Portland State University

ME 492 Capstone Progress Report Winter 2016

Coturnix Quail Egg Embryo Extractor

(c-QEEE)

Sponsorship with

Childs Cancer Therapy Development Institute

Industry Advisor

Dr. Charles Keller, M.D.

Faculty Advisor

Dr. Nathalie Neve, Ph.D.

Submit by

Alex Arnold, Joshua Lake, Robert Lesanovsky, Anne (Man Wai) Ng, Samuel
Rasmussen, Sam Sanford

March 8, 2016
Contents

1 Introduction 3

2 Mission Statement 4

3 Project Schedule 4

4 Product Design Specifications Summary 6

5 External Search 7
5.1 Quail Egg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5.2 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

6 Internal Search 9
6.1 Suction Cup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6.2 Puncturing Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

7 Design Selection & Evaluation 12

7.1 Guillotine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
7.2 Circumscriber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
7.3 Suction-tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
7.4 Design Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

8 Progress on Detailed Design 15

8.1 Handheld Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
8.1.1 Kinematic Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
8.1.2 Prototype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
8.2 Stationary Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

9 Conclusion 17

Appendix A: Bio-safety Criteria 19

Appendix B: Patents 21

Reference 23

1
Executive Summary

The Childrens Cancer Therapy Development Institute conducts research in the fields of
developmental biology and oncology. Their mission is to understand and prove new disease-specific
treatment options for children with cancer. Their research on stopping metastasis is conducted on
fertilized coturnix quail egg embryos due to the ease of manipulation and visualization.
Since the process of removing the embryo from its egg shell is a delicate and time consum-
ing process, the primary goal of this project is to construct a device that is capable of extracting
the contents of a quail egg with higher efficiency than the current method used by the doctors. The
device should have a success rate greater than 50% and should not contaminate the extracted con-
tents. The design requirements of this project are significantly influenced by the project sponsor; Dr.
Charles Keller; the internal customer of this project. The external customers consist of researchers
and interns who determine more specific design elements. A list of questions were surveyed with
the customers in order to identify and prioritize the design elements.
The major accomplishments of the project are: a Product Design Specification (PDS)
document, internal and external research, and concept evaluation and selection. Currently the
project is in the detailed design phase. During external research, existing products and patents
related to separating the egg shell from its internal contents were identified. External research also
consisted of research in embryology of avian species in order to understand how to safely extract
the contents of the egg. Internal research was conducted on two major features: a safe method
to puncture the egg shell and an optimal suction cup design that is capable of holding the egg
shell under applied tension. During the design evaluation phase, suction cups were concluded to
be the most acceptable choice in terms of egg shell separation. Currently the detailed design phase
focuses on designing two prototypes: a handheld device and a stationary device; however, it has
been decided for the handheld device to be the first priority of this project before proceeding to
work on automating the separation process for a stationary device. The working prototype is to be
delivered in June 2016.

2
1 Introduction

The mission of the Childrens Cancer Therapy Development Institute is to translate sci-
entific discovery into clinical trials by understanding and proving new disease-specific treatment
options for children with cancer [2]. Currently, the researchers are exploiting assays to stop metas-
tasis by experimenting on fertilized coturnix quail egg embryos. The accessible embryo of the avian
species yields as an ideal and convenient model for studies in developmental biology and oncology.
It offers easy manipulation and visualization by simply removing portions of the eggshell [4].
In the Childrens Cancer Therapy Development Institute laboratory, the experimentation
employs fertilized quail eggs that have been incubated for 72 hours. Prior to the extraction process,
the slide warmer is set at approximately 37 C, along with wire cutters and a solution of 70% EtOH
prepared.The forceps are sanitized with the EtOH solution and wiped with kimwipes before each
extraction. The initial procedure of the removal implementation method consists of placing the
incubated eggs into the slide warmer with the narrower ends of the egg down for ten minutes, this
allows the yolk to move to the top of the egg. In addition, one of the co-culture well plates is placed
inside the slide warmer.
After the preparation, the egg is held slightly above the empty well plate while keeping
it upright at a tilted angle. The forceps are then used to puncture the shell and cut around the
circumference of the egg until the shell fragment is removed (the shell is slightly pinched in the
process). The entire contents of the egg are poured into the well with the embryo intact. The
successful extraction and survival of the embryo is based on the visibility of the beating heart
facing upward and the yolk sac intact. The procedure is concluded with each plate labeled with the
non-viable embryos covered and marked with an X. The plates are then placed into the embryo
incubator at 37.4 C with 0% of carbon dioxide.
The traditional method is time-consuming, and it yields low embryo survival rates. Our
role in the cancer research project is to implement an efficient and safe process to extract all the
content of the quail egg without any cross-contamination and damages to the embryo. The new
methodology will improve the efficiency and success rate of embryos from the current implementation
process.

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2 Mission Statement

The objective of this project is to design and fabricate an efficient method (ideally a device)
that will safely extract the contents of a fertilized quail egg, and place the contents into a separate
dish with the heart of the embryo being accessible to the researchers. Throughout the process, the
apparatus cannot damage or contaminate the embryo. The time for the apparatus to extract the
contents of the egg should be shorter than the current extraction period employed by the researchers
(approximately one egg per minute). A functioning prototype with a high success rate (e.g. higher
than the current implementation method of 50%) will be delivered in June 2016. A high performance
device will help improve the metastasis experiments and research at the Childrens Cancer Therapy
Development Institute.

3 Project Schedule

A Gantt chart, shown in Fig. 1, is developed to track the progress of the project. The
chart is organized by months ranging from end of December to early June. There are five squares
within each month indicating the five weeks within each month. The dark bar illustrates the seven
engineering design steps, the rectangular box represents more descriptive tasks within each of the
design phases. The diamond symbol represents the milestones or critical tasks that needs to be
accomplish.
Currently, the project is at the concept selection and detailed designing stage. Based on
the external and internal research, methods of suction and puncturing are the major concerns of the
prototype. Several designs of puncturing methods have been developed and evaluated. The next step
of this project is to build a functioning prototype and to troubleshoot any technical problems. After
testing and prototyping, feedback from customers will be evaluated to make additional revisions by
early to mid-April. The Gantt chart is considered to be a living document, meaning there will be
additional changes as the project carries on.

4
Figure 1: A Gantt chart of the quail egg extractor project from December 2015 to June 2016.
Currently, the project is at the concept selection and detailed designing phase.

5
4 Product Design Specifications Summary

The final product design specifications of the quail egg embryo extractor are listed below
in the order of importance (e.g. lowest number indicates the highest priority). The specifications
are based on the feedback and concerns from Dr. Keller and the lab assistants, whom are the end
users of the device.

1. The device must conform to the level two bio-safety practices (refer to Appendix A for a
detailed list of criteria).

2. The quail egg embryos must be successfully removed form the egg and transferred into a
six-well dish with the heart on top at least 90% of the time.

3. The device should be able to process six eggs in less than five minutes.

4. The device should be able to operate continuously for 100 minutes.

5. The device should be adjustable for varies egg sizes.

6. The device should be capable of being cleaned weekly and maintained annually.

7. The lab assistants should be able to operate it easily with little training.

8. The cost must be less than $5,000 for designing and building the device.

9. A fully functional device must be delivered by June 4th , 2016.

10. The device must occupy less than 61107 cm of space.

11. The device should weigh less than 25 pounds.

12. The service life of the device should be five years.

The table space requirement is only for a stationary model, if a hand-held device is devel-
oped, it will need a smaller occupying space in the lab. The prototype will be tested to determine if
the requirements are met. The device is considered successful if all the these requirements are met
and the researchers at the Childrens Cancer Therapy Development Institute are pleased with the
final product.

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5 External Search

5.1 Quail Egg

To develop a device that will successfully remove the internal contents of quail eggs, it is
important to understand the embryology of the avian species during the fertilization period. The
basic structure of a quail egg consists of three major components, the mineral shell, egg albumen,
and the egg yolk [6]. A diagram of the anatomy of a hens egg is shown in Fig. 2 with the appropriate
components labeled. There is no significant difference in anatomy between the anatomy of chicken
and quail egg during fertilization period except the size of the egg. In this project, the eggshell
and membrane of an egg are the primary concerns when developing a puncturing feature of the
prototype. It is important for the device to be robust and be able to pierce through the eggshell
and membrane; however,it should be delicate enough to not damage the embryo of the egg.

Figure 2: The basic structure of a fertilized hens egg with each components labeled [8].

The eggshell strength is a significant mechanical property in designing the eggshell extract-
ing device. The property is typically described using various parameters such as eggshell thickness,
shell stiffness, rupture force,etc. Prelipcean et. al. (2009) has investigated the chemical compounds
and physical properties of 130 fertilized quail eggs, and determined that the two shell membranes
weigh approximately 0.36 to 1.23 grams; indicating that the mineral shell makes up approximately
6.35% to 9.43% of the weight of the whole egg.
In the works of Polat et. al. (2007), the physical parameters and mechanical behaviors
of 40 fertilized quail eggs are determined. The averaged eggshell thickness is determined to be
0.270.02 mm. Fig. 3 illustrates and labels the orientation of a quail egg. The z-axis orientation
onto a fertilized quail egg results in the least required force to break the egg shell, with a value

7
of 6.83 N; while an average force of 9.67 N is required to break the egg shell along the x-axis.
The toughness and deformation are also calculated. With a mean value of 0.0094 mJ/mm2 , 0.029
mJ/mm2 , and 0.0121 mJ/mm2 along the x-front, z-axis, and x-back, respectively. The orientation
of the fertilized quail egg is a significant factor in breaking the eggshells. Based on external searches,
the device will be puncturing the egg along the sides (x-axis) since it requires the least amount of
force to break the egg.

Figure 3: The orientation of quail egg with L, the length, W, the width, fz, the force in the x-axis,
fx, the force in the x-axis [5].

5.2 Applications

There is a variety of developed applications and devices to remove the internal contents of
chicken eggs. Such applications vary from heavy-duty machinery for industrial purposes to hand-
held device for everyday home-use. Among the chicken egg extracting devices, the Egg Breaker
[1] and the Egg Cracker Device [9] have inspired some of the initial design for the quail egg. The
premise of the Egg Breaker is to part the egg in half and empty the content of the egg in one-
single operation. The design the invention consists of a handling bottom frame to hold the egg
and separable members hinged to the frame. As the egg is placed onto the device, the top frame
is pushed together to the bottom frame by the pressure of palm. The top frame then generates a
force that pushes the egg into a projected knife located at the center of the bottom frame, results in
puncturing the egg. The Egg Cracking Device consists of employing a mechanical moment to break
open the eggshell after a puncturing point has been produced. Detailed schematics of the patented
Egg Breaker and the Egg Cracking Device are shown in Appendix B.
Despite the numerous devices to extract the content of chicken eggs, there are several
implementations and methods for extracting quail eggshells. The quail eggshell cutter manufactured

8
by a Japanese company, Korin, is a among the most common device to remove the quail eggshell.
The device, shown in Fig. 4, uses the bases mechanics of a scissor with one distinct feature; a
triangular blade as one of the scissor-arms. The quail egg is placed in the opening slot of the device,
and with a cross-sectional motion, the blade punctures and splits open the quail egg.

Figure 4: A quail egg cutter manufactured by Korin [7].

6 Internal Search

After completing the external search process, the team has decided to focus on two pri-
mary design features: the method of handling the eggshell during embryo extraction, and the shell
puncturing method. Suction cups are chosen as a design feature to hold the egg in its optimal
position and provide a structured support during embryo extraction. A puncturing device is needed
to initiate crack propagation around the circumference of the egg. Several designs of the piercers
are being investigated.

6.1 Suction Cup

Commercial suctions cups are first researched to suit our design. One of the requirements
from the sponsor is that the cups must be flexible to conform to various sizes of eggs, and to be rigid
enough to support a moment applied to the egg. One of the top commercial options is the bellows
cup which works well with various sizes, but is not rigid enough to support a moment. Therefore,

9
custom cups will be made to satisfy both criteria. The design of the prototype is proven to function
during a trip to the research lab by successfully removing an embryo. Although the cup design is
successful, it is not repeatable. Fig. 5 is a photo of a second custom design in the making.

Figure 5: A hand drill in a vice and hacksaw is used to turn a suction cup geometry out of polyester
resin. It will be used as a pattern in a urethane suction cup mold. The mold will be used to cast
silicone rubber cups.

The vacuum needed for the suction cups can be powered either by hand or a motorized
pump. The choice is made to look into both methods and have the end product be either a hand
operated device or an automated process to remove the egg embryo. The hand pump will use the
concept of a manual piston with the main advantages being that it will be quiet and more compact
as it doesnt have a motor. The piston must be large enough to maintain sufficient vacuum. Air
volumes must be calculated for the piston system to function. The main advantage of using a
motorized pump is that the pump can be controlled and a constant vacuum can be maintained.
The disadvantages of this device are that it requires an external energy input and has the potential
to be large and noisy if not designed properly.

6.2 Puncturing Method

Since the extraction process is conducted in a medical research lab, cross-contamination

and sanitation are concerning factors. Therefore, the puncturing tool needs to be self-cleaning or
easily removable to clean between runs. Several designs are being researched to determine the
optimal cutting device to penetrate enough shell to initiate crack propagation and pierce through
the membrane, yet not too deep as to damage the embryo. The designs are: a cushioned hammer

10
with a sharp point, implementing a pure separation force and a lancing device.
The cushioned hammer uses a spring to drive the hammer onto the egg and the cushion
distributes the force and pushes the device back away from the egg. The sharp object is buried in
the cushion and will initiate a crack on the egg. Theoretically, this method will keep the device the
most clean as the cushion could be designed to clean the cutter. A concept illustration is shown in
Fig. 6.

Figure 6: A cushion surrounding the puncturing device distributes the force evenly and wipes the
blade clean after every cycle.

Using a only a separation force, suction cups would apply tension until the egg shell is
ripped in half. Testing with the prototype showed this is not a viable option. A lancing tool,
commonly used for extracting blood samples, will employ a spring to drive and retract a lance into
the egg. A prototype is built and further testing is being done to determine the shape and size of
the cutting lance, as well as the spring force needed to penetrate the shell. The instrument used to
determine this concept is shown in Fig. 7.

Figure 7: This apparatus is used to test puncture force of different piercing tips using a spring and
bolt to adjust the compression.

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7 Design Selection & Evaluation

In this section the initial concepts for removing a quail egg embryo are presented. These
initial concepts are guillotine, circumscriber, and suction-tension. A brief description of each concept
including their advantages and disadvantages are discussed in detail.

7.1 Guillotine

The guillotine method is an approach with simplicity in mind. The idea is to have a blade
quickly slice the tip of the tapered end of the eggshell off completely while the egg is seated in a
cup. Once the end is removed, the contents will fall down into a tray below. Fig. 8 illustrates the
basic concept of this design.

Figure 8: The guillotine blade cuts away the tip of the egg while the embryo falls into a tray. Note,
a curved blade could swipe away the debris.

A benefit of this design is that the mechanism to accomplish this task can be made using a
simple blade, linkage, and spring system with trigger release. The blade can be made wide enough
to cut through many eggs at once, therefore increasing extraction rate. A major problem with this
concept is contamination of the embryos. The cutting process is an invasive procedure that can
deposit contaminants from the blade into the embryo as the blade enters the egg. The blade must
be cleaned and disinfected after every cycle. In addition, the effect of removing a significant portion
of the albumen on the embryo is unknown. More minor problems include shell fragments falling
into the tray below or being scattered and also a large spring loaded blade can create hazards to
the user. The blade can be curved to carry the shell fragments with it during travel to reduce mess.

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7.2 Circumscriber

The circumscriber consists of a method in which a circular crack or cut is made around the
tapered end of the egg shell and the lid is removed so that the embryo can fall out. The possible
mechanisms for making a circular cut are an impact hammer, rotary blade, or piercer array. The
easiest mechanisms of these to construct is the impact hammer and is illustrated in Fig. 9.

Figure 9: An impact hammer is used to create a circumferential crack around the tip of the eggshell
leaves the embryo unharmed.

The advantages of this design are that the embryo would be less prone to contamination
or physical harm and the process is similar to the current method using forceps to snip off the shell
which does work. The disadvantages are that this mechanism could be difficult to automate or make
a fixture for because the egg might need to be inverted, the variability of shell geometry can cause
the circumscriber to malfunction, and there is no built-in method of dealing with shell fragments
and cleanup.

7.3 Suction-tension

In the suction-tension method, two suction cups hold the egg on either end and using a
vacuum, the cups are pulled apart, applying tension to the eggshell. While the shell is under tension,
a puncturing device initiates a crack on the side of the shell and the tension causes the crack to
propagate around the eggs circumference. A moment is applied to each suction cup to break apart
the two halves and release the embryo. Fig. 10 is a general depiction of this method.

13
Figure 10: A crack is initiated by a constant tension and puncturing device. When a moment is
applied, the eggshell opens and the contents fall out.

The advantages of the suction-tension concept are that suction cups conform to many sizes
and shapes of quail eggs, the puncturing device only needs to enter the shell enough to initiate a
crack and split the membrane, the shell remains mostly intact for easier cleanup, and the opening
motion allows easy exit of the embryo. Disadvantages with this design are difficulty coordinating
the required kinematics into a machine, disinfecting and cleaning of the puncturing device between
cycles, and the requirement of a vacuum pump or piston.

7.4 Design Evaluation

A design evaluation matrix is employed to select which design concept to go forward with
in this project. Refer to Table 1 for the design matrix. Each design is scored with 0, 1, and 2 with
respect to each requirement. The meanings of each numerical value are, 0 does not meet, 1 has
the potential to meet, and 2 meets the criteria. The total of each design indicate that the suction-
tension design is an acceptable option with a score of 9. The most important of these requirements
is reliability as requested by the sponsor and end users.

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Table 1: The scores 0, 1, and 2 represent the ability that each design meets the requirement. The
total score indicates that the suction-tension method is the most acceptable design.

Design Requirements & Criteria Guillotine Circumscriber Suction-tension

Simplicity 2 0 1
Low Cost 0 1 2
Short Build Time 0 0 1
Easy to Use 2 0 1
Safety 1 2 0
Sanitary 0 2 1
Reliable 1 1 2
Total 6 6 9

8 Progress on Detailed Design

After deciding to go forward with the suction-tension concept, the possibility of creating
two prototypes are considered. A handheld and a stationary device are being designed. A handheld
version provides a small scale proof of concept for the idea. The stationary device will be designed
to process eggs more rapidly. Both will incorporate the same principle of suction-tension with a
puncturing device. This section will present the details of each design progress.

8.1 Handheld Design

The goal of a handheld version is to complete all the steps necessary to extract the embryo
from the shell while using only one hand to hold and operate the device. The other hand is used for
placing the egg into the suction cups and possibly to clean the puncturing device between cycles.

8.1.1 Kinematic Modeling

The mechanics behind incorporating the actions of holding an egg, creating a vacuum,
applying tension, cracking the egg, and disposing of the shells into one general motion with single
hand operation is a challenging task. In order to explore the details of how a mechanical system
could accomplish this, a CAD program; Autodesk Inventor, is employed. Sketch blocks are created
to simulate each component in 2D and how they interact. Fig. 11 visualizes how this program is

15
used by showing the steps for each critical motion. The major missing component of this design is
the integration of the puncturing device. Minor missing components are springs, vacuum tubing,
and a push button to release the piston.

Figure 11: One complete cycle of the handheld operation. The puncturing device and piston release
button are not yet incorporated into the design.

In step 1, the suction cups are closed by holding the egg in place. In step 2, the handles
are squeezed and the pins engage the linkage that is connected to the cups within the slots. In
addition, the vacuum piston draws air from the cups (tubing connecting to the cups not shown).
In step 3, the handles are closed further, forcing the cups apart while maintaining vacuum. At this
point the puncturing device will crack the egg open. In step 4, the latch on the top of the piston
is released by a push button; while, the piston is pushed by a spring forcing air out of the cylinder
and ejecting the egg shell. In step 5, the handles are opened and the piston is linked and re-engaged
the latch on the piston for the next cycle.

8.1.2 Prototype

In order to confirm that the linkages in a handheld device can properly function and
to troubleshoot the interference, partially functional prototypes are made using Technics Legos.
This process offers the opportunity to experiment with different types of linkage systems such as
employing gears, levers, or combinations of both. Fig. 12 illustrates the two prototypes successfully

16
achieved the range of motion that the handles and suction cups require.

Figure 12: These partially functional prototypes complete the range of motion necessary for the
handheld design. On the left is a design using all gears and on the right is a design using a
combination of gears and levers.

8.2 Stationary Device

The purpose of the stationary device is to process multiple eggs at once transferring the
egg embryos into a six well dish with the operator only having to load the eggs and press a button.
Currently this project is on hold as we have been having trouble getting the suction cups to work
consistently. We decided to focus on the handheld device to get the egg opening process complete
and then work on automating the process for the stationary device built on the same separation
process.

9 Conclusion

As of now the major accomplishments of this project are the internal and external research,
a product design specification report, and the final concept evaluation and selection of designs.
During the concept evaluation and selection phase, it has been decided to develop two designs
simultaneously: a handheld device and a stationary device. Both devices will use suction cups
with a vacuum to hold and open a quail egg. The handheld device will use a manual piston to
create a vacuum while the stationary device will use a constant vacuum supplied by an electric
motor. The project is currently on schedule in the detailed design phase at approximately 40% of
project completion. The major tasks to be completed are to finish the detailed design by developing
a method to puncture the eggshell, to prototype, to perform testing and product validation, to

17
perform any necessary redesigns, and to complete the final documentation. One obstacle that the
team has experienced is the excessive time required to have a specific suction cup design produced
and delivered by a manufacturer. Because of this, the team is in the process of developing and
producing a custom suction cup. Testing is currently underway to determine the required force to
puncture the egg shell using various blades, and the compression force the shell can endure before
failure while being handled by the suction cups. The project sponsor has witnessed the initial
prototype of the suction cups used to extract an embryo in the laboratory setting and is pleased
with the results. If the kinematics of the device perform as designed, with the use of the suction
cups, the device will exceed the time and efficiency requirements set by the sponsor. Until the
detailed design phase is finished and a prototype is produced, it is unclear if the device will meet
the the success rate requirements. This will be determined during the testing and product validation
phase.

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Appendix A: Bio-safety Criteria

Some of the biosafety practices that are applicable to our engineering metrics and con-
straints are summarized in this section. The list of criteria are gathered from Centers for Disease
Control and Prevention. Biosafety Level-2 (BSL-2) is built upon BioSafety Level-1 (BSL-1), which
consists of work involving well-characterized agents not know to cause disease in adult humans and
present minimal potential hazard to laboratory personnel and environment. BSL-2 is suitable for
work involving agents that pose moderate hazards to personnel and the environment. It differs
from BSL-1 such that laboratory personnel have specific training in handling pathogenic agents
and are supervised by scientists competent in handling infectious agents and associated procedures,
access to the laboratory is restricted when work is being conducted, and all procedures in which
infectious aerosols or splashes may be created are conducted in BSCs or other physical containment
equipment.
The following standard, special practices, safety equipment, and facility of the BSL-2 are summa-
rized in the lists below:

1. Agents

1.1. Agents is associated with human disease

1.2. Routes of transmission include per-cutaneous injury, ingestion, mucous membrane expo-
sure

2. Practices

2.1. BSL-1 practice consists of some of the following:

2.1.1. Mouth pipetting is prohibited; mechanical pipetting devices must be used.

2.1.2. Policies for safe handling of sharps, such as needles, scalpels, pipettes, and broken
glassware must be developed and implemented.
2.1.3. Careful management of needles and other sharps are of primary importance. Needles
must not be bent, sheared, broken, recapped, removed from disposable syringes, or
otherwise manipulated by hand before disposal.
2.1.4. Non-disposable sharps must be placed in a hard walled container for transport to a
processing area for decontamination, preferably by autoclaving.
2.2. Decontaminate work surfaces after completion of work and after any spill or splash
of potentially infectious material with appropriate disinfectant.

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 2.3. Decontaminate all cultures, stocks, and other potentially infectious materials before
disposal using an effective method.

2.4. Universal bio-hazard warning signs posted at entrance to laboratory when infectious
agents are present.

2.5. All procedures involving manipulation of infectious materials that may generate an
aerosol should be conducted within a BSC or other physical containment devices.

2.6. Biosafety manual defining any needed waste decontamination or medical surveillance
policies.

3. Primary Barriers & Safety Equipment

3.1. BSCs or other physical containment devices used for all manipulations of agents that
cause splashes or aerosols of infectious materials aerosols of infectious materials.

3.2. Protective laboratory coats, gowns, smocks, or uniforms designated for laboratory use
must be worn while working with hazardous materials.

4. Facilities (Secondary Barriers)

4.1. Biosafety Level-1 consists of the following:

4.1.1. Laboratories should have doors for access control, sink for hand washing, should be
designed so that is can be easily cleaned.

4.1.2. Laboratory furniture must be capable of supporting anticipated loads and uses.
Spaces between benches, cabinets, and equipment should be accessible for cleaning.

4.1.3. Laboratories windows that open to the exterior should be fitted with screens.

4.2. BSCs must be installed so that fluctuations of room air supply and exhaust do not
interfere with proper operations. BSCs should be located away from doors, windows,
that are opened, heavily traveled laboratory areas, and other possible airflow disruptions.

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Appendix B: Patents

This section consists of the schematics of the Egg Breaker and the Egg Cracker Device.
Both of the device have inspired the handheld prototype designs.

Figure B1: The patent design of the Egg Breaker. It consists of several components that will part
the egg in half and empty the content of the egg in one-single operation [1].

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Figure B2: The schematics of patented design of the Egg Cracker Device [9].

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References

[1] Bartell G. S., & Singh, J.D. (1955). U.S. Patent No. 2706507. Washington, DC: U.S. Patent
and Trademark Office.

[2] Childrens Cancer Therapy Development Institute. (n.d.). Retrieved March 05, 2016, from
http://www.cc-tdi.org/#!mission/ctnu

[3] Control for Disease Control and Prevention. December 2009 Section IV-Laboratory Biosafety
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