Lab 05: Photosynthesis

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To submit this assignment, students will complete the Lab Worksheet on pages 7-10, then upload their completed document as a DOC or PDF file in Canvas

 

BIO 101 Lab 08: Photosynthesis

 

Notification: If you have a disability that makes it difficult to complete this lab, please contact your instructor. Please provide your instructor a copy of the Memorandum of Accommodation (MOA) from NVCC Disability Support Services.

 

Objectives:________________________________________________________________

· Determine the effects of light on the rate of photosynthesis

· Determine the absorption spectrum of leaf pigments

 

Background:_______________________________________________________________

Sunlight provides the majority of energy for organisms living in most ecosystems, however only a subset of organisms are capable of harvesting this energy. Plants use their chloroplasts to absorb the energy from sunlight. This energy is then stored in the covalent bonds of glucose, a simple sugar, and can be used by the plant for structural purposes (cellulose), as usable energy (ATP generation), or for energy storage (starch). Animals can eat plants to obtain glucose and produce energy through a process called cellular respiration.

 

The overall reaction for photosynthesis is represented by the chemical equation:

 

6 CO2 + 6 H2O + sunlight → C6H12O6 + 6 O2

 

The entire process is complex and involves many enzymatic reactions. You may notice that the photosynthesis reaction is nearly the exact reverse of cellular respiration. From the equation above the three key elements for photosynthesis to occur are carbon dioxide (CO2), water (H2O), and light. If any of the three are missing from the system then photosynthesis will not occur, and glucose production in the plant will be negatively affected.

 

 

 

 

 

 

 

 

 

 

 

There are two phases of photosynthesis: H:BIO101 Lab Manual editsBIO101 XZ VB AS revision 2017Edited AJS512px-Simple_photosynthesis_overview.svg.png

 

1) In the light-dependent phase chlorophyll molecules located in the thylakoid membrane stacks of the chloroplasts absorb the energy from light resulting in the production of ATP and NADPH.

 

2) In the light-independent reactions (the Calvin Cycle) the energy stored in ATP and NADPH is used to ultimately convert carbon dioxide to sugar. The process of taking carbon dioxide from the air to build carbohydrates is called carbon fixation.

 

 

In solution, CO2 can be converted to carbonic acid (H2CO3) when dissolved in water. The carbonic acid will then release hydrogen ions (H+), causing the pH of the solution to decrease.

 

CO2 + H2O H2CO3 HCO3- + H+

 

Bromothymol blue is a pH indicator that changes color based on the pH of a solution. Bromothymol blue turns yellow at lower pH and blue as the pH is increased. This indicator can be used to track respiration (turns yellow as CO2 is added to the system) or photosynthesis (turns blue as CO2 is consumed).

 

Different colors of Bromothymol blue at the indicated pH conditions

 

Light energy is a small part of the electromagnetic spectrum which is visible to the eye. The wavelength of visible light lies between 380 nm and 760 nm. In order to obtain the energy from light, plants must absorb light energy using pigments, namely chlorophyll a, chlorophyll b, carotene and xanthophyll. These pigments show characteristic colors because they do not absorb all light equally. By measuring the absorbance at different wavelengths, the absorption spectrum of the leaf pigments can be obtained.

Materials:__________________________________________________________________

· Internet

 

 

  

Safety:

Follow all standard laboratory safety procedures.

 

Procedure:________________________________________________________________

 

Experiment 1. Observing photosynthesis (work in groups)

1. In the laboratory, you would add about 60 ml of tap water into a beaker and then add about 1 mL of bromothymol blue to the water in the beaker.

 

2. Using a clean straw, you would then gently blow into the solution until the color of the solution turns yellow.

 

3. You would next fill three test tubes 2/3 full with this yellow-colored solution.

 

4. Then, you would place a 3-inch cutting of Elodea into Tube 1 and another 3-inch cutting of Elodea into Tube 2, making sure that the cuttings were completely immersed in the solution. After this, you would wrap Tube 1 with green film. Tube 3 will be left alone and will not contain a plant cutting.

 

5. Watch the following video about setting up the photosynthesis lab from time 0:00 until 5:46.

https://www.youtube.com/watch?v=GWUawtweJGM

 

Ignore the use of a second plant in the video, we only use Elodea in this laboratory activity. In the video, Tubes 1 and 2 contain Elodea plant cuttings and Tube 1 is wrapped in foil instead of green film.

In our photosynthesis lab experiment, we wrap Tube 1 in green film, which only allows green-colored light to reach the Elodea plant cutting. The green film we use ends up having the same effect as the aluminum foil used in the video.

 

6. In Table 1 in the Lab Worksheet, record the color of the solution before blowing into it in the “before exhaling” column.

 

7. In Table 1 in the Lab Worksheet, record the color of the solution after blowing into is in the “after exhaling column.

 

8. Answer question 1 in the Lab Worksheet

 

9. In the laboratory, you would then place the test tubes in front of a light source such as a fluorescent lamp. You would allow the tubes to be exposed to the light for 1 hour.

 

10. Record the color of the solution in each test tube in Table 2 of the Lab Worksheet

 

11. Make a hypothesis about how the color of bromothymol blue solution in each test tube will change and record the color you expect for each tube in Table 2 in the “Expected Color after 1 hour of light exposure” column.

 

When making a hypothesis, consider why the pH-indicating solution turned color in the first place and what might happen if the substance that caused the color change was removed by a plant performing photosynthesis.

 

12. Watch the following video showing the results of photosynthesis lab from time 0:00 until 2:41.

https://www.youtube.com/watch?v=SZsQG_rPJwQ

In the video, Vial A corresponds to Tube 1 in our photosynthesis lab experiment. Ignore Vial B from the video. Vial C in the video corresponds to Tube 2 in our photosynthesis lab experiment and Vial D in the video corresponds to Tube 3 (the control).

Note that in our photosynthesis lab experiment, we wrap Tube 2 in green film, which only allows green-colored light to reach the Elodea plant cutting. The green film we use ends up having the same effect as the aluminum foil used in the video.

 

13. Using the results diagrammed below, fill in Table 2 in the Lab Worksheet

 

 

14. Record the color you observe for each test tube in Table 2 in the “Observed color” column.

 

15. Answer questions 2 – 3 in the Lab Worksheet.

 

 

Experiment 2. Absorption spectrum of leaf extract (work in groups)

1. In the laboratory, you would first turn on the spectrophotometer and allow it to warm up for 15 min.

 

2. You would then insert the blank cuvette (which contains alcohol) in sample holder marked “B” and the cuvette containing leaf extract in sample holder “1”, making sure that the orientation of the cuvettes is correct.

 

3. Then, you would select a wavelength to measure and calibrate the spectrophotometer by measuring the absorbance of the blank cuvette at this wavelength. You would then see the absorbance reading set to 0.

 

4. Now, you would measure the absorbance of the cuvette containing the leaf extract and record the absorbance in Table 3 in the position corresponding to the wavelength you selected.

 

5. You would repeat steps 3 – 4, changing to a different wavelength each time, until all the wavelengths in Table 3 had been measured. Record absorbance readings in Table 3.

 

6. Watch the following video about using a spectrophotometer from time 0:00 until 4:43.

https://www.youtube.com/watch?v=C-M7EtSuD40

Note that the video uses a tube containing a red liquid, while in our photosynthesis lab, we would use a tube containing leaf extract (which would appear green) as our sample tube and a tube containing alcohol (which would be transparent) as our blank tube.

 

7. Table 3 has been partially filled in for you in the Lab Worksheet. Complete Table 3 by filling in the color of light that corresponds to the grouped wavelengths.

 

8. Answer question 4 by making a Line Graph of the data in Table 3.

 

9. Answer questions 5 and 6 based on your Line Graph

 

10. Answer question 7 based on the results of both Experiment 1 and Experiment 2.

 

BIO 101 Lab 08: Photosynthesis Worksheet

 

Name: __________________________ Section: ______________________

 

Data Analysis and Synthesis Questions:

 

Table 1. pH-indicating dye color

Color before exhaling into beaker Color after exhaling into beaker
   

 

1. Why did the solution in the beaker change color after you exhaled?

 

 

 

 

 

Table 2. Observing Photosynthesis

Tube Color before 1 hour of light exposure Expected Color after 1 hour of light exposure Observed color after 1 hour of light exposure
1

(Elodea + green film)

      
2

(Elodea)

      
3

(no plant)

      

 

2. In this experiment , what is the purpose of the tube without the plant?

 

 

 

 

 

 

3. Explain the color change or lack of color change in the three experimental tubes:

 

a. Tube 1:

 

 

 

 

b. Tube 2:

 

 

 

 

c. Tube 3:.

 

 

 

 

Table 3. Absorbance of Different Wavelengths of Light

Color of Light Wavelength (nm) Absorbance   Color of Light Wavelength (nm) Absorbance
  400 0.58     575 0.09
  425 0.82     600 0.12
  450 0.61     625 0.16
  475 0.44     650 0.23
  500 0.22     675 0.53
  525 0.08     700 0.04
  550 0.06     725 0.05

 

 

 

 

4. Make a Line Graph of the absorbance readings versus the wavelength of light data from Table 3. Be sure to include x-axis and y-axis labels and a chart title.

 

 

 

 

 

 

 

 

 

5. Which wavelength(s) and colors of light are most effectively absorbed by leaf pigments (where are the peaks in your Line Graph)?

 

 

 

 

 

 

6. Which wavelength(s) and colors of light are poorly absorbed (where are the valleys in your Line Graph)?

 

 

 

 

 

 

7. Draw a general conclusion about which color(s) of light are best for plant growth based on your data from both the measurement of absorbance of light in leaf extracts and the observations of CO2 use by Elodea cuttings in different light conditions.

 

 

 

 

 

 

 

 

 

 

BIO 101 Lab 08: Photosynthesis 10
 
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Micro

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Please Pick 2 question to answer from each chapter and provide reference.

2 questions per chapter (chapter 15-20)  10 questions total. Word doc attached.

Chapter 15 

1.Explain the two main features that characterize the third line of host defense mechanisms.

2.Chronic lymphocytic leukemia (CLL) leads to the production of cancerous B cells, and treatment often involves bone marrow transplantation. Based upon your knowledge of lymphocyte development, explain how this procedure can lead to therapeutic effects in some patients.

3.Recently, scientists have been experimenting with using IRA-B cells as a treatment for hospitalized patients in an attempt to prevent them from getting septic infections. Speculate on what the principle behind this might be.

4.Provide an explanation to refute the following statement: Humans would never develop natural immunity to a novel biological agent created in a laboratory.

5.   a.Explain how the anamnestic response is triggered by vaccination. b.Conduct additional research and discuss one current example illustrating how lack of herd immunity within a population has led to localized disease outbreaks in the United States.

Chapter 16

1.Conduct additional research and discuss examples that illustrate how cancer can be both a cause of immune dysfunction and an effect of this process.

2.Summarize the roles of the microbiome and genetics in the development of type I allergic reactions. Discuss how probiotics or gene therapy could be used to alter an individual’s allergic response to antigen.

3.   a.Draw a diagram illustrating whether or not each of the following transfusions would be immunologically compatible. Type A donor into a type B recipient Type B donor into a type AB recipient Type O– donor into a type O+ recipient b.Explain how xenotransplantation might be successful in light of the immune system’s robust ability to recognize foreign antigen.

4.Summarize the role of the immune system in the development of type I diabetes. Propose a strategy that could be used to protect young children from developing an autoimmune reaction, and subsequently type I diabetes, after a viral infection.

5.A patient in your unit exhibits frequent bouts of microbial infections and is found to produce extremely low levels of IgG and IgM antibodies. Your colleague suggests that the patient receive numerous vaccinations against a broad spectrum of common pathogens; you disagree. Why? Explain another treatment that may be beneficial to this patient.

Chapter 17

1.Explain why specimens should be taken aseptically, even when nonsterile sites are being sampled and selective media are to be used, and explain why speed is important in the clinical testing process.

2.In the middle of an outbreak of measles (an infection that is highly communicable), a public health department is using immunologic testing to determine whether sick children have measles or not. If the children test positive, they are quarantined at home, and their contacts are counseled to update their MMR vaccine. In this situation, would you prefer to use a diagnostic test that is highly specific or one that is highly sensitive? Justify your answer.

3.Explain which type of ELISA can be used to determine an individual’s past exposure to a pathogen.

4.You are working at a health clinic, and a woman enters, suspecting that she was exposed to HIV two nights ago. a.Discuss whether or not she can be tested for HIV infection at this point. b.Summarize how you would respond to this patient, providing her with appropriate information regarding testing for HIV infection.

5.Compare and contrast the process of restriction analysis used in traditional DNA fingerprinting with the procedure used in pulse-field gel electrophoresis.

Chapter 18 

1.A young boy was at the playground when he felt a sharp pain on his leg. Upon inspection, his mother realized he had been stung by a bee. They went home and she carefully removed the stinger and washed the site well. Within a week, the site became swollen and painful; a red line appeared at the site, trailing up his leg.

a.Explain what condition the young boy appears to be suffering from and the most likely causative agent involved.

b.Discuss how the microbe may have gained access to the portal of entry.

2.A farmer working on a piece of machinery gets his shirtsleeve caught in a moving piece of the equipment. His shirt is sliced, and a sharp blade covered in mud slices through his upper arm. He attempts to control the bleeding and immediately seeks medical attention. After 3 days, he develops a fever and his arm becomes extremely swollen and painful. Pulling back the bandages, he finds that the wound has become blackened and is leaking a bloody fluid. Microscopic analysis of the fluid reveals the presence of gram-positive bacilli.

a.Discuss what condition the patient is suffering from and the likely causative agent of this infection.

b.Explain how the patient contracted this pathogenic microbe and what virulence factors contributed to the pathogenesis seen at the wound site.

c.In addition to antibiotics, the physician prescribes hyperbaric therapy. Describe what this treatment involves and how it could be therapeutic to this patient.

3.   a.Conduct additional research, and discuss whether “pox parties” represent a safe method of developing immunity to varicella zoster virus.

b.Provide evidence in support of or refuting the following statement: Shingles develops when you are reinfected with varicella zoster virus later on in life.

4.Smallpox has a rich history—from prompting the first vaccine to potential use as a bioterrorism agent. Given what you know about the etiology of the disease and the current state of the world’s immunity to smallpox, discuss how effective (or ineffective) a smallpox biological weapon could be against a human population.

5.Your coworker says that her spouse is in the hospital for elective surgery and his recovery is complicated by two infections: He has chicken pox all over the upper half of his body, and shingles on his legs. What questions will you ask for clarification?

Chapter 19

1.   a.Explain why the nervous system is described as “immunologically privileged,” and discuss whether this provides a beneficial or disadvantageous effect in this system.

b.Discuss the defenses a pathogen encounters as it attempts to gain entry into the nervous system.

2.Conduct research and summarize the causative agent and mode of transmission behind the multistate meningitis outbreak linked to steroid injections that occurred in 2012. How did improper physical and chemical control methods play a major role in this outbreak? Did portal of entry play any role?

3.Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting white-tailed deer and elk. In 2005, venison from a deer testing positive for CWD was served during an annual wild-game dinner in the town of Verona in upstate New York. Based upon your knowledge of vCJD and related spongiform encephalopathies and any additional research you conduct, discuss whether any of the attendees are at risk for developing disease in the future.

4.As you learned in section 8.3, many types of fruits and vegetables can be fermented into alcoholic beverages. Such was the case recently when prisoners in Utah attempted to make an illegal beverage called “pruno”; however, someone added a weeks-old baked potato to the mix, letting a microbe into the party who was clearly uninvited. Consumers of the pruno began to develop difficulty swallowing, vomiting, double vision, and muscle weakness; three required ventilation therapy. No deaths were attributed to the contaminated beverage.

a.What disease were the prisoners suffering from, and what was the causative agent involved?

b.Based upon your knowledge of this disease, what form of treatment was used to successfully avoid the worst outcomes of the disease in these patients?

5.In August 2011, a soldier from Fort Drum in Watertown, New York, tested positive for rabies; he died less than 3 weeks later. Further investigation revealed that he actually became infected when he was bitten by a dog in January of the same year while stationed in Afghanistan. Discuss any risks the soldier posed to his platoon, explaining whether or not this fatal outcome could have been avoided.

Chapter 20 

1.Explain why cases of dengue fever have been observed beyond endemic regions of the world today. Discuss whether or not completely eradicating mosquito (vector) populations from disease-ridden areas is advisable.

2.   a.Discuss whether or

not genetics plays a role in HIV infection, providing at least one example to illustrate your position.

b.Provide evidence in support of or refuting the following statement: An HIV-positive individual will always harbor the virus even if no viral load is detectable by PCR or other methods.

3.Explain why over the years the incidence of HIV infection has declined in the United States while the prevalence of AIDS has increased.

4.   a.Compare and contrast various characteristics of hemorrhagic and non hemorrhagic fever diseases.

b.Provide an explanation for the observed increase in incidence of these zoonotic infections around the world today.

5.Several pathogens in this chapter are listed as Category A bio weapons by the Centers for Disease Control and Prevention (CDC). What characteristics of the pathogens in this chapter make them suited for mass infection with high mortality?

Chapter 15

 

1.Explain the two main features that characterize the third line of host defense mechanisms.

 

2.Chronic lymphocytic leukemia (CLL) leads to the production of cancerous B cells, and treatment often involves bone marrow transplantation. Based upon your knowledge of lymphocyte development, explain how this procedure can lead to therapeutic effects in some patients.

 

3.Recently, scientists have been experimenting with using IRA-B cells as a treatment for hospitalized patients in an attempt to prevent them from getting septic infections. Speculate on what the principle behind this might be.

4.Provide an explanation to refute the following statement: Humans would never develop natural immunity to a novel biological agent created in a laboratory.

 

5. a.Explain how the anamnestic response is triggered by vaccination. b.Conduct additional research and discuss one current example illustrating how lack of herd immunity within a population has led to localized disease outbreaks in the United States.

 

Chapter 16

 

1.Conduct additional research and discuss examples that illustrate how cancer can be both a cause of immune dysfunction and an effect of this process.

 

2.Summarize the roles of the microbiome and genetics in the development of type I allergic reactions. Discuss how probiotics or gene therapy could be used to alter an individual’s allergic response to antigen.

 

3. a.Draw a diagram illustrating whether or not each of the following transfusions would be immunologically compatible. Type A donor into a type B recipient Type B donor into a type AB recipient Type O– donor into a type O+ recipient b.Explain how xenotransplantation might be successful in light of the immune system’s robust ability to recognize foreign antigen.

 

4.Summarize the role of the immune system in the development of type I diabetes. Propose a strategy that could be used to protect young children from developing an autoimmune reaction, and subsequently type I diabetes, after a viral infection.

5.A patient in your unit exhibits frequent bouts of microbial infections and is found to produce extremely low levels of IgG and IgM antibodies. Your colleague suggests that the patient receive numerous vaccinations against a broad spectrum of common pathogens; you disagree. Why? Explain another treatment that may be beneficial to this patient.

 

Chapter 17

 

1.Explain why specimens should be taken aseptically, even when nonsterile sites are being sampled and selective media are to be used, and explain why speed is important in the clinical testing process.

 

2.In the middle of an outbreak of measles (an infection that is highly communicable), a public health department is using immunologic testing to determine whether sick children have measles or not. If the children test positive, they are quarantined at home, and their contacts are counseled to update their MMR vaccine. In this situation, would you prefer to use a diagnostic test that is highly specific or one that is highly sensitive? Justify your answer.

 

3.Explain which type of ELISA can be used to determine an individual’s past exposure to a pathogen.

 

4.You are working at a health clinic, and a woman enters, suspecting that she was exposed to HIV two nights ago. a.Discuss whether or not she can be tested for HIV infection at this point. b.Summarize how you would respond to this patient, providing her with appropriate information regarding testing for HIV infection.

 

5.Compare and contrast the process of restriction analysis used in traditional DNA fingerprinting with the procedure used in pulse-field gel electrophoresis.

 

Chapter 18

 

1.A young boy was at the playground when he felt a sharp pain on his leg. Upon inspection, his mother realized he had been stung by a bee. They went home and she carefully removed the stinger and washed the site well. Within a week, the site became swollen and painful; a red line appeared at the site, trailing up his leg.

a.Explain what condition the young boy appears to be suffering from and the most likely causative agent involved.

b.Discuss how the microbe may have gained access to the portal of entry.

 

2.A farmer working on a piece of machinery gets his shirtsleeve caught in a moving piece of the equipment. His shirt is sliced, and a sharp blade covered in mud slices through his upper arm. He attempts to control the bleeding and immediately seeks medical attention. After 3 days, he develops a fever and his arm becomes extremely swollen and painful. Pulling back the bandages, he finds that the wound has become blackened and is leaking a bloody fluid. Microscopic analysis of the fluid reveals the presence of gram-positive bacilli.

a.Discuss what condition the patient is suffering from and the likely causative agent of this infection.

b.Explain how the patient contracted this pathogenic microbe and what virulence factors contributed to the pathogenesis seen at the wound site.

c.In addition to antibiotics, the physician prescribes hyperbaric therapy. Describe what this treatment involves and how it could be therapeutic to this patient.

 

3. a.Conduct additional research, and discuss whether “pox parties” represent a safe method of developing immunity to varicella zoster virus.

b.Provide evidence in support of or refuting the following statement: Shingles develops when you are reinfected with varicella zoster virus later on in life.

 

4.Smallpox has a rich history—from prompting the first vaccine to potential use as a bioterrorism agent. Given what you know about the etiology of the disease and the current state of the world’s immunity to smallpox, discuss how effective (or ineffective) a smallpox biological weapon could be against a human population.

5.Your coworker says that her spouse is in the hospital for elective surgery and his recovery is complicated by two infections: He has chicken pox all over the upper half of his body, and shingles on his legs. What questions will you ask for clarification?

 

Chapter 19

1. a.Explain why the nervous system is described as “immunologically privileged,” and discuss whether this provides a beneficial or disadvantageous effect in this system.

b.Discuss the defenses a pathogen encounters as it attempts to gain entry into the nervous system.

 

2.Conduct research and summarize the causative agent and mode of transmission behind the multistate meningitis outbreak linked to steroid injections that occurred in 2012. How did improper physical and chemical control methods play a major role in this outbreak? Did portal of entry play any role?

 

3.Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting white-tailed deer and elk. In 2005, venison from a deer testing positive for CWD was served during an annual wild-game dinner in the town of Verona in upstate New York. Based upon your knowledge of vCJD and related spongiform encephalopathies and any additional research you conduct, discuss whether any of the attendees are at risk for developing disease in the future.

 

4.As you learned in section 8.3 , many types of fruits and vegetables can be fermented into alcoholic beverages. Such was the case recently when prisoners in Utah attempted to make an illegal beverage called “pruno”; however, someone added a weeks-old baked potato to the mix, letting a microbe into the party who was clearly uninvited. Consumers of the pruno began to develop difficulty swallowing, vomiting, double vision, and muscle weakness; three required ventilation therapy. No deaths were attributed to the contaminated beverage.

a.What disease were the prisoners suffering from, and what was the causative agent involved?

b.Based upon your knowledge of this disease, what form of treatment was used to successfully avoid the worst outcomes of the disease in these patients?

 

5.In August 2011, a soldier from Fort Drum in Watertown, New York, tested positive for rabies; he died less than 3 weeks later. Further investigation revealed that he actually became infected when he was bitten by a dog in January of the same year while stationed in Afghanistan. Discuss any risks the soldier posed to his platoon, explaining whether or not this fatal outcome could have been avoided.

 

Chapter 20

1.Explain why cases of dengue fever have been observed beyond endemic regions of the world today. Discuss whether or not completely eradicating mosquito (vector) populations from disease-ridden areas is advisable.

 

2. a.Discuss whether or

not genetics plays a role in HIV infection, providing at least one example to illustrate your position.

b.Provide evidence in support of or refuting the following statement: An HIV-positive individual will always harbor the virus even if no viral load is detectable by PCR or other methods.

 

3.Explain why over the years the incidence of HIV infection has declined in the United States while the prevalence of AIDS has increased.

 

4. a.Compare and contrast various characteristics of hemorrhagic and nonhemorrhagic fever diseases.

b.Provide an explanation for the observed increase in incidence of these zoonotic infections around the world today.

 

5.Several pathogens in this chapter are listed as Category A bioweapons by the Centers for Disease Control and Prevention (CDC). What characteristics of the pathogens in this chapter make them suited for mass infection with high mortality?

 
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UMUC Biology 102/103 Lab 4: Enzymes

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Download a plgrism free answer from here

 

 

Your Full Name:

UMUC Biology 102/103

Lab 4: Enzymes

INSTRUCTIONS:

 

·         On your own and without assistance, complete this Lab 4 Answer Sheet electronically and submit it via the Assignments Folder by the date listed in the Course Schedule (under Syllabus).

·         To conduct your laboratory exercises, use the Laboratory Manual located under Course Content. Read the introduction and the directions for each exercise/experiment carefully before completing the exercises/experiments and answering the questions.

·         Save your Lab 4 Answer Sheet in the following format:  LastName_Lab4 (e.g., Smith_Lab4).

·         You should submit your document as a Word (.doc or .docx) or Rich Text Format (.rtf) file for best compatibility.

Pre-Lab Questions

 

  1. How could you test to see if an enzyme was completely saturated during an experiment?

 

  1. List three conditions that would alter the activity of an enzyme. Be specific with your explanation.

 

  1. Take a look around your house and identify household products that work by means of an enzyme. Name the products, and indicate how you know they work with an enzyme.

 

 

Experiment 1: Enzymes in Food

This experiment tests for the presence of amylase in food by using Iodine-Potassium Iodide, IKI. IKI is a color indicator used to detect starch. This indicator turns dark purple or black in color when in the presence of starch. Therefore, if the IKI solution turns to a dark purple or black color during the experiment, one can determine that amylase is not present (because presence of amylase would break down the starch molecules, and the IKI would not change color).

concept_tab_2

Materials

(1) 2 oz. Bottle (Empty)
(1) 100 mL Graduated Cylinder
30 mL Iodine-Potassium Iodide, IKI
Permanent Marker
Ruler
2 Spray Lids
30 mL Starch (liquid)
*Cutting Board

  

*2 Food Products (e.g., ginger root, apple, potato, etc.)
*Kitchen Knife
*Paper Towel
*Saliva Sample
*Tap Water

*You Must Provide

 

Procedure:

  1. Remove the cap from the starch solution. Attach the spray lid to the starch solution.
  2. Rinse out the empty two ounce bottle with tap water. Use the 100 mL graduated cylinder to measure and pour 30 mL of IKI into the empty two ounce bottle. Attach the remaining spray lid to the bottle.
  3. Set up a positive control for this experiment by spraying a paper towel with the starch solution. Allow the starch to dry for approximately one hour (this time interval may vary by location).
  4. In the mean time, set up a negative control for this experiment. Use your knowledge of the scientific method and experimental controls to establish this component (hint: what should happen when IKI solution contacts something that does not contain starch?) Identify your negative control in Table 1.

Note: Be sure to space the positive and negative controls apart from each other to prevent cross-contamination.

  1. When the starch solution has dried, test your positive and negative controls. This step establishes a baseline color scale for you to evaluate the starch concentration of the food products you will test in Steps 7 – 11. Record your results in Table 1.
  2. Select two food items from your kitchen cabinet or refrigerator.
  3. Obtain a kitchen knife and a cutting board. Carefully cut your selected food items to create a fresh surface.
Figure 3: Sample set-up.
Figure 3: Sample set-up.
  1. Gently rub the fresh/exposed area of the food items on the dry, starch-sprayed paper towel back and forth 10 – 15 times. Label where each specimen was rubbed on the paper towel with a permanent marker (Figure 3).
  2. Wash your hands with soap and water.
  3. Take your finger and place it on your tongue to transfer some saliva to your finger. Then, rub your moistened finger saliva into the paper towel. Repeat this step until you are able to adequately moisten the paper towel.

    Note: You should always wash your hands before touching your tongue! Alternatively, if you do not wish to put your hands in your mouth, you may also provide a saliva sample by spitting in a separate bowl and rubbing the paper towel in the saliva. Be sure not to spit on the paper towel directly as you may unintentionally cross-contaminate your samples.

  4. Wait five minutes.
  5. Hold the IKI spray bottle 25 – 30 cm away from the paper towel, and mist with the IKI solution.
  6. The reaction will be complete after approximately 60 seconds. Observe where color develops, and consider what these results indicate. Record your results in Table 1.
Table 1: Substance vs. Starch Presence
Substance Resulting Color Presence of Starch?
Positive Control: Starch Dark Purple Yes
Negative Control : Cellulose Brownish red color  No
Food Product: Apple Dark Purple  yes
Food Product: Potato Dark Purple  yes
Saliva: Amylase Brownish red color  No

 

Post Negative Control -Lab Questions

1.      What were your controls for this experiment? What did they demonstrate? Why was saliva included in this experiment?

2.      What is the function of amylase? What does amylase do to starch?

3.      Which of the foods that you tested contained amylase? Which did not? What experimental evidence supports your claim?

 

4.      Saliva does not contain amylase until babies are two months old. How could this affect an infant’s digestive requirements?

 

5.      There is another digestive enzyme (other than salivary amylase) that is secreted by the salivary glands. Research to determine what this enzyme is called. What substrate does it act on? Where in the body does it become activated, and why?

 

6.      Digestive enzymes in the gut include proteases, which digest proteins. Why don’t these enzymes digest the stomach and small intestine, which are partially composed of protein?

 

Experiment 2: Effect of Temperature on Enzyme Activity

Yeast cells contain catalase, an enzyme which helps convert hydrogen peroxide to water

Figure 4: Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen.
Figure 4: Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen.

and oxygen. This enzyme is very significant as hydrogen peroxide can be toxic to cells if allowed to accumulate. The effect of catalase can be seen when yeast is combined with hydrogen peroxide (Catalase: 2 H2O2 → 2 H2O + O2).

In this lab you will examine the effects of temperature on enzyme (catalase) activity based on the amount of oxygen produced. Note, be sure to remain observant for effervescence when analyzing your results.

 

Materials

(2) 250 mL Beakers
3 Balloons
30 mL 3% Hydrogen Peroxide, H2O2
Measuring Spoon
Permanent Marker
Ruler
20 cm String

  

3 Test Tubes (Glass)
Test Tube Rack
Thermometer
Yeast Packet
*Hot Water Bath
*Stopwatch

*You Must Provide

 

Procedure

  1. Use a permanent marker to label test tubes 1, 2, and 3. Place them in the test tube rack.
  2. Fill each tube with 10 mL hydrogen peroxide. Then, keep one of the test tubes in the test tube rack, but transfer the two additional test tubes to two separate 250 mL beakers.
  3. Find one of the balloons, and the piece of string. Wrap the string around the uninflated balloon and measure the length of the string with the ruler. Record the measurement in Table 2.
  4. Create a hot water bath by performing the following steps:
    1. Determine if you will use a stovetop or microwave to heat the water. Use the 100 mL graduated cylinder to measure and pour approximately 200 mL of water into a small pot or microwave-safe bowl (you will have to measure this volume in two separate allocations).
    2. If using a stovetop, obtain a small pot and proceed to Step 4c. If using a microwave, obtain a microwave-safe bowl and proceed to Step 4e.
    3. If using a stove, place a small pot on the stove and turn the stove on to a medium heat setting.
    4. Carefully monitor the water in the pot until it comes to a soft boil (approximately 100 °C). Use the thermometer provided in your lab kit to verify the water temperature. Turn the stove off when the water begins to boil. Immediately proceed to Step 5.

      CAUTION: Be sure to turn the stove off after creating the hot water bath. Monitor the heating water at all times, and never handle a hot pan without appropriate pot holders.

    5. If using a microwave, place the microwave-safe bowl in the microwave and heat the water in 30 second increments until the temperature of the water is approximately 100 °C. Use the thermometer provided in your lab kit to verify the water temperature. Wait approximately one minute before proceeding to Step 5.
  5. Place Tube 1 in the refrigerator. Leave Tube 2 at room temperature, and place Tube 3 in the hot water bath.

Important Note: The water should be at approximately 85 °C when you place Tube 3 in it. Verify the temperature with the thermometer to ensure the water is not too hot! Temperatures which exceed approximately 85  °C may denature the hydrogen peroxide.

  1. Record the temperatures of each condition in Table 2. Be sure to provide the thermometer with sufficient time in between each environment to avoid obscuring the temperature readings.
  2. Let the tubes sit for 15 minutes.
  3. During the 15 minutes prepare the balloons with yeast by adding ¼ tsp. of yeast each balloon. Make sure all the yeast gets settled to the bulb of the balloon and not caught in the neck. Be sure not spill yeast while handling the balloons.
  4. Carefully stretch the neck of the balloon to help ensure it does not rip when stretched over the opening of the test tube.
  5. Attach the neck of a balloon you prepared in step 8 to the top of Tube 2 (the room temperature test tube) making sure to not let the yeast spill into the test tube yet. Once the balloon is securely attached to the test tube lift the balloon and allow the yeast to enter the test tube. Tap the bulb of the balloon to ensure all the yeast falls into the tube.
  6. As quickly and carefully as possible remove the Tube 1 (cold) from the refrigerator and repeat steps 9 – 10 with Tube 1 using a balloon you prepared in step 8.
  7. As quickly and carefully as possible remove Tube 3 (hot) from the hot water bath and repeat steps 9 – 10 with Tube 3 using a balloon you prepared in step 8.
  8. Swirl each tube to mix, and wait 30 seconds.
  9. Wrap the string around the center of each balloon to measure the circumference. Measure the length of string with a ruler. Record your measurements in Table 2.
Table 2: Balloon Circumference vs. Temperature
Tube Temperature (°C) Balloon Circumference (Uninflated; cm) Balloon Circumference (Final; cm)
1 – (Cold)      
2 – (RT)    
3 – (Hot)    

 

 

Post-Lab Questions

1.      What reaction is being catalyzed in this experiment?

2.      What is the enzyme in this experiment? What is the substrate?

3.      What is the independent variable in this experiment? What is the dependent variable?

4.      How does the temperature affect enzyme function? Use evidence from your data to support your answer.

 

5.      Draw a graph of balloon diameter vs. temperature. What is the correlation?

6.      Is there a negative control in this experiment? If yes, identify the control. If no, suggest how you could revise the experiment to include a negative control.

 

7.      In general, how would an increase in substrate alter enzyme activity? Draw a graph to illustrate this relationship.

8.      Design an experiment to determine the optimal temperature for enzyme function, complete with controls. Where would you find the enzymes for this experiment? What substrate would you use?

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Kukai’s TenKukai’s Ten Mindsets (Japanese Buddhist Philosophy)Mindsets (Japanese Buddhist Philosophy)

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Kukai’s background and summary of Shingon Buddhism

In the Japanese philosophy and humanitarian history, there has been a series of religious people who have taken part in sharing and teaching about different humanitarian ideas and theories. One of these individuals was the Buddhist tantric monk Kukai. Kukai was born in 774 in a sea-village known as the Gbu-ga-ura a village that is not far from the Tadotsu, in Sanuki. The boy Kukai was born into a family called Saiki and his father, was Yoshimichi who was a nobleman from the ancient emperor descendent. The birth of Kukai is said to be miraculous in nature. The reason is because before conception; his mother saw a vision in her dream of a saintly monk coming to her house and monk using supernatural powers made Kukai’s mom pregnant. Again, when the boy Kukai was born, his hands were held together as if he was in prayers (Casal, Ugo Alfonso, 1959). Kukai was baptized to a name Henshg King which means “worldwide shining diamond.” Locally, during his growing up, he was also given a nickname that people used to call him “TotGmono” which means “out treasure.”

Kukai started his mission when he was young, and he could he have visions like being seated on eight-petalled lotus flower having a dialogue with the Buddha. Kukai joined the University when he was twelve years in preparations for his career in Confucianism. However, being unsatisfied with the career, Kukai privately studied Taoism because it was the job that he felt was in alignment with his reasoning. In great curiosity to learn the truth, Kukai left his city at a tender age and became a member of the Buddhist temple as an acolyte. His great passion in Buddhism made him to mode clay images of Buddha. With the images, he established a shrine from where he could do his Adoration to Buddha. This great step of faith in Buddha subjected Kukai to trials of the devil as he was continually tried. However, it is believed that Buddha protected him from the trials and tribulations and he was granted special power to overcome spiritual battles. His great commitment and adoration led to his ordination as a Buddhism priest by priest Gonso of Iwabuchidera (Casal, Ugo Alfonso, 1959).

As a priest, Kukai worked together with Ambassador Fujiwara, and together they traveled to different countries like China between the years 778-818. Upon his return to Japan, Kukai founded an independent Buddhist institution by the name Heian Buddhism which focused on teaching about the truth. In his works at the Heian Buddhism ministry, Kukai engaged in a lot of teachings which among them was the understanding of human mind where he came up with ten different mindsets development  and the different personal characteristics and behaviors associated with the different mindsets. According to Kukai’s teachings through his school of thought, the ten different mindset stages that individuals go through before they can achieve full consciousness of issues as they are naturally.

The philosophy claimed that more one moves to another step the more developed one’s brains get, and the more real the world becomes (Casal, Ugo Alfonso, 1959). Notably, each stage has its specific characteristics and behaviors that a person tends to show. Kukai’s state of mind teachings came to be taught and known a big number of people, and they are instructions that are known to have realized massive respect and value from the Japanese people. However, opposition towards Kukai’s view of mindset was highly present from some Ryuichi who was a Buddhist revisionist. Nevertheless, Kukai’s teachings came to be recognized as the Japanese Buddhist philosophy.

Apart from the teachings, priest Kukai did a lot of Buddhism services to people like communicating with different gods like the god of rain, soil, the sun, and harvest among many other gods and goddesses. Kukai constantly did a lot of supernatural powers that made the people believe in his powers and consider him a saint. The reason is that it was hard in Buddhism to communicate and have a response from the gods without being a saint. Also, for people to believe in his exorcism Buddhism, he also had to write a lot of literature and philosophies. However, this essay shall focus on Kukai’s ten mindsets successfully articulate the state of mind achieved through his school of thought.

Kukai’s ten mindsets

According to Dalisay, (2014); Priest Kukai who was born like a saint established ten different mindsets. The various mindset stages have a lot to do with humanity and a person’s state of mind. Each mindset has its specific characteristics and behaviors that a person shows when he/she belongs to that particular mindset category. Progressing to different mindset stages comes along with different features and behaviors. The following table shows all the different mindsets and their respective characteristics and behaviors.

Level of mind Characteristics and behaviors
1.      Unstable goatish mind Driven by instincts and needs for security, sex, and food
2.      Foolish abstinent mind Strives to be ethical and moral
3.      Childlike fearless mind Seeks to believe in an eternally unchanging god or salvation doctrine
4.      Mind of selfless aggregates Seeks personal liberation
5.      Mind free of karmic seeds Understands the process of conditioning, slowing destroying ignorance
6.      Compassionate Mahayana mind Has great compassion as it sees the consciousness in nature
7.      Mind awake to the unborn Realizes the void nature of both objects and mind itself
8.      Mind of the single way Realizes that all things in the world are contained in a single thought in mind
9.      Mind of ultimate no-self nature Penetrates all things but still limited by duality
10.  Secret sublime mind Breaks through duality, fully realizing the true nature

 

Stage 1: unstable, goatish mind

According to Kukai, the unstable, goatish mind is the first step of mindset and according to Kukai is the toughest step in the entire development process. The reason is that it is the stage that presents individuals with the crudest and bleak visions of life. This is in regards to personal paths to follow and the starting point. Moreover, it is the stage when a person is presented the most challenging and complicated life visions that determine who a person is. Nevertheless, it is the mindset step that has a profound honesty starting point (Dalisay, 2014). The reason is that, despite a person feeling that he/she is sophisticated; the first level of mind is always familiar and strangely real. The danger about this stage is that instincts normally drive a person. So if the instincts are misleading, it is likely for one to have an entirely wrong mindset. The reason is that, in this step, people tend to act like sheep, and hence they tend to show ignorance and stupidity.

Stage 2: foolish abstinent mind

According to Kukai, a foolish abstinent mind is the second stage of mind development. In this stage, self-awareness hastens, and it is at this stage that a person realizes that there is more to life than just following the fickleness on the untrained and ordinary mind. Kukai also stated that it is at this stage that a person starts refraining from over-indulgence as well as harmful actions hence making a person start turning away from the first level of mind and instead starts seeking for something more meaningful in life (Casal, Ugo Alfonso, 1959). This stage was also named as gudo jisai-shin, and a person starts to restrain oneself from self-instincts. The reason is that the stage of mind development makes one realise the different foolish things that one was engaging in and the desire to stop them is triggered.

Stage 3: Childlike fearless mind

This is a stage of mind development a person seeks to believe in an eternally unchanging god or salvation doctrine. During this third stage of mind development, Kukai stated that a person in this state is normally wearied with human suffering and it is for this reason that the person starts to seek the peace of dwelling in heaven with the only thing being salvation (Casal, Ugo Alfonso, 1959). It is this mind that first awakens the need for religion because one in this mindset development stage makes a person to feel like a child and hence increased need to seek comfort for the mother’s embrace. Unfortunately, a person in this stage fails to realize that the mother too is subject to death, old age, and sickness and hence the only option is to believe in a forever spiritual doctrine or unchanging god. The need is even more desperate when the mind is at this stage, and one performs the four mental concentrations together with the six practices (Matsuda, 2003). The reason is that these practices increases the disliking of the world below and instead increases the need to love and gain more knowledge about heaven (Taoism and Hinduism).

According to Kukai, in this third stage of mind development, a person starts to have amazing spiritual visions. However, these visions are only practicable in another world and not the present world that the individual is in. However; a person’s vision of what can be achieved expands to infinite even though the confidence involved makes it possible for the vision to happen in life or fail to occur in this life.

Stage 4: Mind of selfless aggregates

According to Kukai’s philosophy, the fourth stage of mindset development makes one see oneself as impermanent, but the person still thinks that the five aggregates have a real existence. These five aggregates include; perception, mental activity, consciousness, form, and will. The realization of the five aggregates, a person’s mind can think more clearly and realize the notion of a permanent ego is in the real sense unreal. The realization further makes a person see the need and benefits of the eightfold anticipation as well as the six supernatural powers (Dalisay, 2014). This means that the development of the brain makes the mind to recognize the existence of components and denies a permanent ego any form of recognition. According to Kukai, a person meets Buddhadharma for the first time in the fourth stage of mindset. It is the stage that people tend to practice all the core principles and values taught by Shakamuni. More so, the system of practice is normally characterized by the three mysteries which are; Samadhi, sila, and prajana. This stage is also the personal obstacle overcoming stage and awakens a person’s abilities to handle challenges (Matsuda, 2003).

Stage 5: Mind free of karmic seeds

The fifth stage of mind development makes a person destroy all the arrogance and ignorance which are said to have been planted by the bad karma. Just like in the fourth stage of brain development, in this stage also the brain lacks any form of compassion towards other beings (Casal, Ugo Alfonso, 1959). This means that in as much a lot of arrogance is uprooted; other mental weaknesses develop hence hindering total effectiveness on how the brain works.

Stage 6: Compassionate Mahayana mind

According to Kukai in his Japanese Buddhist Philosophy, the compassionate Mahayana mind is the level of mind where the mind becomes compassionate about all living things in the world. The mind becomes conscious of all things despite the realization that all objects are void and hence the mind tends to see all objects as real (Dalisay, 2014). Kukai stated in his philosophy that in the entire brain development process, this is the first time that great compassion takes place and one takes the action of granting aid to other people who might be in need of any help.

Stage 7: Mind awake to the unborn

According to Kukai, when the mind gets into this level of development, it is the time that an individual gets into an actual realization about the void nature of both the mind and objects. In this stage, Kukai elaborates that with the unborn, he means the false predictions that are made clear by the absolute emptiness that one feels in mind and objects (Matsuda, 2003). It is the stage of mind awakening to face the truth, and the mind in this stage becomes unproduced. More so, it is the stage that the mind comes to the realization that no dramas exist beyond the mind.

Stage 8: The mind single way

According to the philosophy, the eight step of mind development leads to a single way of thinking. Kukai defined mind single was as the way to the truth. The reason is because he believed that the brain can only perceive issues from one single way for the truth to be determined. According to Kukai, this level of mind development can understand and acknowledge that the world of delusion and enlightenment is the world of mind and matter, an all possible world, and all thoughts are contained in one individual mind (Casal, Ugo Alfonso, 1959). In this stage of mind development, the mind can come together and put more efforts and concentration on the true issues and minimizes the void felt in the seventh stage of mind development. The reason is that according to Kukai, the brain can consolidate and narrow down the wide perception to a narrow perception hence making things to have meaning and purpose,

Stage 9: Mind of ultimate no-self nature

According to Kukai’s Japanese philosophy, the mind in the ninth stage of development has gone through different stages, and the mind can push through all things around it. Nevertheless, the brain is still limited by its duality, and this is something that further hinders the brain from functioning or perceiving issues from a perfect point of view (Dalisay, 2014). The reason is that when the mindset is limited to duality, it means that the mind is being affected by two different issues hence compromising the way of perceiving problems.

Stage 10: secret sublime mind

This is the last stage of mindset, and in this stage, the mind has already gone through all the process, and the stage brings the brain to a breakthrough. The reason is that the mind has been able to follow illusions, comes to realize and overcome them among many other challenges. It is for this reason that Kukai stated that this stage of mind development is marked with a breakthrough of the duality and finally the mind can realize the true nature (Matsuda, 2003). With the duality challenge overcome, the much searched for truth is eventually realized, and the mind can settle after obtaining the truth.

Treatise on the Ten Stages of the Development of Mind

Just like everything else in the world that develops, the mind also has different stages of development. It is by going through all the stages of development that the mind is grown fully and can distinguish between good and bad or something acceptable and things that are not acceptable (Dalisay, 2014). When one is young, the mind is not fully developed, and it means that as one grows up, the mind continues to grow as well as go through the different stages. First, when the mind is young, there is a lot of confusion and the confusion is the one making one to feel the void or the emptiness. The reason is that one from the onset normally has a purpose and before the purpose is obtained it is hard for one to get the real world. Thus, one ends up going through different stages and satisfaction is realized when the brain becomes fully developed. The reason is that this is the time that from one’s thinking; things in life starts making sense and more clear. It is also the time one is sure of the feelings and it like the reality dawns hence making one have a better sense of direction and purpose.

This difference in characteristics and behaviors that come with each stage of mind development according to Kukai is the reason that makes people who belong to different mind development stages to behave differently from each other. The latter is in the sense that the actions that one engages in according to how the brain is receiving and interpreting information are different from the actions that another person who belongs to another brain development level would show (Casal, Ugo Alfonso, 1959). For example, a person whose mind is in the first stage of development would innocently act on instincts. On the other hand, a person who has already passed the first stage of brain development and now is in the third stage of mind development would not act by instincts but would apply morals and ethics earned in stage two and also act in a manner that will please the heavens. The reason is that this stage makes a person to seek salvation as the best way out.

Social cycles

From the different stages of mind development and the different actions associated with the stages, the social cycles are created. The people from the same level of mind development tends to behave in the same manner and share similar characteristics something that makes them belong to the same social cycle (Matsuda, 2003). The more reason is that they are likely to get along easily because of the similarities in behavior and characteristics as compared to dealing with an individual with an entirely different way of perceiving issues and behaving. However, the different social groups are important because when they come together, they form the society. This means that according to Kukai, the community is made of people from different levels of brain development and social cycles (Casal, Ugo Alfonso, 1959). In as much, each human being that makes up a social cycle or society has his/her characteristics, so it is the same case with the society. In most cases, the members of the community come together and form different laws and traditions that help the people despite from the fact they belong to different mind levels; they can interact and live together in harmony. In the world today, there are high-minded societies which have already gone through the maturing process like the European Union, and the low-minded societies that are still in the development process yet to mature like the warring nations.

The warring nations and countries practicing discrimination and other malpractices are classified as low-minded societies because they have not reached the maturity stage where all malpractices would lose meaning (Matsuda, 2003). This means that these societies have not been able to break through the limitedness of duality, and hence they continue being held back. After the breakthrough, the society is also able to perceive issues from a positive point of view, and the malpractices become irrelevant hence making the members of the society even without being commanded to act responsible and argue from an ethic, moral, and religious point of view.  The latter is in the sense that going through the development process helps the society to continue learning new things hence changing perception and way of thinking.

 

Work cited

https://logisticsofbelonging.com/2016/12/20/kukais-writing-praxis/

Casal, Ugo Alfonso. “The Saintly Kôbô Daishi in Popular Lore (AD 774-835).” Folklore Studies (1959): 95-144; https://web.archive.org/web/20070927213255/http://www.nanzan-u.ac.jp/SHUBUNKEN/publications/afs/pdf/a117.pdf

Dalisay Jr, Juan. “Understanding Humanity: An Analysis of Human Minds and Societal Patterns.” (2014); http://gurukul.edu/wp-content/uploads/2014/05/Understanding-Humanity-Jan-20141.pdf

Matsuda, William J. The founder reinterpreted: Kukai and Vraisemblant narrative. Diss. The University of Hawaii at Manoa, 2003; https://scholarspace.manoa.hawaii.edu/bitstream/10125/7110/2/uhm_ma_3075_r.pdf

 

 
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