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Grade 10 STE- RESEARCH 4

Quarter 2 – Module 5:
Rules on Potentially Hazardous
Biological Agents

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Grade 10 STE- Research 4
Alternative Delivery Mode
Quarter 2 – Module 5: Rules on Potentially hazardous Biological Agents
First Edition, 2020

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Published by the Department of Education – Schools Division of Sorsogon

Schools Division Superintendent – Jose L. Doncillo, CESO V
Asst. Schools Division Superintendent – Ma. Jeany T. Abayon
Curriculum Implementation Division Chief – Rolando F. Embile
Education Program Supervisor (Science) – Michelle H. Guadamor

DEVELOPMENT TEAM OF THE MODULE

WRITER: JASMIN P. NACION

EDITOR: BELLA M. ALIPIO

REVIEWERS: JANETH B. DELLOMAS

WELIMEN C. OSEO

MICHELLE H. GUADAMOR

LAYOUT ARTISTS: ERNESTO B. GILE JR.

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GRADE 10 STE- RESEARCH 4

Quarter 2 – Module 5:
Rules on Potentially Hazardous
Biological Agents

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 Most Essential Learning Competency

Conduct an enhanced/continuing research project

(maybe done in different research institutions, constant
consultation with experts should be practiced).

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 Rules on Potentially Hazardous
Biological Agents

Introduction

In the previous modules, you were able to

enumerate some rules in conducting scientific
research project and devices and to conduct an
enhanced or continuing research project by
incorporating the rules applied to them. In this
module, you will be able to learn some legal duties/
rules that apply to anyone undertaking studies with
“biological materials/agents. This part of the guide
gives an overview of the rules in dealing potentially
hazardous biological agents, and a more detailed
explanation of the requirements of the most
important regulations that are applicable to
research projects.

Most Essential Learning

Competency & Objectives

In this module, you will conduct an enhanced/continuing research project.

Specifically, you should be able to:

1. Discuss the rules on Potentially Hazardous Biological Agents.
2. Apply the rules on Potentially Hazardous Biological Agents to Research
Projects conducted in School/Home/Regulated Research Institution.

Duration: 5 meetings

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 Pre-Test

Before we start our lesson, try to answer these

five (5) questions below.

Directions: Read each question carefully and choose the letter corresponding to the
correct answer.

1. The following forms are required with Potentially hazardous Biological Agents
EXCEPT ____.
a. Human Participants Form 4
b. Qualified Scientist (2), when applicable
c. Regulated Research Institution Form (1C) - when applicable
d. Human and Vertebrate Animal Tissue Form (6B) – for all studies
involving tissues and body fluids.

2. Which source of biological agents cause allergic reactions to living organisms?

a. Body fluids c. mould spores
b. Animal waste d. skin contact

3. In which Biosafety Levels can students’ researchers be allowed to conduct their

research study in a laboratory?
a. BSL 1 AND 2 c. BSL 3 and 4
b. BSL 3 d. BSL 4

4. The following lists are the authorized persons that will approve all researches
dealing with Potentially Hazardous Biological Agents (PHBA) before
experimentation EXCEPT ______.
a. Designated Supervisor c. SRC, IBC, or IACUC
b. Qualified Scientist d. School heads

5. Which of the following guidelines/rules is NOT intended in the collection and

examination of fresh/frozen tissue and/or body fluids?
a. It must be considered in Biosafety level studies.
b. It must be conducted in BSL-1 laboratory.
c. It must be considered Biosafety Level 2.
d. It must be supervised by a Qualified Scientist or trained designated
Supervisor

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 How did you find the test? Don’t worry
about its results. As you go on with this
module, you will learn and find out if your
answers are correct. This only checks how
far you are about dealing biological
agents.

Learning Activities

E licit/Engage
Let us get started by understanding what
biological agent is and its biological hazards
risk groups and sources. This will identify if
your research study falls on this category.

Biological agents are everywhere, and while they may be very beneficial for life,
they may also be hazardous. This are relevant to many trades and occupations and
the population (working or not) facing the risks of exposure like in this Covid-19
pandemic. Researchers dealing with this is a must and deals it seriously for some of
us know a little about the risk and exposure to biological agents. This topic may include
the risks, groups of biological hazards classification, requirements for research works
and control.

Biological agents are living organisms or products of living organisms. These

include viruses, bacteria and fungi and can enter the body by inhalation, ingestion or
by absorption through the skin, eyes, mucous membranes or wounds.

As you go on with your research project, you will observe that not all research
works follow the guidelines. There are some topics that are related to a certain
guidelines in dealing or conducting a research work specifically in different
institutions/home or school.

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 Activity 1

Identifying Potentially Biological Agents

Let us check if your research work falls on this category.

Research Title/Topic:
______________________________________________________________
_____________________________________________________________

A. Biological agents may be used deliberately, but most of the time workers are
exposed to them unintentionally. Thus, infection at the workplace can occur via
different sources such as (Advisory committee on Dangerous Pathogens, 2003)
listed below.

Write a tick mark if your research study defines any of these sources.
Sources of Infection Yes No
Does your research study can generate infections coming
from
1. blood and other body fluids?
2. human bodies, animal carcasses and raw meat?
3. human or animal waste products such as faeces
and urine?
4. respiratory discharges such as coughs and
sneezes?
5. skin contact?
6. Allergic and toxic reactions can occur via:
a. moulds or moulds spores?
b. Dust, mites, feathers, animal hairs, pollen?

B. Below is the level of infection risk of the biological agents (if there is). Identify if
your research project falls under this classification. Write a tick mark under the
column of your response.
Level of Infection Risks Yes No
Does your research
1.unlikely to cause harm disease?
2. a. can cause human disease and might be hazard
to workers/researchers?
b. unlikely to spread to the community?
c. usually effective prophylaxis or treatment
available?
3. a. can cause severe human disease and present a
serious hazard to workers/researchers?

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 b. It may present a high risk of spreading to the
community and there is no treatment available?
4. a. can cause severe human disease and is
serious hazard to workers/researchers?
b. It may present a high risk of spreading to the
community and there is no treatment available.

How did you find your answers?

I hope that through the previous activity, you have

already identified if your research falls on this category and
must follow the guidelines on Potentially Hazardous Biological
Agents that will be discussed as we go on in this module.

Only scientists, doctors and laboratory staff are aware

of this risk not only that they are invisible but they are in this
field. This may explain why risk assessment is difficult to
undertake. Researchers working in this category needs
supervision to control contamination, to have reliable data and
be safe while working on their research study.

You may now have a clearer vision of where your research project is headed.
Your project may require pre-approval from regulatory institutions/agencies or experts
before you can proceed. As student researchers you will be guided by your adult
sponsors.

E xplore
Let us find out whether your research paper has passed
the requirements. It should be noted that we are going to
identify the pathogens into groups to identify the level of
infection. The following activities will help you
conceptualize the guidelines in dealing with potentially
biological hazardous agents.

Identifying the experimental units and its risk is a hard task. When researchers
fall on this category, probably some of them revise or change their samples and levels
of study to not spend much more money, time and effort. But a researcher with a heart
continue the study in order to give new information to every living being.

The first activity gives you a hint if your research deals with biological agents.
A tick from a YES column provides you an information that you will follow the
guidelines of Potentially Hazardous Biological Agents. Kindly inspect your answers

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from Activity 1. If it is confirmed then you may ask the assistance of an adult
sponsors/research teachers or any SRC’s of the school. Level of infection from 1 to 4
denotes the group of biological hazards respectively.

Activity 2

In or Out?

Reefer to the previous activity, kindly identify the sources of infection in your
research study: Write your answer in the blank provided for.
Note: Students can describe their study if it does not fall in this category:

a. Research Title:
__________________________________________________
__________________________________________________
b. Source/s of infections (Optional):
___________________________________________________
___________________________________________________

c. Level of infection (Optional): Group No. ________________________

The planning stage of a project is often the most critical and time-extensive. It
involves goals-setting, project ideation, brainstorming, prototyping, background
research (a literature review and/or patent search for prior art), needs assessment,
identifying a mentor or supervisor for the project and obtaining any approvals
necessary if your project will be working with human participants or vertebrate animals.

Activity 3
Some examples of workplaces are with potential exposure to biological agents:
Kindly give examples of a research study in this category:

1. Food Production:
______________________________________________________________
_____________________________________________________________.
2. Agriculture: __________________________________________________

3. Health care: __________________________________________________

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 Activity 4

Safety Signs

Controlling the risk of infection is relatively straightforward. It is usually simple

but effective hygiene measures. Recently, dealing with Covid-19 patients provides
additional measures such as wearing of PPE.
The following signs may be used when biological risks cannot be eliminated.
Describe the meaning of each sign.

A B C D

A. _________________________________________________
B. _________________________________________________
C. _________________________________________________
D. _________________________________________________

Activity 5

When doing microbiological research study, you are

working with potentially hazardous biological agents
(PHBAs). You will need to follow all the rules that you have
encountered in this module. A special committee will
review your plan, determine its risk level, and approve
your project before beginning an experimentation- these
are the SRC’ (Scientific Review Committee, teachers,
designated supervisors, and qualified scientists who are
willing to review your project.

Answer the activity as concise as possible. As you answer this activity, focus
on the research project that you are working on. Complete the information needed on
Potentially Hazardous Biological Agents Risk Assessment Form (6A)

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 E xplain
Now, you have identified the guide for Potentially
Hazardous Biological Agents. Researchers like you
are permitted to do research projects with potentially
hazardous biological agents meeting the conditions
and rules describe below which were designed to
protect you and to ensure that we follow the
regulations and guidelines of federal and international
biosafety with the aid and supervision of SRC’s, Adult
Sponsors and research regulated Institutions.

When dealing with potentially hazardous biological agents, it is the

responsibility of the student and all of the adults involved in a research project to
conduct and document a risk assessment on Form (6A) to define the potential level of
harm, injury or disease to plants, animals and humans that may occur when working
with biological agents. The risk assessment determines a biosafety level which in turn
determines if the project can proceed, and if so, the laboratory facilities, equipment,
training, and supervision required.

All projects involving microorganisms, recombinant DNA technologies and

human or animal fresh/frozen tissues, blood or body fluids must adhere to the rules
below AND, depending on the study, to the additional rules in Section A, B or C.

Rules for ALL Studies with Potentially Hazardous Biological Agents

(PHBA)

1. Prior review and approval is required for the use of potentially hazardous
microorganisms (including bacteria, viruses, viroids, prions, rickettsia, fungi, and
parasites), recombinant DNA (rDNA) technologies or human or animal fresh/frozen
tissues, blood, or body fluids.
2. An affiliated fair SRC, an IBC or an IACUC must approve all research before
experimentation begins. The initial risk assessment determined by the student
researcher and adults supervising the project must be confirmed by the SRC, IBC
or IACUC.
3. Experimentation involving the culturing of potentially hazardous biological agents,
even BSL-1 organisms, is prohibited in a home environment. However, specimens
may be collected at home as long as they are immediately transported to a
laboratory with the BSL containment determined by the affiliated fair SRC.

4. Research determined to be at Biosafety Level 1 (BSL-1) must be conducted in a

BSL-1 or higher laboratory. The research must be supervised by a trained
Designated Supervisor or a Qualified Scientist. The student must be properly
trained in standard microbiological practices.

5. Research determined to be a Biosafety Level 2 (BSL-2) must be conducted in a

laboratory rated BSL-2 or above (commonly limited to a Regulated Research

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 Institution). The research must be reviewed and approved by the Institutional
Biosafety Committee (IBC) if the Regulated Research Institution requires the
review. For a high school BSL-2 laboratory, the SRC must review and approve.
The research must be supervised by a Qualified Scientist.

6. Students are prohibited from designing or participating in BSL3 or BSL-4 Research.

7. Laboratory studies designed to culture known clinically significant multidrug

resistant organisms (MDROs) must have a written justification for usage and be
conducted at a Regulated Research Institution laboratory with a minimum of BSL-
2 containment and documented IBC review and approval. Representative
examples include, but are not limited to the following known agents: MRSA
(Methicillin Resistant Staphylococcus aureus), VISA/VRSA (Vancomycin
Intermediate or Resistant Staphylococcus aureus), VRE (Vancomycin-Resistant
Enterococci), CRE (Carbapenem Resistant Enterobacteriacae), ESBLs (Extended
Spectrum Beta-Lactamase producing gram negative organisms), and fungi (yeasts
or molds) with known resistance to antifungal agents.

8. Insertion of antibiotic resistance markers for the clonal selection of bioengineered

organisms is permitted. However, students may not genetically engineer
organisms with multiple drug resistant traits, nor intentionally select for such
organisms through passage in culture, with the intended purpose of investigating
the pathology, development, or treatment of antibiotic-resistant infections.
Insertion of antibiotic-resistance traits or selection of organisms expressing traits
that may affect the ability to provide effective treatment of infections acquired by
humans, animals, or plants is strictly prohibited.

9. Extreme caution must be exercised when selecting and sub-culturing antibiotic-

resistant organisms. Studies using such organisms, including BSL-1 organisms
that may have originally been exempt from prior SRC approval, require at least
BSL-2 containment.

10. The culturing of human or animal waste, including sewage sludge, is considered a
BSL-2 study.

11. Naturally-occurring plant pathogens may be studied (not cultured) at home, but
may not be introduced into a home/ garden environment.

12. All potentially hazardous biological agents must be properly disposed at the end of
experimentation in accordance with their biosafety level. For BSL 1 or BSL 2
organisms: Autoclave at 121 degrees Celsius for 20 minutes, use of a 10% bleach
solution (1:10 dilution of domestic bleach), incineration, alkaline hydrolysis,
biosafety pick-up and other manufacturer recommendations are acceptable.

13. Any proposed changes in the Research Plan/Project Summary by the student after
initial local or affiliated fair SRC approval must undergo subsequent SRC or IBC
review and approval before such changes are made and before experimentation
resumes.

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14. The following forms are required:
a. Checklist for Adult Sponsor (1), Student Checklist (1A), Research
Plan/Project Summary, and Approval Form (1B)
b. Regulated Research Institution Form (1C) - when applicable
c. Qualified Scientist (2), when applicable
d. Risk Assessment (3), when applicable
e. PHBA Risk Assessment Form (6A), when applicable
f. Human and Vertebrate Animal Tissue Form (6B) – for all studies involving
tissues and body fluids.

A. Additional Rules for Projects Involving Unknown Microorganisms

Studies involving unknown microorganisms present a challenge because the

presence, concentration and pathogenicity of possible agents are unknown. In
science fair projects, these studies typically involve the collection and culturing of
microorganisms from the environment (e.g. soil, household surfaces, skin.)

1. Research with unknown microorganisms can be treated as a BSL-1 study under

the following conditions:
a. Organism is cultured in a plastic petri dish (or another standard sterile non-
breakable container) and sealed.
b. Experiment involves only procedures in which the petri dish remains sealed
throughout the experiment (e.g., counting presence of organisms or
colonies).
c. The sealed petri dish is disposed of via autoclaving or disinfection under the
supervision of the Designated Supervisor.

2. If a culture container with unknown microorganisms is opened for any purpose,

(except for disinfection/disposal), it must be treated as a BSL-2 study and involve
BSL-2 laboratory precautions.

B. Additional Rules for Projects Involving Recombinant DNA (rDNA)

Technologies

Studies involving rDNA technologies in which microorganisms, plants and/or

animals have been genetically modified require close review to assess the risk
level assignment. Some rDNA studies can be safely conducted in a BSL-1 high
school laboratory with prior review by an SRC.

1. All rDNA technology studies involving BSL-1 organisms and BSL-1 host vector
systems, including commercially available kits, must be conducted in a BSL-1
laboratory under the supervision of a Qualified Scientist or Designated Supervisor
and must be approved by the SRC prior to experimentation. Examples include
cloning of DNA in E. coli K–12, S. cerevesiae, and B. subtilis host vector systems.
2. An rDNA technology study using BSL-1 agents that may convert to BSL-2 agents
during the course of experimentation must be conducted entirely in a BSL-2 facility.
3. All rDNA technology studies involving BSL-2 organisms and/or BSL-2 host vector
systems must be conducted in a Regulated Research Institution and
approved by the IBC prior to experimentation, where applicable.
4. Propagation of recombinants containing DNA coding for human, plant or animal

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 toxins (including viruses) is prohibited.
5. All genome editing studies that include alteration of germline cells, insertion of gene
drives, use of rapid trait development systems (RTDS®), etc., should be
categorized as a BSL-2 study and must be conducted at an RRI and approved by
the IBC from the institution. Qualified scientists are expected to ensure that student
research protocols address appropriate intrinsic and extrinsic containment
precautions.
6. Introduction or disposal of non-native, genetically-altered, and/or invasive species
(e.g. insects or other invertebrates, plants, vertebrates), pathogens, toxic
chemicals or foreign substances into the environment is prohibited. Students and
adult sponsors should reference their local, state and national regulations and
quarantine lists.

C. Additional Rules for Projects with Tissues and Body Fluids, including Blood
and Blood Products

Studies involving fresh/frozen tissue, blood or body fluids obtained from humans
and/or vertebrates may contain microorganisms and have the potential of causing
disease. Therefore, a proper risk assessment is required.

1. Research involving human and/or non-human primate established cell lines and
tissue culture collections (e.g., obtained from the American Type Culture
Collection) must be considered a BSL-1 or BSL-2 level organism as indicated by
source information and treated accordingly. The source and/ or catalog number of
the cultures must be identified in the Research Plan/Project Summary.

2. If tissues are obtained from an animal that was euthanized for a purpose other than
the student’s project, it may be considered a tissue study.
a. Use of tissues obtained from research conducted at a Regulated Research
Institution requires a copy of the IACUC certification with the name of the
research institution, the title of the study, the IACUC approval number and date
of IACUC approval.
b. Use of tissues obtained from agricultural/aqua cultural studies require prior
SRC approval.

3. If the animal was euthanized solely for the student’s project, the study must be
considered a vertebrate animal project and is subject to the vertebrate animal rules.
(See vertebrate animal rules.)

4. The collection and examination of fresh/frozen tissue and/or body fluids, (not
including blood or blood products; see rule 8) from a non-infectious source with
little likelihood of microorganisms’ present must be considered Biosafety level 1
studies and must be conducted in a BSL-1 laboratory or higher and must be
supervised by a Qualified Scientist or trained Designated Supervisor.

5. The collection and examination of fresh/frozen tissues or body fluids or meat and
meat by-products NOT obtained from food stores, restaurants, or packing houses
may contain microorganisms. Because of the increased risk from unknown
potentially hazardous agents, these studies must be considered biosafety level 2

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 studies conducted in a BSL-2 laboratory under the supervision of a Qualified
Scientist.

6. Human breast milk of unknown origin, unless certified free of HIV and Hepatitis C,
and domestic unpasteurized animal milk are considered BSL-2.

7. All studies involving human or wild animal blood or blood products should be
considered at a minimum a Biosafety level 2 study and must be conducted in a
BSL-2 laboratory under the supervision of a Qualified Scientist. Known BSL3 or
BSL-4 blood is prohibited. Studies involving domestic animal blood may be
considered a BSL-1 level study. All blood must be handled in accordance with
standards and guidelines set forth in the OSHA, 29CFR, Subpart Z. Any tissue or
instruments with the potential of containing blood-borne pathogens (e.g. blood,
blood products, tissues that release blood when compressed, blood contaminated
instruments) must be properly disposed after experimentation.

8. Studies of human body fluids, where the sample can be identified with a specific
person, must have IRB review and approval, and informed consent.

9. Any study involving the collection and examination of body fluids that may contain
biological agents belonging to BSL-3 or BSL-4 is prohibited.

10. A project involving a student researcher using their own body fluids (if not cultured)
a. can be considered a BSL-1 study,
b. may be conducted in a home setting,
c. must have IRB review if the body fluid is serving as a measure of an effect of an
experimental procedure on the student researcher (e.g., Student manipulates
diet and takes a blood or urine sample). An example of a project not needing
IRB review would be collecting urine to serve as a deer repellent.
d. must receive prior SRC review and approval prior to experimentation.

11. Studies involving embryonic human stem cells must be conducted in a

Registered Research Institution and reviewed and approved by the ESCRO
(Embryonic Stem Cell Research Oversight) Committee.

Exempt Studies (no SRC pre-approval required)

The following types of studies are exempt from requiring SRC pre-approval as
listed below, but may be subject to additional rules dependent upon the design of the
project. Student researchers and adult sponsors are required to refer to sections A, B,
and C of this section to review additional rules for projects that involve unknown
organisms, recombinant DNA (rDNA) technologies, tissues, fluids, blood, or blood
products before deciding upon a final biosafety level (BSL) designation for projects.

1. The following types of studies are exempt from prior SRC review, but require a Risk
Assessment Form 3:
a. Studies involving protists and archaea.
b. Research using manure for composting, fuel production, or other non-
culturing experiments.

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 c. Commercially-available color change coliform water test kits. These kits
must remain sealed and must be properly disposed.
d. Studies involving decomposition of vertebrate organisms (such as in
forensic projects).
e. Studies with microbial fuel cells.

2. The following types of studies involve BSL-1 organisms and are exempt from prior
SRC review and require no additional forms:
a. Studies involving baker’s yeast and brewer’s yeast, except in rDNA studies.
b. Studies involving Lactobacillus, Bacillus thuringiensis, nitrogen-fixing, oil-
eating, and algae-eating bacteria introduced into their natural
environment. (Not exempt if cultured in a petri dish environment.)
c. Studies involving water or soil microbes not concentrated in media conducive
to their microbial growth
d. Studies of mold growth on food items if the experiment is terminated at the
first evidence of mold.
e. Studies of slime molds and edible mushrooms. f. Studies involving E. coli
k–12 (and other strains of E. coli used solely as a food source for C.
elegans) that are performed at school and are not subject to additional
rules for recombinant DNA studies or use of antibiotic resistant
organisms.

Exempt Tissues (no SRC pre-approval required)

1. The following types of tissue do not need to be treated as potentially hazardous
biological agents:
a. Plant tissue (except those known to be toxic or hazardous)
b. Plant and non-primate established cell lines and tissue culture collections
(e.g., obtained from the American Type Culture Collection). The source
and/or catalog number of the cultures must be identified in the Research
Plan/Project Summary.
c. Fresh or frozen meat, meat by-products obtained from food stores,
restaurants, or packing houses and pasteurized milk or eggs. d. Hair,
hooves, nails and feathers e. Teeth that have been sterilized to kill any
blood-borne pathogen that may be present. f. Fossilized tissue or
archeological specimens. g. Prepared fixed tissue

International Rules: Guidelines for Science and Engineering Fairs 2018 – 2019,
student.societyforscience.org/intel-isef Page 15

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 Potentially Hazardous Biological Agents Risk Assessment
Use this information to complete PHBA Risk Assessment Form (6A)

Risk assessment defines the potential level of harm, injury or disease to

plants, animals and humans that may occur when working with biological
agents. The end result of a risk assessment is the assignment of a biosafety
level which then determines the laboratory facilities, equipment, training, and
supervision required. Risk assessment involves:

1. Assignment of the biological agent to a risk group

2. Studies involving a known microorganism must begin with an initial

assignment of the microorganism to a biosafety level risk group
based on information available through a literature search.

3. The study of unknown microorganisms and the use of fresh tissues

relies on the expertise of the supervising adult(s).

4. Determination of the level of biological containment available to the

student researcher to conduct the experimentation. (See “Levels of
Biological Containment” for details.)

5. Assessment of the experience and expertise of the adult(s)

supervising the student.

6. Assignment of Biosafety level for the study based on the risk group
of biological agents, level of biological containment available and
the expertise of the Qualified Scientist or Designated Supervisor
who will be supervising the project.

7. Documentation of review and approval of study prior to

experimentation:

a. If a study is conducted at a non-regulated site (e.g. school),

the SRC reviews the Research Plan/Project Summary. reviews
the institutional forms provided and documents SRC approval
(Form(6A)).
b. If the study was conducted at a Regulated Research
Institution, and was approved by the appropriate institutional
board (e.g. IBC, IACUC), the SRC.
c. If a PHBA study was conducted at a Regulated Research
Institution but the institution does not require review for this type
of study, the SRC must review the study and document approval
on Form 6A that the student received appropriate training and
the project complies with Intel ISEF rules.

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Classification of Biological Agents Risk Groups
Biological agents, plant or animal, are classified
according to biosafety level risk groups. These
classifications presume ordinary circumstances
in the research laboratory, or growth of agents
in small volumes for diagnostic and
experimental purposes.
BSL-1 risk group contains biological agents that
pose low risk to personnel and the
environment. These agents are highly unlikely
to cause disease in healthy laboratory workers,
animals or plants. The agents require Biosafety
Level 1 containment. Examples of BSL-1
organisms are: Agrobacterium tumefaciens,
Micrococcus leuteus, Neurospora crassa,
Bacillus subtilis.
BSL-2 risk group contains biological agents that
pose moderate risk to personnel and the
environment. If exposure occurs in a laboratory
situation, the risk of spread is limited and it
rarely would cause infection that would lead to
serious disease. Effective treatment and
preventive measures are available in the event
that an infection occurs. The agents require
Biosafety Level 2 containment. Examples of
BSL-2 organisms are: Mycobacterium,
Streptococcus pneumoniae, Salmonella
choleraesuis.
BSL-3 risk group contains biological agents that
usually cause serious disease (human, animal
or plant) or that can result in serious economic
consequences. Projects in the BSL-3 group are
prohibited.
BSL-4 risk group contains biological agents that
usually produce very serious disease (human,
animal or plant) that is often untreatable.
Projects in the BSL-4 group are prohibited.
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Levels of Biological Containment
There are four levels of biological
containment (Biosafety Level 1–4). Each
level has guidelines for laboratory facilities,
safety equipment and laboratory practices
and techniques.
BSL-1 containment is normally found in
water-testing laboratories, in high schools,
and in colleges teaching introductory
microbiology classes. Work is done on an
open bench or in an appropriate biosafety
hood. Standard microbiological practices are
used when working in the laboratory.
Decontamination can be achieved by
treating with chemical disinfectants or by
steam autoclaving. Lab coats and gloves are
required. The laboratory work is supervised
by an individual with general training in
microbiology or a related science.
BSL-2 containment is designed to maximize
safety when working with agents of
moderate risk to humans and the
environment. Access to the laboratory is
restricted. Biological safety cabinets (Class 2,
type A, BSC) must be available. An autoclave
should be readily available for
decontaminating waste materials. Lab coats
and gloves are required; eye protection and
face shields must also be worn as needed.
The laboratory work must be supervised by
a scientist who understands the risk
associated with working with the agents
involved.
BSL-3 containment is required for infectious
agents that may cause serious or potentially
lethal diseases as a result of exposure by
inhalation. Projects in the BSL-3 group are
prohibited.
BSL-4 containment is required for
dangerous/exotic agents that pose high risk
of life-threatening disease. Projects in the
BSL-4 group are prohibited.
 CHECKLIST: Requirements for ISEF research plans involving PHBAs
adapted from: https://member.societyforscience.org/document.doc?id 642

The following items must be covered in the procedure and risk assessment sections
of your research plan. BE SURE you have included each item in this list before you
submit your plan for review:

Procedure: discuss media and microbes you will use, and how many samples
you'll test, what incubation method you'll use, where you will do this study, how you
will collect data.

Procedure: Give source of agent, source of specific cell line, etc. (Where are you
getting your PHBA? Is it a specific strain? Who will order it or collect it and what form
will it come in?)

Risk Assessment: Describe Biosafety Level Assessment process and resultant

BSL determination (see the section below for more information about this).

Risk Assessment: Detail safety precautions (You will be trained in basic

microbiological lab safety practices, and you can also find tutorials online which will
help you understand what safety precautions should be followed when in the lab and
when you start and finish experimenting).

Risk Assessment: Discuss methods of disposal (All potentially hazardous

biological agents must be properly disposed at the end of experimentation in
accordance with their biosafety level. For BSL 1 or BSL 2 organisms: Autoclave at
121 degrees Celsius for 20 minutes, OR use of a 10% bleach solution (1:10 dilution
of domestic bleach) are acceptable).

Finally, you are done in understanding the Rules and Guidelines on

Potentially Hazardous Biological Agents (PHBA). Risk Assessment for this research
topic is more challenging and if not followed might lead to unreliable information and
maybe hazardous to those working with this study. Unlike other dangerous
substances, biological agents are able to reproduce in a very short period of time at
any varied temperatures. Supervision while working with the related studies is a
must and to surely come up with a new and valid information.

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 E laborate/Extend
I hope that you are now well-equipped in dealing
with biological agents. You are provided with the list of
extra requirements that must be included in your
research plan as you will review to improve or enhance
your study. Thus, you learned how to assess your work
following the ISEF Guidelines. In this activity, you will
apply this guidelines by determining the risk
assessments of potentially hazardous biological agents
involved and approval of the study.

Activity 6
Directions: Write A if the statement involves some of the risk assessment of
Potentially Hazardous Biological Agents and B if it is about documentation of review
and approval of study prior to experimentation. Write your answers on your answer
sheets.

__1. Determination of the level of biological containment available to the student

researcher to conduct the experimentation.
__ 2. Assessment of the experience and expertise of the adult(s) supervising the
students.
__ 3. Reviewing of the Research Plan by SRCs if a study is conducted at a non-
regulated site.
__ 4. A letter from an institutional representative stating that review was not required
must be obtained if ta PHBA was conducted at a regulated research institution
but they do not require review for this type of study.
__ 5. The study of unknown organisms and the use of fresh tissues relies on the
expertise of the supervising adult(s).
__ 6. Assignment of biosafety level for the study based on risk group of biological
agent, level of biological containment available and the expertise of the
Qualified scientist/Designated Supervisor who will be supervising the project.
__ 7. Studies involving a known biological microorganism must begin with an initial
assignment of the microorganism.

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 Vocabulary List

Biological Agent
A microorganism, cell culture, or human endoparasite, whether or not
genetically modified, which may cause infection, allergy, toxicity or otherwise
create a hazard to human health (Control of Substance Hazardous to Health
Regulations (COSHH).

[Hazard] Group
Group in relation to a biological agent, means one of the four Risks or Hazard
groups specified as classification of Biological Agents.

Microorganism
A microbiological entity, cellular or non-cellular, which is capable of replication
or of transferring genetic materials.

PPE (Personal Protective Equipment)

It includes a laboratory coats, protective gloves, etc, which must be supplied
and used where exposure cannot be adequately controlled by other means.

Risk Assessment
It defines the potential level of harm, injury or disease to plants, animals and
humans that may occur when working with biological agents.

Post-Test

E valuate
Directions: Read each question carefully and choose the letter corresponding to the
correct answer.

1. The following statement below are the reasons why risk assessment in
biological agents is more challenging EXCEPT ____.
a. Because of the diversity of these agents.
b. Because pathogenic microorganism is may be extremely hazardous at
low concentration and at a very short time.
c. Because biological agents are not visible for the naked eye.
d. Because occupational exposure limits have been set.

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2. Which of the following groups of biological hazards inherited by an organisms
may present a risk of spreading to the community but with effective available
treatment?
a. Group 1 c. Group 3
b. Group 2 d. Group 4

3. Which of this protection from risks pertains to the determination of the

concentrations, routes of exposure, potential for absorption, frequency and
duration of exposure estimates the doses to which workers/researchers are or
may be exposed?
a. dose (concentration) c. hazard identification
b. exposure assessment d. risk or characterization

4. The following types of studies do not require approval prior to SRC review, but
require a Risk Assessment Form 3 EXCEPT ___.
a. Fresh or frozen meat, meat by-products obtained from food stores
b. Plant tissue
c. Studies with microbial fuel cells.
d. Studies of mold growth on food items if the experiment is terminated at
the first evidence of mold.

5. Below is a basic example of a laboratory protocol in conducting research related

to Potentially Hazardous Biological Agents (PHBA).

Which of the following is the correct order for quantifying bacterial growth with
known/potential antibiotics?

1. Make nutrient broth media.

2. Quantify growth by absorbance at 540 nm at 72 hours incubation, and CFU count
at 96 hours.
3. Inoculate each 25ml tube with 20uL of E. coli (strain K12, obtained from Carolina
Biological Supply Co, 1 vial freeze-dried, started according to standard
protocol, BSL-1.
4. Subculture E. coli, 20uL each into 30 tubes per treatment, three treatments
(amoxicillin, caffeine, vanilla .1M) after incubation at 37degC for 24 hours
5. After 72 hours, subculture to nutrient agar plates (30 per treatment) and take
also 1000 uL for spectrophotometry (index of cell counts based on light
absorbance).

a. 1 – 4 – 3 – 2 -5 c. 1 - 3 - 4 - 2 - 5
b. 1 – 5 - 2 – 3 -4 d. 1 – 2 – 3 – 4 -

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 Answer Keys

Pre-Test Answer Keys

1. A
2. C
3. A
4. D
5. C

Learning Activities Answer Keys

Activities 1 (A & B), 2, 3, and 5

Answers may vary depending on the topic of the student researcher.
Activity 4
A. The blue signs indicate an obligation to wear protective overalls (PPE).
B. The red signs signify that smoking is not permitted. There is a presence
of a particular danger.
C. The blue signs indicate the obligation to wear a protective mask (e.g. face
mask).
D. The red signs point out that it is forbidden to drink or eat.
Activity 6:
1. A 5. A
2. A 6. A
3. B 7. A
4. B

Post-Test Answer Keys

1. D
2. C
3. B
4. C
5. B

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 References

E-FACTS, “Risk Assessment for Biological

Agents. (2020):53_risk_assessment_biological_agents_ [SHARED].pdf

Crest-Jane Goodall Science Symposium,” Supplemental Guidelines &

Requirements for Your ISEF Research Plan. (August
2016): PHBAsuppl2016_2017.pdf

MMS MDLADLANA, Occupational Health and Safety Act, 1993 (ACT NO. 85
of 1993), “Hazardous Biological Agents Regulations”

International Rules: “Guidelines for Science and Engineering Fairs 2018 –

2019”, student.societyforscience.org/intel-isef

The University of Reading, “Control of Biological Hazards, safety Guide

Number 14 Part 2 Legal Duties” (2020), SG_14 Part 2(Jan. 6),
_legalduties.pdf

Potentially Hazardous Biological Agents, Index, “Rules for All Studies with

Potentially Hazardous Biological Agents:www.societyforscience.org>isef

K16.cuny.edu>sites>2019/01.pdf

www.sciencebuddies.org>biological_agents

www.mnsu.edu>pdf>old

What are Biological Agents? hsa.ie

Laboratory Hazards: Chemical, Physical and Biological Agents in Laboratory,

Interfocus

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