Portland State University

ME 493 Capstone Progress Report Spring 2016

Coturnix Quail Egg Embryo Extractor

(c-QEEE)

Sponsorship with

Child’s 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

June 3, 2016

Contents

Executive Summary 2

1 Introduction 3

2 Mission Statement 4

3 Product Design Specifications Summary 5

4 Top-level Final Design Alternatives 6

5 Final Design: Handheld Device 8

5.1 Suction Cups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

5.2 Vacuum Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5.3 Puncturing Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5.4 Mechanical Levers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

6 Final Design Evaluation 11

6.1 Performance& . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

6.2 Structural Integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

6.3 Manufacturability & Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

7 Conclusion 14

Appendix A: Bio-safety Criteria 15

Appendix B: External Research Summary 17

Appendix C: Internal Research Summary 18

Appendix D: Detailed Product Design Specifications 19

Appendix E: CAD Drawings 21

Appendix F: Bill of Materials 26

Appendix G: Operational Manual 27

1

Executive Summary

The Children’s Cancer Therapy Development Institute researches developmental biology

and oncology of cancers in children. Their goal is to develop and prove new treatments for stopping

metastasis of cancers in children. They conduct their experiments on fertilized coturnix quail egg

embryos. The current method of removing the embryo from the egg shell is difficult and time

consuming, the project is to build a device that can transfer the contents of a quail egg to a six

well dish faster than the current rate of one per minute, with a higher success rate than the current

50% and without contaminating the contents. Dr. Charles Keller is the internal customer for this

project and will significantly influence the design requirements. The researchers and interns are the

external customers and they will help determine more specific design elements. We conducted a

survey of the customers to determine and prioritize the design elements.

The documentation requirements for this project are a Product Design Specifications doc-

ument, a progress report, and a final report. Currently the project is in the phase of finalizing

the product to the end user. During external research, existing products and patents related to

separating the egg shell from its internal contents are identified. External research also consists of

researching in embryology of avian species in order to understand how to safely extract the contents

of the egg. Internal research are 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 are concluded to be the most acceptable

choice in terms of egg shell separation. The detailed design phase focuses on designing the handheld

device, which consists of four major components: the mechanical lever, two suction cups, puncturing

mechanisms, and a vacuum system. The working prototype is delivered the end users.

2

1 Introduction

The mission of the Children’s 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 [1]. 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 [2].

In the Children’s 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.

3

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 Children’s Cancer Therapy

Development Institute.

4

3 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 withstand high temperature of 120�C in an autoclave.

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

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

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

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

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

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

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

11. The device must occupy less than 61⇥107 cm of space.

12. The device should weigh less than 25 pounds.

13. 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 Children’s Cancer Therapy Development Institute are pleased with the

final product.

5

4 Top-level Final Design Alternatives

Three different designs are developed in the design phase. To decide between the top-level

design alternatives, a design evaluation matrix is used, as shown in Table 1. Each design is scored

with a numerical value between 0 to 2 with respect to each requirement. The meaning of each

number are: 0 does not meet, 1 has the potential to meet, and 2 meets the criteria. The totals 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. The

two other methods, guillotine and Plan B, are discussed for why they were not the top choice.

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 Plan B Suction-tension

Simplicity of Design 2 2 1

Low Cost 0 2 2

Short Manufacturing Time 0 1 1

Easy to Operate 2 1 1

Safety 1 1 0

Sanitary 0 1 1

Reliable 1 0 2

Total 6 8 9

The guillotine method, shown in Fig. 1, is a simple and fast method to remove the embryo

from the egg. The design would have springs pull a specially designed blade across the bottom of

the egg allowing the yolk to fall freely into a cup below and discarding the remains in a receptacle.

The main advantages of the guillotine method are its ease of use and the ability to process all 6 eggs

at one time. The main problem, aside from the lowest score, was believed to be how unsanitary the

process would be.

6

Figure 1: 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.

For the Plan B, a purely mechanical device is designed with simplicity in mind. The main

function of Plan B is to hold an egg firmly in the egg holder, lower it down onto a blade rigidly

fixed to the base and then pulled apart for the removal of the embryo. Inside the base plate are

compression springs to push the egg holder back up after the embryo is removed. A 3D model

assembly is shown in Fig. 2.

Figure 2: A 3D model of Plan B, with the major components labelled.

7

5 Final Design: Handheld Device

The final design consists of the suction cups, vacuum system, puncture device, and me-

chanical levers. These components are what primarily interacts with the quail egg and are the focus

of the final design description.

5.1 Suction Cups

The required function of the suction cups is to provide an adequate seal on multiple sizes

and shapes of quail eggs and to restrict the egg from moving and rotating during egg shell separation.

Forming a seal around any size and shape of quail egg are accomplished with a conical shaped suction

cup as shown in Fig. XX. This conical shape allows a seal to form around any diameter below 1",

and all quail eggs have a location where its diameter is below 1". The cups are made of silicone with

a shore hardness of 20A. Silicone is chosen for its capability to withstand high temperatures above

120�C. The shore hardness is an appropriate hardness to allow the suction cup to conform around

irregularities that are common on quail eggs. Early designs of suction cups has had complications

with being pulled out from the suction cup holder during egg separation. This is prevented by

adding a barb feature on the outer wall of the suction cup.

Figure 3: A section view of the suction cup design drawn in Solidworks showing features a.) small

lip to grip the egg in the correct position before vacuum is applied b.) gap that allows the suction

cup to expand around larger sizes of eggs c.) barb feature to prevent the suction cup from being

pulled out of the suction cup holder.

8

5.2 Vacuum Pump

The vacuum pump provides constant suction to the cups to hold the egg in place. The

final pump provides 7.8 PSI using 12 W at 12 V. There is a vacuum trap connected before the

pump in case the egg breaks and is sucked into the tubing. The pump does not have a regulator

due to the maximum pressure being needed to hold the egg during the cracking open of the quail

egg; however, the egg does sometime break from the suction. A single switch operates the pump

and is installed on the pump holder requiring the operator to use it during the quail egg opening

phase. The vacuum pump and trap are shown in figure below.

Figure 4: The 12V vacuum pump and the operating switch shown in the right. The vacuum trap

is shown in the left, with the reverse valve. The vacuum trap is manufactured from barb fittings,

silicon tubing, and a clear glass jar.

5.3 Puncturing Device

The method of puncturing the quail egg shell in order to initiate crack propagation is

designed to be integrated into the handheld device so that it could be performed one handed. One

of the handles of the handheld device are designed to incorporate a trigger that could be pulled with

the user’s index finger as shown in Figure 5. This pulls on a cable that is attached to the trigger

that then pulls up on a swinging arm attached to one of the suction cup holders as shown in Fig.

6. Mounted to the swinging arm is a razor which is used to create the puncture on the underside

9

of the quail egg. After the eggshell has been successfully punctured, the trigger is released, and a

spring connecting the swinging arm to the back of the suction cup holder causes the arm and the

razor to retract. The contents of the eggs are free to fall into the six well dish after the handles are

compressed.

Figure 5: a.) Handheld prototype with integrated puncture device, b.) close-up of the puncturing

mechanism attached to the suction cups. c.) frontal view of the suction cups and the blade.

5.4 Mechanical Levers

The levers are identified to be Class 1 and Class 3 levers with the top set connected to the

bottom by linkages as shown in the figure below. The levers are classified by the force and resistance

required, and the position of the fulcrum. Class 1, and 3 indicate the fulcrum and the efforts are

loaded in the middle of the lever; meaning that the force is applied on one side of the lever, and the

resistance on the other.

10

Figure 6: Caption

The purpose of these levers is to decrease the mechanical advantage so when the top levers

(handles) are squeezed together, the cups will spread apart far enough that a quail egg can be

placed between them. After the handles are released, suction is applied and the egg has had the

initial puncture, the handles are squeezed again to break the quail egg open. The low mechanical

advantage helps get the eggshells apart fast enough to let the egg yolk drop into the six well dish

unobstructed.

6 Final Design Evaluation

The method by which this product is evaluated is by testing the final prototype in the

cc-tdi lab with six fertilized incubated quail eggs. Only six were tested because the majority were

unsuccessful extractions.

6.1 Performance&

The puncturing was done by hand in the same method that the mechanical puncturing

device operates. The other elements of the device including separator, suction cups, and vacuum

were used. Six eggs were tested and only one embryo survived. With the puncturing technique of

11

going straight into the shell, the embryo was ruptured every time. The one occurrence where the

egg survived, the blade was inserted at a shallow angle thus confirming that the current puncturing

method does not work.

Each of the suction cups attached to the separator retract unevenly and this makes it

difficult to seat each egg properly between the cups. In addition when an egg fractures in such a

way that the yoke gets sucked into the silicone tubing the cleaning process takes several minutes to

ready the device for the next egg.

6.2 Structural Integrity

The two components of this section are the structural integrity of the device and the eggs as

well. The handheld device is made of high temperature materials (aluminum, Mylar, stainless steel,

and silicone) so that it can be sanitized in an autoclave at 120 degrees Celsius with experiencing

corrosion or warping. The device was not tested in the autoclave to confirm its resilience.

The structural integrity of the eggs is tested when they are inserted into the suction

cups and the clamping force creates a compressive pressure on the shell. Four out of the six eggs

survived the compressive forces even after being punctured from the side. The remaining two eggs

were crushed as a result of the crack initiation.

6.3 Manufacturability & Assembly

The machined parts are made from 6061-T6 aluminum plate of thicknesses 18 ,

14 , and 1 inch

plates. This material is chosen because it may be machined using a variety of tools such as milling,

laser cutting, and water jet. For this prototype the aluminum was machined using a band-saw,

hand drill, and hand files. The fasteners and plumbing parts are ordered from online distributors.

To evaluate this manufacturing process the number of man-hours logged on the construction time

using the previously mentioned tools is ten hours.

The final prototype consists of 124 individual parts and 18 unique parts. Most of the parts

are Mylar washers used for spacing in each pivot joint. Because of the many small parts used for

assembly, care must be taken when assembling the prototype by laying the components out on a

clean flat surface and using a step by step procedure. The figure below shows the general process of

assembling the device. To be broad, first the arm linkages are assembled, then they are mounted to

the base plates, and the plumbing and springs are attached. This assembly process takes a single

person 15 minutes to complete.

12

Figure 7: General procedure of assembling the final product of the quail egg embryo extractor.

From step one to two, the mechanical levers and the suction levers are assembled. In step three and

four, the base plates are assembled on the front and back sides of the device. Lastly, in step five

and six, the suction cups and silicon tubing are connected to the device.

The cost for materials for produce the final prototype consisting of separator, vacuum, and

the puncture device is $291.58. This includes shipping costs and extra materials left over. The total

amount of money spent for the project including research and development is $579.22.

13

7 Conclusion

The final design of the quail egg embryo extractor fell short of meeting the PDS require-

ments in terms of embryo extraction success rate. To meet the requirements, the puncturing method

would have to be improved and the vacuum generation would have to be automatically regulated

to prevent the vacuum from pulling out the contents of the quail egg. It should also be noted that

the yolk sac of fertilized quail eggs are thinner and more sensitive than the yolk sac of unfertilized

quail eggs. For this reason testing should have been done mostly on fertilized quail eggs to be able

to prove in earlier stages if the device was functioning correctly or not.

14

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.

15

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.

16

Appendix B: External Research Summary

The preliminary external research performed was focused on existing devices that per-

formed a similar task as well as research of quail egg anatomy. It was found that there are several

devices that perform the same operation on chicken eggs but few were found specifically for quail

eggs. There are many automated machines that are designed to remove chicken egg yolks on a

large scale. The team did not have the time or experience to create such an automated process for

quail eggs so the focus of the external search were on handheld devices that extracted on egg at

a time. One such device that does this for chickens eggs, shown in Figure 8 below, is called the

EZ cracker. This design relies on mechanical advantage to open the egg shell. A problem with

applying this design the the opening of quail eggs is that the EZ cracker applies a compressive force

to the top of the egg shell with a wedge in order to create a puncture on the bottom of the egg with

razors. Due to the fragility and positioning of the quail embryo this method would not be viable.

After researching quail embryo anatomy it is found that the embryo will float to the top of the egg

depending on how the egg is held.

Figure 8: The Ez Cracker, the egg cracker shown in motion.

Although the puncturing method that the EZ cracker uses would not work for this project,

the concept of a handheld device is attractive. A similar method could be implemented that would

leave the top of the egg undamaged while still initiating a puncture on the bottom of the egg.

17

Appendix C: Internal Research Summary

Based on the external research, two major design features are focused during the engineer-

ing design process; the method of handling the eggshell during embryo extraction, and the eggshell

puncturing method. Suctions cups are chosen as a primary design feature to hold the egg in its

optimal position and to provide a structured support during embryo extraction.

One of the primary requirement for the design of the suction cups are to be flexible to

conform to various egg sizes, and to be rigid enough to support the egg. Customized suction cups

are made from silicone rubber to fit the two requirements. In additional, a motorized vacuum pump

is employed to supply and maintain a constant suction.

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

and sanitation are concerning factors. Several designs are researched to determine the optimal

cutting device to penetrate the shell to initiate a crack propagation and to pierce through the

membrane. The designs are, a cushioned hammer with a sharp point, to implement a pure separation

force, and a lancing device–similar to the medical device used for taking blood samples. A prototype

is built to evaluate the optimal size and shape of the cutting tool, as well as the spring force required

to penetrate the shell.

18

Appendix D: Detailed Product Design Specifications

The elements of the product design specifications are organized into the table below in or-

der of decreasing importance (e.g. the top indicates the most importance, and bottom indicates the

least).This sequence ranking order was determined by the customer’s feedback using the question-

naire and the team’s judgement.The design specifications shaded in pink indicates high significance,

and the yellow and green represent medium and low importance, respectively.

19

Figure 9: Detailed PDS of the quail egg embryo extractor.

20

Appendix E: CAD Drawings

.25 B

.01

E.0

05

AD

.01

A

B

2X P

.13 B

.005

.01

A

B L

.08

A

C

UO

SF

.02

ISO

MET

RIC

VIEW

FOR

REFE

REN

CE O

NLY

HAN

DLE

LH

SCAL

E 1:

1

Figure 10: CAD drawing and geometric tolerance for the handle of the quail egg embryo extractor.

21

.25 B

.005

E.0

05

A

D.0

1 A

B

4X P

13/

64 B

.002

.01

A

B L

.08

A

P .1

3 B

.005

L.0

1 A

B

C

C

UO

SF

.02

ISO

MET

RIC

VIEW

FO

R RE

FERE

NCE

ON

LY

LEVE

R SC

ALE

1:2

Figure 11: CAD drawing and geometric tolerance for the lever of the quail egg embryo extractor.

22

SECT

ION

A-A

SCAL

E 1

: 1

SECT

ION

B-B

SCAL

E 1

: 1

A A

B B

1.50

B.0

1E

.01

A

D.0

1 A

B

P 1

.00 B

.002

H.0

1 A

B

C

F.0

02

A B

C

V.0

05

C

4X P

.15 B

.005

.01

A

B C

L

.08

A

B IS

OM

ETRI

C VI

EWFO

R RE

FERE

NCE

ON

LY

UO

SF

.02

CUP

HO

LDER

SCAL

E 1:

1

Figure 12: CAD drawing and geometric tolerance for the two cup holders of the quail egg embryo

extractor.

23

E .0

1

A

.50 B

.01

D 0

O

A

B

H .0

1 A

C

2X P

13/

64 B

.005

.01

A

B C

L

.08

A

B

2X P

3/1

6 B

.005

.01

A

B C

L

.08

A

B

UO

SF

.02

ISO

MET

RIC

VIEW

FOR

REFE

REN

CE O

NLY

(1.1

4)(1.8

0) FLAT

PAT

TERN

FOR

REFE

REN

CE O

NLY

CUP

BRAC

KET

SCAL

E 1:

1

Figure 13: CAD Drawing and geometric tolerance for he two cup bracket of the quail egg embryo

extractor.

24

UO

SF

.02

E .0

1

A

.50 B

.01

D0O

A

B

2X P

13/

64 B

.005

.01

A

B L

.008

A

C

ISO

MET

RIC

VIEW

FO

R RE

FERE

NCE

ON

LY

LIN

K SC

ALE

2:1

Figure 14: CAD drawings and geometric tolerance for the linkages of the quail egg embryo extractor.

25

Appendix F: Bill of Materials

The materials needed to manufacture and assemble the embryo extractor is summarized

below. With a running total of $579.22.

Figure 15: Bill of materials corresponding to the different parts of the embryo extractor.

26

Appendix G: Operational Manual

To operate the quail egg extractor, the following procedures are taken into First, the

suction cups are open by squeezing the handles together and a quail egg is placed within the suction

cups. Next, as shown in Fig. XX.2., the switch is pressed to activate the vacuum pump. This

ensures that the egg is held in place properly. Then to begin the extraction process, a sterilized

carbon steel blade is employed. The blade is inserted through the eggshell and membrane in a linear

motion. Finally, the device is held over the six-well dish and the content of the egg is released by

squeezing the handles. It is noted that additional suction may be needed prior to the release of the

embryo to ensure a crack is propagated along the diameter of the shell.

Figure 16: 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.

27

References

[1] Children’s Cancer Therapy Development Institute. (n.d.). Retrieved March 05, 2016, from

http://www.cc-tdi.org/#!mission/ctnu

[2] Ketelaere, B.D., Govaerts, T., Coucke, P., Dewil, E., Vissche, J., Decuypere, E., & Baerde-

maeker, J.D. (2002, 05). Measuring the eggshell strength of 6 different genetic strains

of laying hens: Techniques and comparisons. British Poultry Science, 43 (2), 238-244.

doi:10.1080/007166012012454

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

Level Criteria Microbiological and Biomedical Laboratories (BMBL) 5th. Ed.

from http://www.mun.ca/biolog/desmid/brian/BIOL3530/DEVO_03/ch03f13.jpg

28