broken, or otherwise manipulated by hand before disposal Broken glassware should not be handled directly by hand, remove by brush and dustpan, tongs, or forceps

4. TRANSPORT of BIOHAZARDOUS MATERIALS

Disposal Containers used for all discarded plates, tubes, clinical samples and other contaminated materials Special boxes are provided for sharps (e.g. syringes, slides, broken glass) to minimize risk of injury Avoid overfilling contaminated materials should be autoclaved before disposal

4. TRANSPORT of BIOHAZARDOUS MATERIALS

The primary infectious agent container, must be placed in an unbreakable second container that can be sealed using a screw-top tube or a plastic bag Transport disposal containers with caution to the autoclave room

CONCLUSION

Microbiology safety is largely in the hands of the laboratory worker, who even in the remote areas with few resources can achieve a considerable measure of protection by maintaining a high standard of personal hygiene and care, and by good laboratory practice.