Experiment name :Bio-safety and blood-borne pathogen training program

Principle :
The principle of laboratory biohazard and biosafety is a comprehensive framework designed to
ensure the safe handling of biological agents within laboratory settings. It is grounded in the
recognition that biological agents, including bacteria, viruses, fungi, and other microorganisms,
can pose risks to the health of laboratory personnel, the surrounding community, and the
environment. This principle encompasses several fundamental elements. First and foremost,
laboratories must conduct a thorough risk assessment before commencing any work with
biological agents. This assessment helps to determine the level of risk associated with the
specific agents, procedures, and laboratory conditions. It is a critical step in categorizing the
laboratory work into appropriate biosafety levels .

Biosafety
Biosafety refers to the measures and practices designed to prevent the unintentional release of
potentially harmful biological agents, such as microorganisms or genetically modified
organisms, into the environment or to protect individuals from exposure to these agents. It
encompasses a range of procedures, safety equipment, and protocols that are used in laboratories,
healthcare settings, and other environments where biological materials are handled. Biosafety
levels (BSL) are used to categorize the level of containment and safety measures required, with
BSL-1 being the lowest risk and BSL-4 the highest. Biosafety is crucial in preventing the spread
of diseases and maintaining the safety of researchers and the general population.
Biosecurity
Biosecurity refers to measures and practices implemented to prevent the spread of infectious
diseases and protect against biological threats. It encompasses a wide range of actions, from
controlling access to restricted areas and laboratories to protocols for handling and storing
potentially hazardous biological materials. Biosecurity is essential in various fields, including
agriculture, healthcare, and research, to mitigate the risks associated with the accidental release
or deliberate misuse of dangerous pathogens and biological agents.
Key features of a biosecurity plan should include:
● facility security
● inventory of pathogens and
● emergency protocols for security incidents.

Biohazard
A biological hazard, or biohazard, is a biological substance that poses a threat to the health of
living organisms, primarily humans. This could include a sample of a microorganism, virus or
toxin that can adversely affect human health.
Example of biohazards:

● Human blood and blood products.These pathogen such as HIV and hepatitis B ,can be
transmitted through contact with infected blood .
● Human body fluids, including semen, vaginal secretions, amniotic fluid, and pleural
fluid.
● Microbiological wastes, including discarded specimen cultures, discarded live and
attenuated viruses, and disposable culture dishes.
● Pathological waste, including waste biopsy materials, human tissues, organs and body
parts, and anatomical parts from autopsy, surgeries, and procedures.
● Sharps waste, including used syringes, scalpels, broken glass that is contaminated with
blood or potentially infectious material.

● Respiratory pathogen : These pathogen such as influenza virus and the SARS –CoV -2
virus,can be transmitted through the air .

Biohazardous agents can be classified into several groups:

● Bacteria
● Viruses
● Fungi
● Parasites
 ● Zoonotic Pathogens
● Prions
● Toxins
● Recombinant DNA
● Cell Lines
● Human blood and tissue

A laboratory-acquired infection (LAI) is an infection that is acquired in a laboratory.

Laboratory-acquired infections due to a wide variety of bacteria, viruses, fungi, and parasites
have been described. Although the precise risk of infection after an exposure remains poorly
defined, surveys of laboratory-acquired infections suggest that Brucella species, Shigella species,
Salmonella species, Mycobacterium tuberculosis, and Neisseria meningitidis are the most
common causes. Infections due to the bloodborne pathogens (hepatitis B virus, hepatitis C virus,
and human immunodeficiency virus) remain the most common reported viral infections, whereas
the dimorphic fungi are responsible for the greatest number of fungal infections.

Types of infectious agents

Agent Description

Microorganisms, or microbes are extremely diverse microscopic organisms,

Microorganisms including archaea and bacteria, which are commonly grown and studied in
laboratories and can infect laboratory animals and laboratory workers.

Viruses infect all types of life forms, from animals and plants to microorganisms,
including bacteria and archaea, and are commonly passaged and studied in
Viruses
laboratories, which can directly or indirectly cause zoonotic infection of laboratory
animals and laboratory workers.

Parasites may include various types of protozoa, worms and ectoparasites which
Parasites are studied in laboratories and which can cause the infection of laboratory workers
and animals.

Routes of transmission
 Route Description

This occurs when microbes, viruses and/or parasites are transmitted

through direct contact with laboratory workers, or contaminated surfaces,
Mucous membranes
or items such as vials, devices, and/or equipment, to the host's mucous
membranes.

This occurs with the dissemination of either airborne droplet nuclei of

evaporated droplets containing microorganisms or viruses, or dust
particles containing the infectious agent, are transmitted via the host's
Respiratory tract respiratory tract. Microorganisms carried in this manner can be dispersed
widely by air currents and may become inhaled by the infected host
within the same room or over a longer distance from the source host,
depending on environmental factors.

This occurs with the oral intake of an infectious agent through a pipette,
Gastrointestinal tract
smoking or eating, infecting the host through their gastrointestinal tract.

This occurs with a vector, such as mosquitoes, flies, rats, and other
vermin transmit microbes, viruses and/or parasites to a host through a
Percutaneous inoculation
bite or scratch; or the transmission of an agent carrying an infectious
pathogen through a needle or syringe.

Laboratory-Acquired Infection Chart (Till Date):

Organism Route of Transmission % of Cases

Neisseria meningitidis Inhalation, contact with 5-10%
mucous membranes
Shigella species Ingestion, contact with 15-20%
mucous membranes
Brucella species Percutaneous inoculation, 20-30%
inhalation
Mycobacterium Inhalation 10-15%
tuberculosis
 Salmonella species Ingestion, contact with 10-15%
mucous membranes
Hepatitis B virus Percutaneous 5-10%
(HBV) inoculation, contact with
mucous membranes
Hepatitis C virus Percutaneous inoculation 2-5%
(HCV)
Human Percutaneous inoculation < 1%
immunodeficiency
virus (HIV)
Other bacteria Percutaneous 10-15%
inoculation, inhalation,
contact with mucous
membranes
Other fungi Inhalation, contact with 2-5%
mucous membranes
Other viruses Inhalation, contact with 5-10%
mucous membranes
Other parasites Ingestion, contact with 1-2%
mucous membranes

Route of transmission of laboratory-acquired infection(LAI)

Microorganisms can enter the body through accidental inoculation, ingestion, mucous
membrane, direct contact or aerosols. In laboratory-acquired infections the route may not be
the same as when the disease is acquired naturally. The dose or number of organisms required
to initiate infection is often difficult to ascertain and depends on the route of exposure. It is not
unreasonable to expect that any person who works with pathogenic microorganisms will be
more likely than members of the community to become infected. There is evidence that some
organisms cause more laboratory infections than others and that the incidence of infections
varies according to the nature of the work and the health status of the worker. Attention must
be given to ways in which laboratory workers may become infected.

Biological agent Infectios dose Route of inoculation

Escherichia coli 108 ingestion
Escherichia coli 0157:H7 10 ingestion
Bacillus cereus >or=105 ingestion
Camphylobacter jejuni <or =500 ingestion
Treponema pallidum 57 intradermal
Francisella tularensis 10 Inhalation ,ingestion
Bacillus anthracis 8-503 Inhalation,ingestion
Mycobacterium tuberculosis and <10 Inhalation
bovis
Coxiella burneti 10 inhalation
Salmonella typhi 105 ingestion
Shigella flexneri 180 ingestion
Treponema pallidum 57 intradermal
Vibrio cholerae 108 ingestion
Yersinia pestis 100-500 Ingestion,inhalation
Smallpox virus (v.major) 10-100 Ingestion,inhalation(rare)
poliovirus 2 ingestion
Influenza A2 virus <790 inhalation
Venezuelan encephalitis virus 1 subcutaneous
Hepatitis A virus 10-100 Ingestion ,intravenous
adenovirus >150 intranasal
Respiratory syncytial virus >100-640 intranasal
Plasmodium falciparum 10 intravenous
Histoplasma capsulatum 10(mice) inhalation

*Dose is the number of micro-organism otherwise indicated. These data are collected from different
peer reviewed resources. For discussion about the definition of "infectious dose" see: Johnson B. OSHA
Infectious Dose White Paper. Applied Biosafety 2003; 8(4): 160-165.

Biohazard risk assessment:

Biological risk assessment is one of the key principles of biosafety. It is the process
used to identify the hazardous characteristics of an infectious organism, the activities
that could lead to exposure, the chances of contracting a disease after an exposure and
the consequences of an infection.

The World Health Organization (WHO) defines the risk groups

as:
WHO Risk Group 1 (no or low individual and community risk) - A microorganism that
is unlikely to cause human disease or animal disease.

WHO Risk Group 2 (moderate individual risk, low community risk) - A pathogen that
can cause human or animal disease but is unlikely to be a serious hazard to laboratory
workers, the community, livestock or the environment. Laboratory exposures may
cause serious infection, but effective treatment and preventative measures are
available and the risk of spread of infection is limited.

WHO Risk Group 3 (high individual risk, low community risk) - A pathogen that
usually causes serious human or animal disease but does not ordinarily spread from
one infected individual to another. Effective treatment and preventive measures are
available.

WHO Risk Group 4 (high individual and community risk) - A pathogen that usually
causes serious human or animal disease and that can be readily transmitted from one
individual to another, directly or indirectly. Effective treatment and preventive
measures are not usually available.

Risk Disease Severity Individual Comunity Biological agent

Group Risk Risk
Risk Group Unlike to cause Low Individual low community Bacillus subtilis
1 diseade Naegleria gruberi
Infectious canine
hepatitis virus
E. coli
Risk Group Effective treatment Moderate low community Measles virus
2 is available Individual Salmonellae
Toxoplasma spp.
Hepatitis B Virus
Risk Group Severe disease High Individual Moderate M. tuberculosis St.
3 Treatment possible community risk Louis encephalitis
virus Coxiella
burnetti
Risk Group Severe to fetal case High Individual Ebola Zaire Sin
4 Treatment not Nombre virus Rift
possible Valley Fever

Cell line :

Cell line assessment is a process to evaluate the quality of a cell line for use in research or
production. It includes a variety of tests and procedures to assess the cell line's authenticity,
purity, growth characteristics, genetic stability, and functional properties. Cell line assessment
is important for a number of reasons:
 To ensure that the cell line is the correct cell type and has not been contaminated with
other cell lines.
  To identify any changes in the cell line that may have occurred over time, such as
genetic instability or loss of function.
 To ensure that the cell line is suitable for the intended research or production
application.

Laboratory animals:
Work with animals poses a variety of hazards including exposure to infectious agents (zoonotic
diseases), animal bites and scratches. In addition there are numerous additional regulatory
requirements such as those of the Canadian Council for Animal Care. These are covered in a
separate course designed for those who work with animals .
Control of biohazards (containment levels )
The containment levels range from the lowest risk of biosafety level 1 (BSL1) to the highest risk
of biosafety level 4 (BSL4)
BSL-1 (Basic Level):

 Suitable for work with low-risk agents that do not cause disease in healthy adults.
 Standard laboratory practices and basic personal protective equipment (PPE) are
sufficient.
BSL-2 (Moderate Level):.
 BSL-2 facilities are commonly used for handling pathogens like E. coli, Staphylococcus,
or hepatitis viruses.
BSL-3 (High Level):
 PPE may include respiratory protection (e.g., N95 masks) in addition to lab coats, gloves,
and eye protection.
 Commonly used for pathogens like tuberculosis bacteria, certain strains of the influenza
virus, and the bacterium responsible for anthrax.
BSL-4 (Maximum Level):
 BSL-4 facilities have the highest level of containment, with stringent entry and exit
procedures, controlled airflow, and full-body suits with integrated air supplies for
personnel.
 Examples of agents worked with at BSL-4 include Ebola virus, Marburg virus, and
smallpox virus.

Biosafety success:
Biosafety success can be measured by various factors that indicate effective management and
control of biohazards. Here are some indicators of biosafety success:
1. Incident Prevention:Few or no incidents involving the accidental release of biohazards,
infections, or exposures among personnel.
2.Compliance with Regulations: Adherence to local, national, and international biosafety
regulations and guidelines.
3.Safety Culture: A strong culture of safety within the organization, where all personnel
understand the importance of biosafety and actively participate in safe practices.
4.Effective Training: Comprehensive and ongoing training programs that ensure all personnel
are well-informed about biohazards and safety procedures.
5.Proper Equipment and Facilities: Adequate infrastructure, containment facilities, and
equipment that meet or exceed required biosafety levels.
Signs and Levels:
If a person enters an area where biohazardous waste is present, they need to be aware that
they are going to be at risk. With a biohazard sign in place, you are making sure everybody who
sees it is taking extra caution around the contaminated area. All laboratories assigned a
containment level of 2 or greater, must have a biohazard warning sign posted on the outside of
all entry doors giving the containment level and the name of the contact person or laboratory
supervisor. All containers, equipment and storage units used with biological agents, as well as
biohazardous wastes must have biohazard labels.
Human Pathogen:
Human blood and body fluids are a potential source of pathogenic microorganisms that may
present a risk to workers who are exposed during the performance of their duties. The sources
include:
• Blood
• Semen
• Vaginal secretions
• Other body fluids (cerebrospinal, amniotic, synovial)
• Tissue cultures
• Organ cultures
• Infected experimental animals
The pathogens of primary concern are:
• the human immunodeficiency virus (HIV),
• hepatitis B virus (HBV),
• Hepatitis-C virus (HVC) and can also include any other sexually-transmitted or other disease
pathogenic to humans .

General laboratory practice:

General laboratory practices are essential for maintaining safety, accuracy, and consistency in a
laboratory setting. Here are some key practices to follow:
1. Personal Protective Equipment (PPE): Always wear the appropriate PPE, including lab coats,
gloves, safety goggles, and any other necessary protective gear.
2. Lab Safety: Familiarize yourself with the specific safety protocols and emergency procedures
in your lab. This includes knowing the location of safety equipment like eyewash stations and
fire extinguishers.
3. Chemical Handling: Handle chemicals with care. Read labels, understand their properties,
and follow the recommended storage and handling procedures. Always label and date
containers.
4. Equipment Use: Use laboratory equipment and instruments according to instructions and
with proper training. Calibrate and maintain equipment regularly.
5. Waste Disposal: Follow guidelines for proper disposal of hazardous waste and chemicals.
Segregate waste as required and dispose of it in designated containers.
6. Record Keeping: Maintain accurate and detailed records of experiments, procedures, and
results. This is essential for reproducibility and accountability.
7. Sample Handling: Properly handle and store samples to avoid contamination and maintain
sample integrity.
8. Hygiene: Maintain good personal hygiene and avoid eating, drinking, or applying makeup in
the lab. Wash hands before and after working in the lab.
9. Labeling: Clearly label all containers, samples, and equipment with relevant information,
including names, dates, and content.
10.Proper Pipetting: Use pipettes and micropipettes accurately to prevent errors in volume
measurements.
11. Ventilation: Ensure proper ventilation in the lab, especially when working with volatile or
hazardous substances.
12.Emergency Preparedness: Know the location of emergency exits, emergency contact
information, and how to respond to spills, fires, or other emergencies.
13.Cleanliness: Keep the laboratory clean and organized. Regularly clean workspaces and
equipment to prevent contamination.

Adhering to these general laboratory practices is crucial for the success of experiments, the
safety of personnel, and the integrity of research conducted in the lab. Specific practices may
vary depending on the type of laboratory and the nature of the work being performed.

Biological safety cabinet :

A Biological Safety Cabinet (BSC) is a specialized laboratory equipment designed to provide a
controlled, sterile, and safe environment for working with biological materials, such as bacteria,
viruses, and cell cultures. It helps protect both the researcher and the surrounding environment
from potential contamination. BSCs use high-efficiency particulate air (HEPA) filters to filter the
incoming and outgoing air, creating a clean and controlled airflow. There are different classes
and types of BSCs, each serving specific purposes and offering varying levels of protection.

BSCs are classified into four different types based on the level of protection they provide:

Biosafety Cabinet Containment Level Protection Provided Examples of Use

Class
Class 1 basic User and Low-risk biological
environment agents, such as
Bacillus subtilis and
non-pathogenic
strains of Escherichia
coli
Class 2 moderate User and product Moderate-
environment risk biological agents,
And product such as HIV, measles
virus, and
Staphylococcus
aureus
Class 3 high User and High-risk biological
environment and agents, such as
 product Mycobacterium
tuberculosis, Brucella
species, and SARS-
CoV-2
Class 4 Maximum User and Extremely high-risk
environment and biological agents,
product such as Ebola virus,
Marburg virus, and
Nipah virus

Decontamination
Decontamination in public health refers to the process of removing, neutralizing, or destroying
contaminants, such as harmful microorganisms, chemicals, or radioactive materials, to prevent
their spread and reduce health risks to the public. It's an essential component of emergency
response, disease control, and environmental protection. Decontamination methods can
include:
1. Physical cleaning
2. Chemical decontamination
3. Biological decontamination
4. Radiological decontamination
Organism Severity Recommended Disinfection Method Low-risk organisms a low-level
disinfectant, such as 70% ethanol Soap and water, followed by Moderate-risk organisms A
medium-level disinfectant, such as glutaraldehyde or bleach High-risk organisms A high-level
disinfectant, such as peracetic acid or vaporized hydrogen peroxide

Organism Severity Recommended Disinfection Method

Low risk organism Soap and water, followed by a low-level
disinfectant, such as70% ethanol
Moderate risk organisms A medium-level disinfectant, such as
glutaraldehyde or bleach
High risk organism A high-level disinfectant, such as peracetic
acid or vaporized hydrogen peroxide

Mainly of 2 types :
1.Disinfection
2.Sterilization

1.Disinfection is the killing of infectious agents outside the body by direct exposure to
chemical or physical agents. However, chemical disinfectants only work if surfaces have
been thoroughly cleaned first to remove grease and other dirt.
2.Sterilization

Sterilization is the process of eliminating all forms of microbial life, including bacteria, viruses,
and spores, to prevent the transmission of infections. There are various methods of
sterilization, each with its own advantages and disadvantages. Here are some common types of
sterilization:
• Autoclaving: Autoclaving is one of the most widely used methods of sterilization. It involves
exposing items to high-pressure steam at temperatures typically around 121°C (250°F) for a
specific period. This effectively kills bacteria, viruses, and spores.
Dry Heat Sterilization: This method uses hot air to sterilize items. It requires higher
temperatures (typically 160-180°C) and longer exposure times compared to autoclaving. Dry
heat is suitable for heat-resistant items that can withstand the high temperatures.
• Ethylene Oxide (ETO) Sterilization: Ethylene oxide gas is used to sterilize items that are
sensitive to heat and moisture. It's commonly used for medical devices, pharmaceuticals, and
some food products. However, ETO is potentially toxic and requires thorough aeration to
remove residual gas.
• Chemical Sterilization: Chemical agents like hydrogen peroxide, peracetic acid, and
formaldehyde can be used to sterilize surfaces, equipment, and some medical devices. These
chemicals kill or inactivate microorganisms but must be used with care due to potential health
hazards.
• Filtration: For heat-sensitive liquids and gases, filtration through membrane filters with small
pores can be used to remove microorganisms. This method is often employed in the
pharmaceutical and biotechnology industries.
• Boiling: Boiling water for an extended period can be used to sterilize items, but it is less
reliable than other methods, as some heat-resistant spores may survive.
• Sterilizing Chemical Solutions: Chemical agents like glutaraldehyde are used to sterilize
medical instruments and endoscopes by immersing them in the solution for a specific duration.
It's important to select the appropriate sterilization method based on the type of items to be
sterilized, their heat and moisture sensitivity, and the desired level of microbial reduction. Each
method has its own limitations and safety considerations, so it's crucial to follow established
protocols and guidelines to ensure effective sterilization
Different types of disinfectant preparation
here are various types of disinfectant preparations, each with its own ingredients and uses.
Some common types include:
1. Alcohol-Based Disinfectants:These contain ethanol or isopropyl alcohol and are effective
against a wide range of pathogens. They are commonly used for sanitizing hands and surfaces.
2. Bleach-Based Disinfectants: These solutions contain sodium hypochlorite and are effective
against bacteria, viruses, and fungi. They are used for disinfecting surfaces and can be diluted to
various concentrations.
3. Quaternary Ammonium Compounds (Quats): Quats are often found in disinfectant sprays
and wipes. They are effective against various microorganisms and are frequently used in
healthcare settings.
4. Hydrogen Peroxide-Based Disinfectants:Hydrogen peroxide can be used as a disinfectant
when properly diluted. It is effective against a wide range of microorganisms and can be used
on surfaces and for wound care.
5. Iodine-Based Disinfectants: Iodine solutions, like povidone-iodine, are used as antiseptics and
disinfectants. They are effective against bacteria, viruses, and fungi and are used in healthcare.
6. Phenolic Disinfectants: Phenol-based disinfectants are effective against various
microorganisms. They are often used in healthcare and industrial settings.
7. Chlorine Dioxide Disinfectants: Chlorine dioxide is used for water disinfection, and it can also
be used to disinfect surfaces.
8. Silver-Based Disinfectants: Some products use silver ions as a disinfectant, particularly in
healthcare settings.
9. Acidic Disinfectants: These disinfectants are effective against mineral deposits and scale.
They are often used in cleaning and disinfecting bathrooms and kitchen areas.
10. Natural Disinfectants:Some people opt for natural disinfectants like vinegar or essential oils
(e.g., tea tree oil) for milder disinfecting purposes.

It's important to use disinfectants according to their intended purpose, follow manufacturer
instructions, and consider safety precautions, such as proper ventilation and protective
gear when necessary.
Waste management
Waste management in the lab is essential to protect the health of laboratory personnel, the
public, and the environment. Laboratory waste can contain a variety of hazardous materials,
including chemicals, biological agents, and radioactive materials. If not properly managed, this
waste can pose a serious risk to human health and the environment.
There are a number of key principles of laboratory waste management:
Segregation: Laboratory waste should be segregated at the source to prevent
crosscontamination and to facilitate proper treatment and disposal.
Minimization: Laboratory waste should be minimized by reducing the amount of waste
generated and by recycling materials whenever possible.
Safe handling and storage: Laboratory waste should be handled and stored in a safe manner to
prevent exposure to laboratory personnel, the public, and the environment.
Proper treatment and disposal: Laboratory waste should be treated and disposed of in a
manner that is safe and environmentally sound.

Discussion:
Microbiology laboratories are special, often unique, work environments that poses risks to
people both in or near them. There have been numerous cases of laboratory acquired
infections and also cases in which harmful microbes have been released into the environment.
Through these cases biosafety has been improved and will continue to improve. However
regular updates must be made to all procedures ensuring their relevance.
All components of biosafety are important and must be followed. Projects that require specific
laboratory conditions must never be done without those conditions being put in place.
Biosafety levels must be observed strictly. Laboratory personnel must also be trained in
biosafety protocols before they begin working in the laboratory. Incidents in the laboratory
must be reported immediately and handled by following the proper procedures. Regular
monitoring and maintenance must also be done to ensure that all safety measures are being
followed. This will help mitigate risks which are already considerably high and will ensure the
safety.