My portfolio

Carol A. Hefley
chefley@okstate.edu 1205 East Hartford Street Broken Arrow, OK 74012 918-402-6625 EDUCATION: Bachelor of Science, Biological Sciences Oklahoma State University Stillwater, Oklahoma Expected graduation date: May 2015 Associate of Science, Biotechnology, Psychology Tulsa Community College, Tulsa, Oklahoma Graduation date: May 2010 RELEVANT EXPERIENCE: Biological Research Internship May 2012 to present Consortium (TABERC) Oklahoma State University Center for Health Sciences, Tulsa, Oklahoma Studied the possible causative agent of Morgellons Disease Grew bacteria and fungi from samples of Morgellons patients and compared with control samples Performed PCR( Polymerase Chain Reaction) to quantify the sample size

Research Technician August 2012 to present Oklahoma State University, Stillwater, Oklahoma Studied morphologies of Monarch butterflies AWARDS: NSF URM Research Scholar (awarded through OSU) NSF S-STEM scholar (awarded through OSU) References upon request Conducted surveys for milkweed plants and Monarch butterflies Observed and recorded Monarch caterpillar movement and host plant preference

Career statement My name is Carol Saylor-Hefley. I was born in Seguin Texas and moved to Tulsa when I was 9. While in high school I was involved in drama, the debate team, show-choir, and was also elected president of FCCLA- Family Career and Community Leaders of America. I graduated in May 2007. Following high school I attended Tulsa Community College as a Tulsa Achieves student. After taking basic undergraduate courses, I received two associates degrees in liberal arts and psychology in 2009. I then enrolled in the biotechnology program. I received another Associates degree in Biotechnology in the fall of 2012. While in Biotechnology courses at TCC, I applied for and received a scholarship through the TABERC organization, which provided me with an internship and the opportunity to research Morgellons disease at OSU-CHS. I am currently attending OSU Stillwater campus. I am a S-STEM and URM scholar which will allow me to graduate with a bachelor degree in biological sciences in 2015. I plan to pursue a PhD in a molecular biology in the future.

Writing assignment

Critical thinking This paper was one of the longest papers I have ever written and it was on a subject I knew absolutely nothing about prior to the assignment. This assignment got almost 20 hours of my life and I am proud of my research put into it. This addresses almost all the departmental objectives in the research and communication skills area which are: Understand the scientific method and its application to the life sciences; understand the concept of scientific peer review and how to access peerreviewed literature; demonstrate the critical thinking ability to summarize and evaluate basic information on biological Systems published in the scientific literature; and demonstrate the ability to present scientific information clearly and concisely orally and in writing. This assignment challenged these areas and I learned a tremendous amount of knowledge in this area of these objectives.

Research proposal This power point presentation of a research proposal is far from perfect but I learned a tremendous amount useful information. Organizing my thoughts in such a way to be able to propose a certain type of research was a challenged, but I enjoyed it and dint realize I could actually do it at the time. This assignment addresses quite of few learning objectives such as understanding the relatedness and distinctions among specialized fields of study within the life sciences as they relate to career options; ; and appreciate standards of responsible use of organisms and environments in research as well as in the application of knowledge that comes from that research. Within this proposal I actually had a chance to perform this research in the lab which makes this artifact even more important.

Carol Hefley Dr. Baum

What do you do if you have a bacteria that you think might be Escherichia coli. What is the first step in the process of characterization? The first step is growing it on agar, but what agar do you use?

In the Hiroshi article it explains how MacConkey agar has a role in Escherichia coli growth. Escherichia coli is a very common bacteria. To grow it on agar you have to do some background information on the specific bacteria and what it needs to grow. The Fijikawa article talks about the comparison of growth in pouched foods and gives a lot of detail what Escherichia coli is.

The question is what kind of agar do you use when trying to characterize Escherichia coli? My hypothesis is the agar in which Escherichia Coli will grow will be Nutrient agar . Nutrient agar has all the basic nutrition bacteria needs to thrive and grow and is not selective for any specifiic bacteria type.

We will experiment with nutrient agar and MacConkey agar. The methods that will be used first is inoculating a agar with a plastic loop. Streak for isolation with nutrient agar plate with the Ecoli.

Take out inoculated Ecoli plate and make sure all colonies look the same. Then take another plastic loop and inoculate a MacConkey plate plate. Put in 37 incubator for 24 hours.

Take MacConkey agar plate out of incubator. Growth= Gram negative bacilli and Escherichia coli is a gram negative bacilli.

One limitation to this experiment is that if you have growth on MacConkey agar you do prove that you have a gram negative Bacilli BUT there is a lot of different gram negative bacilli bacteria in the world. So further research would need to be done to guarantee that Escherichia coli is the bacteria you are growing.

The significance of this is that when you have a bacteria you need to characterize you start with agar. This is the first microbial technique you would perform. Figuring what kind of agar the bacteria will grow on is first step to figuring put what test do next on the flow chart.

Hiroshi, F. and S, Morozumi. 2007. Modeling Surface Growth of Escherichia coli on agar plates, Applied Environmental Microbiology, NCBI Pubmed http:/Pwww.ncbi.nlm.nih.gov/pmc/articles/P MC1317346/ Fijikawa, H . K. Yano and S. Morozumi. 2006. Model comparison of Escherichia coli growth in pouched food, NCBI Pubmed http://www.ncbi.nlm.nih.gov/pubmed/16862 989

Technical instructions

This assignment is a huge accomplishment that took over 20 hours to complete. This helped me with organizing my thoughts and being able to identify what scientific procedure I wanted to do and then write step by step instructions for someone to be able to replicate. Yet again I didnt get the grade I was shooting for but I feel this assignment shows how much time and effort I put into this assignment. This assignment also covered several departmental objectives. Such as understanding the relatedness and distinctions among specialized fields of study within the life sciences as they relate to career options; ; and appreciate standards of responsible use of organisms and environments in research as well as in the application of knowledge that comes from that research.

How to Characterize Bacteria from a Sample

The goal is to grow bacteria from your sample (dust example in this procedure) and then characterize it by determining if it is gram positive or gram negative. Reading and performing this procedure will help you identify what kind of cell walls your bacteria have see figure 3. It is important for you to figure out what kind of cell wall because this tells you if it gram positive or negative and by knowing this you can start a flow chart. A flow chart shows you what steps to follow to continue the investigation of your bacteria species see figure1. This, of course, is of interest when furthering research. Charactering bacteria from a sample is your first step to figure out what is growing on or in your sample. The procedure takes three days and must be done in a lab in a bio-safety cabinet level 1 see figure 2. The audiences for these instructions are scientist, professors, students, lab owners and lab assistants. Keep in mind after those three days you will know if sample has gram negative bacteria growing in a sample! You have to do this procedure in a lab and you must wear a lab coat goggles and gloves at all times see figure 5. You will have to have controls in this experiment meaning that for every inoculated plate you will have a control so you will be able to rule out accidental contamination see figure 6. Figure 1 Figure 2

Bacteria

Figure 3

Gram negative

Gram positive
Figure 4 Lab coat, Goggles and Gloves Must be worn

Materials
Sample Test tube Gram scale Test tube rack

Vortex machine 10 ml plastic dropper 10ml Deionized water (DI water) 5 Plastic inoculating loops 37C Incubator *2 Nutrient agar in a Petri dish *2 MacConkeys agar plate in a Petri dish

Figure 2

** Quantity of theses agar plates depends how much bacteria grows from your sample. In his procedure it is assumed just one colony is being characterized. Figure 6 MacConkey agar Nutreient agar

Getting your sample ready

1. Put test tube on rack and with 10 ml plastic dropper put 10ml of DI water in tube.
TIP- Wear coat goggles and gloves as at times when working with microbes there is an increased risk of contamination.

2. Weigh out 0.1 grams of your sample and put it in the prepared test tube. See figure-7

Figure-7 3. Take test tube out of rack and place on Vortex. Vortex for 3 minutes then put back in rack. See figure 8

Figure-8 4. Label 1 nutrient agar plate with your name, date and sample identification code. 5. Vortex your sample for 3 more minutes 6. With 10 ml dropper, take 0.5 ml of your now contaminated water and place it in the middle of the labeled nutrient agar plate.

7. Take one inoculating loop and smear evenly throughout the plate. See figure 9

TIP-Make sure sample is evenly dispersed on the plate.

Figure 9 8. Put lid on agar plate and put in 37C incubator ( place additional agar plate in incubator as control to make sure there is no bacteria already on the plate)

Streaking for isolation

9. Check control plate to make sure there is no growth. Analyze the inoculated nutrient agar plates. Count how many different morphologies you have within the plate. TIP- when you analyze the plate you look at the round bacteria colonies and count how many of them are different see figure-10

Figure 10a

Figure 10 b These arrows point out all the different morphologies in figure 10b figure 10a. has only one morphology. 10. Label additional nutrient agar plates, corresponding to the number you counted in the inoculated plate (this procedure assumes one). 11. To streak for isolation draw 3 lines evenly spaced between each other on the plate in other word, draw three lines to make sections and divide the plate. See figure 11.

Figure 11 . 12. Take new plastic inoculating loop pick up one of the colonies with the loop, start at the first line A, running loop in-between A and B. Throw inoculating loop away in biohazard trash. 13. Turn the nutrient agar plate to wear you are between B and C lines. Take a new inoculating loop and drag from between B and A lines and streak a single squiggly line downward toward line C.

14. Turn the nutrient agar plate to where your between the C and A line . Use new inoculating loop and drag from in-between C and B and make a squiggly line downward towards the line A. 15. Your plate will now be divided into three sections see figure 8.ut the nutrient agar plate in the 37incubater along with a non inoculated agar plate for control for 12-24 hours. See figure 12 a&b

Figure 12a

Figure 12b

Characterizing your bacteria

16. Take out plates from 37 incubator from previous day and analyze. 17. There should be only one kind if bacteria on the plate. The morphology of the colonies should all look the same, if so move onto step 17 if not got back to step 9.

18. Take new inoculating loop and pick up a few of the colonies and streak them on the MacConkey Agar plate. Make sure to thoroughly smear the bacteria on the plate figure 13.

Figure 13 19. Put plate in 37 incubator for 12-24 hours.

Figure 13

Day 3 20. Get out inoculated MacConkey agar plate from previous day and analyze. If there is any growth on this plate you just characterized your first bacteria to be gram negative!

Further Explanations

Clean up informationAll agar plates, inoculating loops and plastic 10 ml pipettes need to be thrown away in biohazard trash! Test tube that held the sample needs to be autoclaved. Frequently asked questionsQ: How do know that growth on MacConkey agar means it gram negative?

A:MacConkey agar is selective for gram negative bacteria. It is specifically made to only grow gram negative bacteria. Q:Why do you have to streak for isolation how come you cannot just take from first agar plate? A: This is because in the first agar plates you have many bacteria thats growing the purpose for isolation is to separate the different ones so you can characterize the different bacteria. This is only the first step in characterizing bacteria using several other protocols you can figure out exactly what bacteria you have grown from your sample. Q:What if there is no growth on MacConkey agar plate? A:If there is no growth on MacConkey agar plate then you do not have a gram negative bacteria you have gram positive bacteria.

Trouble shooting adviceIf you are not having any growth at all with the first agar plate try to vortex sample again and plate on another nutrient agar plate. If after the second attempt and you still didnt grow anything then the agar plates could be too old, talk to the scientist that runs the lab for newer Nutrient agar plates. Sources for additional informationhttp://www.antimicrobialtestlaboratories.com/Difference_Between_Gram_Positive_and_Gram_N egative_Bacteria.htm

Product specificationsThe Agar plates need to be stored in refrigerator before inoculated. All other materials in this procedure can be stored at room temperature.

Additional artifact

This assignment is a huge accomplishment for me. Through TABERC internship and OSU-CHS I have had the privilege to research Morgelllons disease. I was ask to present my research in front of a number of people representing Oklahoma colleges. This was one of my very first presentations and I was very nervous but so proud after I researched put it together and presented.
Morgellons PowerPoint for TABERC internship

Morgellons: Is This Mysterious Skin Condition of Fungal or Bacterial Origin?

Carol Hefley -TCC Dr. Randy Wymore OSU CHS Lindsey AllenResearch Associate

What is Morgellons Disease

Cutaneous Skin Symptoms:

Crawling, Biting, Stinging sensations Microscopic Fibers Persistent Skin Lesions rashes and sores

Psychosomatic Symptoms include: Depression, anxiety, paranoia, fatigue

TABERC Summer 2012 OU Schusterman Learning Center August 2, 2012

images_sizedimage_218140556.jpg timesocket.com

The Dilemma: The Question

Is this a real disease, a psychosomatic disorder, or a combination of both?

Morgellons patients complain about tiny fibers under their skin claiming it feels like they've rolled around in fiberglass

morbo-di-morgellons.jpg swinehoax.blogspot.com

PCImorgpic3-206x300.png morgellonsitchyrash.com

http://www.fox23.com/news/local/story/Morgellons-A-medicalmystery/npmHr2UdK0WIjjjhhgkeqQ.cspx

Persons who suffer from this unexplained demopathy sometimes report various noncutaneous symptoms such as generalized fatigue, difficulty concentrating short-term memory loss and depressed mood.
CDC report

morgellons1-300x257.jpg cure-skin-parasites.com

Importance of this Research

Why this research is important.

Patients have voiced their opinions and are suffering. They have explained their conditions and how much pain they are in. Patients have been waiting for the condition to be addressed.

http://www.google.com/imgres?imgurl=http://morgellonswatch.com/images/morgellons_usa.jpg&imgrefurl=http://morgellonswatch.com/2006/07/11/fuzzballs/&h=384&w=5 12&sz=69&tbnid=IdgW8p9cdz5HM:&tbnh=98&tbnw=131&prev=/search%3Fq%3Dmorgellons%2Bkid%2Bpatient%2Bpictures%26tbm%3Disch%26tbo%3Du&zoom=1&q=morgellons+kid+pati ent+pictures&usg=__SUfXTHHakbSudcOfUGudTb6Wb_Q=&docid=Qwx61-3tIsb38M&hl=en&sa=X&ei=J20QUMsOIXs8wSY74HwAw&ved=0CEsQ9QEwAg&dur=4290

sites.google.com/site/drjoneskids/tick-bornediseases/morgellons&h=384&w=512&sz=19&tbnid=dlfbf-oLSdoPM:&tbnh=89&tbnw=118&prev=/search%3Fq%3Dmorgellons%2Bkid%2Bpatient%2 Bpictures%26tbm%3Disch%26tbo%3Du&zoom=1&q=morgellons+kid+patient+pi ctures&usg=__v4Atvz7J2U2tt1OIyfdjXasaEZQ=&docid=nBkzOqTfSk5WM&hl=en&sa=X&ei=J20

Thousands of people have been self diagnosed.

PCI_morgpic2-300x285.png morgellonsitchyrash.com hercampus.com

Experimental Approach

Methods:

Analyzed the microbiological differences in environmental samples collected from Morgellons and control households. We then investigated how these differences possibly correlated to the cause of the disease.

Isolation of bacteria We placed weighed dust sample in water. We plated samples on the following agars for isolation:
Mueller Hinton, Nutrient, O/F Basal (with Dextrose), Mannitol Salt, Mannitol Yolk Polymixin, Tryptic Soy (with sheep blood), and MacConkeys.

Agar Types

Cultivation

Image

Time

Agar Types

Cultivation

Image

Time

Mueller Hinton Agar

For antimicrobial susceptibility

24 hr at 37C

O/F Basal Media (with 10% Dextrose)

Dextrose

24 hr at 37C

Nutrient Agar

Cultivating wide variety of microorganisms

24hr at 37C

Mannitol Salt Agar

Mannitol and coagulase

24hr at 37C

General Bacterial Agar

Oxidizing/Fermenting Agars

Agar Types

Cultivation

Image

Time

Gram positive rod

Mannitol Yolk Polymixin Bacillus cereus 24 hr at 37C

Gram Stain Results:

Gram negative rod

Tryptic Soy Agar (with 5% sheep blood)

Hemolysis of Blood

24-48 hr at 37C

Gram positive cocci

MacConkey Agar Gram Negative Bacteria 24 hr at 37C

Gram negative cocci

Eleventy billion

Selective Bacterial Agars

Methods (cont):
Following isolation -> gram stain bacterial colonies. Once determined if gram positive/negative and rods/cocci, follow with appropriate testing Catalase or Oxidase API Testing

The oxidase test indicates the presence of the enzyme cytochrome oxidase, also presumptively identify Neisseria species and lastly to characterize gram negative bacilli.
chsweb.lr.k12.nj.us 243 180 - Oxidase Test

http://iws2.collin.edu/dcain/CCCCD%20Micro/catalase_test.htm

chapt8-figure04.gif cdc.gov 400 242 - Figure 4 is a picture showing negative and positive catalase test results.

A positive result means the organism has the enzyme catalase and can break down hydrogen peroxide to oxygen and water.

API testing

API tests:

Examples of infections:

Listeria 50 CH (B) Staph Strep NE 20 E NH Coryne

Listeriosis Food poisoning Staph infections Strep throat Cholera and Whooping cough Salmonella and E. coli Influenza Pharyngitis

Fungal Agars
Agar Type Test Image Test Length

Sabouraud Dextrose Agar

Cultivation of fungi

Up to one week

Malt

API testing

Fungal Agar Types

Isolating and cultivating yeast and molds for food, and for yeast and mold stock cultures

Up to one week

Analytical Techniques
Once isolated individual fungal colonies, run samples through following processes:

DNA Extraction Quantification Polymerase Chain Reaction Agarose gel electrophoresis DNA concentration and clean up Quantification Sequencing BLAST

Sample

Quantification of Cleaned and Concentrated Extracted Fungal DNA

Isolation (grams) Type of Agar Sab Dex Sab Dex Malt Malt Sab Dex Malt Sample purity (260/280) 1.947 1.915 1.93 1.84 2.033 1.791 .05CT White Mucoid .01PT Large White Mucoid .05 CT White Side Mucoid .05CT Fluffy White Cloud .05CT White Mucoid sub-unit .05CT Malt Yellow Mucoid 4.317 7.117 6.017 3.717 5.117 2.717

Concentration (ng/l)

1 2 3 4 5 6

Results of Fungal Sequencing

Sample 1 2 Isolation .05CT White Mucoid .01PT Large White Mucoid Type of Agar Sab Dex Sab Dex Species Aureobasidium pullulans Aureobasidium pullulans
API

Gram Stain

Methods Overview

3
4 5 6

.05 CT White Side Mucoid

.05CT Fluffy white Cloud .05CT White Mucoid sub-unit .05CT Matl Yellow Mucoid

Malt
Malt Sab Dex Malt

Aureobasidium pullulans
Alternata arborescens Aureobasidium pullulans Aureobasidium pullulans

Bacteria

BLAST

Fungi

Bacteria

Fungi

There were many differences visualized between control and Morgellons samples as regards to bacteria. Control samples contained much more variation within and quantity of bacteria in comparison with Morgellons samples.

Alternata arborescens and Aureobasidium pullulans species were found in the experimental samples. Fungal samples from control households were not sequenced.

Summary of Results 2 different experimental samples vs controls

We dont know it all yet. The cause still remains a mystery. We do know there are differences in the environmental samples between controls & affected populations, and that Morgellons is a real pathology of unknown cause and not psychiatric.

Future Directions
Some things to look at in more detail to see if there is connection.

Individuals

Funding Sources:

Follow up on other opportunistic fungi. Further sequencing Test samples on other agars Viral tests Get more patient samples from different geographical locations to compare. repeat tests Optimize incubation times Genetic analysis

Randy Wymore, PhD Lindsey Allen, BS Jay Bullard

OSU CHS David Wallace, PhD TABERC

PhD

Charles E. Holman Foundation Diana Spencer PhD Julie Marino PhD Kent Teague PhD Tom Glass PhD

http://singularityhub.com/2010/12/27/dna-sequencing-for-110-the-price-ion-torrents-commercial-sequencer-arrives/

Acknowledgements

References
Anna C Shore Angela S Rossney, Brian O'Connell seline M Herra, Derek J Sulivan, Hilary Humphreys and Davic C coleman. "Detection of Staphyloccal Cassette Chromosome mec-Associated DNA Segments in Multiresistant Methicillin Suseptible Staphylococcus Areus (MSSA)and idenification of Staphlycoccus Epidermitis crrAB4 in both Methicillin-Resistant S. areus and MSSA." Agents And Chemotherapy (2008): 4407-4419. Center For Disease Control. "Morgellons- Creating a New Disease." Science and Medicine. 2012. John Bennet, M.D. redacter. "Report of the External Peer review of CCID's Unexplained Dermopathy (UD) Project." Center For Disease Control, 2009. Morehead, Wayne. "Forensic Inters: Force Multioliers in the Crime Lab." Forensic Science Policy and Management: An International journal (2011): 2:3, 118-134. Natalia VanDuyn, Raja Settivari, Garry Wong and Richard Nass. "SKN-/Nrf2 Dopamine Neuron Degerenation In a Caenorhabditis elegans Model of Methylmercury Toxicity." Toxicological Sciences (2010): 613-624. Pearson ML, Selby JV, Kats KA, Cantrell V, Braden CR, et al. "Clinical, Epidemiologic, Histopatholgic and Molecular Features of an Unexplained Dermopathy." PLus One (2012): volume 7. Sylvio Redanz, Kerstin Stander, Andreas Podbieiski and Bernard Kreikemeyer. "a five Species Transcriptome Array for Oral Mixed-Biofilm Studies." PLos One (2011): 6-12.

Over all reflectionAll of my artifacts cover a wide variety of department objectives. The first writing assignment fulfills the being able to formulate evidencebased arguments on matters of science that generate controversy. I spent a number of hours on this assignment and I learned a lot from it. Same as with my proposal it met quite a few departmental objectives as well such as understanding the relatedness and distinctions among specialized fields of study within the life sciences as they relate to career options; ; and appreciate standards of responsible use of organisms and environments in research as well as in the application of knowledge that comes from that research. After this assignment I really got a grasp of how much work researching and conducting an experiment was a lot of work. Writing a proposal to do such an experiment was even more challenging. This assignment also helped me research career options that I will pursue soon after graduation. My technical writing instructions assignment took my science knowledge to the test when I had to write out a specific protocol that will give me the same results over and over again. The learning objectives I learned from this were appreciate standards of responsible use of organisms and environments in research as well as in the application of knowledge that comes from that research and being able to articulate the relationships among these core disciplines and how application of an integrative approach to life science research contributes to our understanding of the natural world. I also would apply these same last two objectives with my TABERC internship through OSU this was how I got started and in research and the first presentation I gave I am very proud of this presentation and I feel with more practice I can be beneficial to career job in the near future.