Lab 6. Energy use

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Lab 6: Follow the instructions and complete the assignment below. Submit your answers through the Lab 6 Assignment on Blackboard.

Lab 6 1

Lab 6. Energy use

In this week’s lab, you will visualize metabolism in a living organism, evaluate some scientific claims regarding metabolic processes, and use your own scientific and mathematical thinking skills to personally evaluate a “popular” weight loss mantra. Part 1: Sugar Metabolism in Yeast As we are learning this week, living organisms harvest energy from “food” through cellular processes contributing to an organisms “metabolism”. These processes involve the transfer of energy from of carbon based molecules (that were originally produced during photosynthesis) to a more readily useable form (most commonly, ATP), and the carbon is released as waste. This part of the lab will demonstrate the importance of sugar for the metabolic processes of the fungal organism, Saccharomyces cerevisiae, commonly known as baker’s yeast. Yeast are a single-celled type of fungi that humans use and interact with every day. Beneficial applications of these organisms are diverse, ranging from cooking to bioremediation, while some species are also responsible for causing illnesses like athlete’s foot and ringworm. Interestingly (and fortunately for us), yeast can effectively harvest energy from sugar in the absence of oxygen, and this is precisely what we will be observing today. This process is somewhat similar to the aerobic respiration that our (human) cells undergo, in that both processes break down sugar molecules releasing carbon waste; however, no oxygen is required for fermentation. This is why yeast are sometimes called anaerobic organisms. Materials: for this activity, you will need:

 Ruler, able to measure centimeters.

 Marker/tape for labeling

 4 sandwich or quart (or larger) size sealable ziploc bags (if you are able to splurge on bags that you trust will seal, versus the cheaper ones with questionable sealing abilities, do so- it will be worth it).

 4 packages Bakers Yeast (available at any grocery store in the baking aisle)

 Table Sugar (~2 tablespoons or 2 sugar packets; sugar substitute will not work)

 Warm water (4 cups)

 1 tbs measuring spoon for measuring sugar

 1 cup measuring cup for measuring water Experimental Set Up: A. Label your Ziploc bags. Use caution; do not tear or poke a hole in the bag(s)

1: Yeast + Water 2: Yeast + Water 3: Yeast + Water + Sugar

 

 

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4: Yeast + Water + Sugar B. Add 1 package of yeast to each ziploc bag. C. Add 1 tablespoon of sugar to yeast in ziploc bags 3 and 4 only. D. Carefully add 1 cup of warm water to each ziploc bag (one at a time is fine).

Eliminate as much air from the bag as possible before sealing and mix carefully.

 Try to dissolve all the solid clumps in the water, but be gentle with the yeast, and try not to damage the bags.

 The less air you have in the bags at this point, the better your results will be. See image:

 Manage your time carefully here, you don’t want too much time to go by between activating (adding the water) the different treatments.

E. Start your timer and check the seal of each bag for good measure (leaks = messy clean up).

F. Measure the height of the Ziploc bag in centimeters (cm). To do this, hold the ruler up

vertically next to the ziploc bag, and record how “tall” the bag is; the distance between the top of the bag and the bottom (surface of the table is fine). Record your measurement in the provided Yeast Metabolism Data table (below). Also note in the table any observations you have about each treatment (color, bubbles, anything else you notice). This is your time 0 measurement.

G. Every 5 minutes for 45 minutes, gently mix solutions inside bags, and repeat measurements.

 Use caution as you approach and pass 45 minutes; the bag may burst (= messy!)

H. After the final, 30-minute, measurement, calculate the change in Ziploc bag height for each treatment by subtracting time 0 (starting) height measurement from the time 30 height measurement (of the same sample). The difference between these values gives you actual increase in height for each treatment. For example, if your time 0 height was 2cm, and your time 30 height was 10 cm, that treatment would have increased by 8 cm. Fill these values in the Change in Height row (labeled H) of the Yeast Metabolism Data table, below.

I. Determine the average change in height for each condition Yeast without or with sugar. To do this, add the values determined for the Change in Height for treatments 1 and 2, and divide this number by 2. This is your average change in height for the minus sugar condition. Repeat this step for the values obtained for treatments 3 and 4 to determine the average plus sugar height change. Fill these values in the Average Height Change row (labeled I) of the Yeast Metabolism Data table, below.

 

 

 

Lab 6: Follow the instructions and complete the assignment below. Submit your answers through the Lab 6 Assignment on Blackboard.

Lab 6 3

Note: this experiment can also be performed with balloons attached to the top of ~16oz small spout plastic bottles, as seen in the image (20oz soda or water bottles work well, after they’ve been rinsed thoroughly of course). The visual effect of this set up is much better than with Ziploc bags, but more materials are needed (4 balloons, 4 bottles, funnel for transporting ingredients to bottles, etc….) if you are able to/want to repeat the experiment this way, I highly recommend it (it’s a lot more fun). Show your friends and family your new party trick  Yeast Metabolism Data:

Expired Time

Treatment 1: Yeast+Water

Treatment 2: Yeast+Water

Treatment 3: Yeast+Water+Sugar

Treatment 4: Yeast+Water+Sugar

Height in cm

Observations Height in cm

Observations Height in cm

Observations Height in cm

Observations

0 minutes

 

 

5 minutes

 

 

 

 

10 minutes

 

 

 

 

 

15 minutes

 

 

 

 

20 minutes

 

 

 

 

 

25 minutes

 

 

 

 

30 minutes

 

 

 

 

35 minutes

 

 

 

 

 

Lab 6: Follow the instructions and complete the assignment below. Submit your answers through the Lab 6 Assignment on Blackboard.

Lab 6 4

40 minutes

 

 

 

 

45 minutes

 

 

 

 

(H) Height Change:

Treatment 1: Yeast+Water

Treatment 2: Yeast+Water

Treatment 3: Yeast+Water+Sugar

Treatment 4: Yeast+Water+Sugar

(I) Avg Height Change

Yeast (minus sugar): Yeast + Sugar:

When you are finished, answer the following questions:

1. Describe your observed results of the yeast metabolism experiment (include observations and average change for each treatment)? Were these the results you were expecting? Is your average an accurate representation of your treatment data? Why/why not? 2. Based on what you learned this week and the conditions that the yeast cells were in during this experiment, which metabolic process did the yeast undergo? What gas was produced? How do you know? Can humans carry out this process, and if so, what purpose does it serve in human cells? 3. If you were to compare the results of this experiment from several different people, assuming that they all implemented the procedure in the exact same way, would you expect each person to get exactly the same results? Why or why not? In your answer discuss possible sources of variation in this experiment. 4. The sugar that was added to the ziploc bags represents the “food” source for the yeast. Where did the energy that the yeast extracted from the sugar originally come from? Explain how you know this. 5. When you make bread, if you just mix flour, sugar and water, the dough does not rise, and the bread will be flat and hard. If you include yeast in the bread dough, then the dough rises and the bread is bigger and fluffier. Use your results from the yeast metabolism experiment to explain how the yeast helps the bread dough to rise.

6. Discuss how this yeast metabolism experiment relates to the material that we learned this week (and previous weeks!). Use specific examples.

 

 

 

Lab 6: Follow the instructions and complete the assignment below. Submit your answers through the Lab 6 Assignment on Blackboard.

Lab 6 5

Part 2: Is Lost Weight Really Lost? In the next part of this week’s lab, we will read about some research that used mathematical evidence to answer this very question, but also collected some shocking data about what the general public understands about cellular respiration and human metabolism. Below you will find links to read the original, primary, research article, and a few additional summary articles generated for the popular media based on the original. Original Research Article: http://www.bmj.com/content/bmj/349/bmj.g7257.full.pdf Take some time to review the original article first. Don’t be intimidated. For a scientific article, the language is fairly easy to understand for a non-scientist. That being said, don’t worry if you don’t understand every word. Take notes while you read and try to get the general idea of:

 What is the main point of the study? What was the study trying to find out?

 What are the main conclusions, their results/findings?

 How does the study apply to you, and what we’ve learned this week (and this semester)? After you’ve familiarized yourself with the original article, follow the other links to review the 6 summary articles. As you are reading each, take notes. Consider/evaluate each of the following.

 What is/are the main point/s of the article?

 Are the main points of the article consistent with the original research study? Is the article appropriately using information from the original study, or skewing it/making a new point?

 Do you notice anything questionable about the summary article, for example in terms of disclosures, conflicts of interest, echo chamber, etc…. remember our Lab 1materials!

 Is the source reputable? Remember our Week 1 materials! Summary Article 1: https://www.medicalnewstoday.com/articles/287046.php

Summary Article 2: https://www.scientificamerican.com/article/when-you-lose-weight-wher/

Summary Article 3: https://www.npr.org/sections/health-shots/2014/12/16/371210831/when-

you-burn-off-that-fat-where-does-it-go

Summary Article 4: https://www.sciencedaily.com/releases/2014/12/141216212047.htm

Summary Article 5: http://theconversation.com/when-we-lose-weight-where-does-it-go-91594

Summary Article 6: https://www.beachbodyondemand.com/blog/where-does-fat-go-when-you-

lose-weight

When you are finished, answer the following questions: 7. Compared to the original metabolism research article, which summary article do you find to be the most accurate? Which summary article do you find to be the least accurate? Explain your answer, providing at least 2 valid reasons why for each.

 

 

Lab 6: Follow the instructions and complete the assignment below. Submit your answers through the Lab 6 Assignment on Blackboard.

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8. Which metabolism summary article source (publisher) do you find to be the most reputable/trustworthy? Which summary article source (publisher) do you find to be the least reputable/trustworthy? Explain your answer, providing at least 2 valid reasons why for each. 9. Based on the original metabolism research study, when you lose weight, how does the matter leave your body? Identify in what all the forms that the matter is in as well as the percent of each form. With your response, state which article(s) you used to answer and why you chose to use this article as your reference. 10. It is several years in the future, and you are home visiting family for Thanksgiving. During Thanksgiving dinner, your brother is bragging about some of his recent weight loss accomplishments. He says “since he’s shed these 45lbs, that’s 20kg you know, I have all this extra energy”. He takes it even further, saying “as the weight comes off, it transforms right into energy!”. Based on the original research study, explain why this belief may seem logical, but is in fact, wrong. Include in your answer evidence from the original study that illustrates that the majority of people are incorrect in this assumption (hint: look at the figures). 11. It is several years in the future, and you are home visiting family for Thanksgiving. During Thanksgiving dinner, your brother is bragging about some of his recent weight loss accomplishments. He says “since he’s shed these 45lbs, that’s 20kg you know, I have all this extra energy”. He takes it even further, saying “as the weight comes off, it transforms right into energy!”. Based on the original research study, offer your brother a better, scientifically and quantitatively accurate, explanation to his observed phenomenon. Include numerical, quantitative data specific to your brother’s case to support your argument. For this, you must consider how much weight he has lost and based on the article, tell him exactly where that weight went. 12. After completing this week’s course material, you are talking with a friend, who is also taking this class. Your friend mentions that they find it super interesting how a simple, single celled organism, such as yeast can function so similar to us (only in certain ways of course). You ask what they mean, and they say “Well, if you think about it- in the yeast experiment we just did, they “exhaled” the carbon-based product of metabolism, just like we do!” Is your friends statement correct? Why/why not?

Part 3: Is it Really That Simple? It seems obvious, especially after viewing the summary articles in Part 2 of this lab, that we (humans) tend to have a fixation (no pun intended) on diets, fat, and weight. New diets (or lifestyle programs, if we want to use more current terms) seem to come out, one right after another, each claiming to be the next best way to provide quick, permanent, weight loss. However, the researchers behind the original article that we read in Part 2 of this lab argue that weight loss simply represents a balance between intake an output of matter; that to lose weight, you must consume fewer calories than your body uses. The question we will answer in this part of our lab is, is it really that simple? Specifically, as you calculate your own metabolic

 

 

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Lab 6 7

rate and compare it with your typical daily caloric intake, you will put the “eat less, move more” weight loss claim to the test. Before we begin, remember calories are a measurement of energy and because one calorie is a very small unit, food calories are usually measured in units of 1,000 calories, called kilocalories (abbreviated kcal). Also note, although we are limiting our range of study in this exercise to calories only, the skills and information that you will glean here are directly applicable and relevant. To determine your daily energy expenditure/consumption, or metabolic rate, you will incorporate two components: your basal metabolic rate (BMR) and additional calories expended (on top of the cell maintenance/survival processes). Let’s start with your basal metabolic rate (BMR). It is important to note that BMR varies according to the following components (and some others). This experimental procedure takes all these factors into consideration.

 Body style: a tall, thin person has a higher BMR than a short, stout person

 Age: the younger the person, the more likely it is that cell division is occurring; therefore, BMR is higher for younger persons than for older persons

 Sex: males have a higher BMR than females because males have a greater percentage of

muscle tissue
 
 A. To calculate your BMR, use the formula below that is most appropriate for your inherited

(chromosomal based) gender. To do this, you will also need the following information:

 Your weight in pounds (lbs)

 Your height in inches (in)

 Your age in years

Resources: http://www.height- converter.com/

 

Female: BMR = 655 + (4.354 X weight in lbs) + (4.569 X height in inches) – (4.7 X age in years) Male: BMR = 66 + (6.213 X weight in lbs) + (12.69 X height in inches) – (6.8 X age in years)

 

 

Lab 6: Follow the instructions and complete the assignment below. Submit your answers through the Lab 6 Assignment on Blackboard.

Lab 6 8

My BMR = _____ kcal. Next, we will use the “activity multiplier” to determine your total caloric expenditure (actual metabolic rate).

To give you an idea how daily activity impacts overall metabolic rate, review the figure. This figure shows the time required to “burn” 4 different caloric values through 3 different activity levels. B. Multiply your BMR (determined in the previous step) by the appropriate activity factor from the list (below) to determine your total caloric expenditure (actual metabolic rate).

 sedentary (desk job, with little or no exercise) = BMR X 1.2

 lightly active (light exercise, 1-3 days/week) = BMR X 1.4

 moderately active (moderate exercise, 3-5 days/week) = BMR X 1.6

 very active (intensive exercise, 6-7 days/week) = BMR X 1.7

My total caloric expenditure (BMR times the selected activity multiplier) = _____ kcal. C. Determine the number of calories for all the food you consume in a single day.

 Select a typical day when you eat your normal number of meals (with fairly average food choices) and record everything that you eat (including amounts and brand names). Consider using the food diary provided below to keep your records.

 Use the following websites to look up food caloric values. You may also find caloric info for specific foods on the food product wrapping or on manufacturers website. Note that preliminary research comparing calorie “calculators” has identified these two within the most accurate (use caution with others). o https://www.webmd.com/diet/healthtool-food-calorie-counter o https://www.myfooddiary.com/?network=g&keyword=food%20calorie%20counter

&matchtype=p&device=c&devicemodel=&adgroup=1037681552&position=1t1&cre ative=273779895484&gclid=Cj0KCQjw45_bBRD_ARIsAJ6wUXREPZgR4ZO9L5ZnsHV H3wK5iNtSeppegjULpzoEDfJVb1QzsGmlhnEaAh3gEALw_wcB

 If you have trouble finding information, use your best estimate. My total caloric intake over the recorded 24-hour period was ______ kcal.

D. Calculate your energy balance as: total kcal consumed – total kcal expended = ____ kcal

E. Return to your actual metabolic rate (energy expenditure calculations), above and

recalculate what your total calorie expenditures would be if you increased your activity

 

 

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Lab 6 9

multiplier by one level (for example, from light to moderate activity). If you were already at the highest activity level, recalculate for one level lower. My updated caloric expenditure (BMR times the updated activity multiplier) = _____ kcal.

F. Use this updated metabolic rate to recalculate an updated (hypothetical) energy balance, as

updated energy balance = total kcal consumed – updated total kcal expended = ____ kcal Our last step in this part of our lab is too evaluate if our calculations make any (real) sense. G. Click the link; visit the website

https://www.choosemyplate.gov/MyPlatePlan to get your USDA recommended calorie intake values. Click start on the “Get Your MyPlate Plan” widget.

H. When prompted, enter/fill in your

 Age

 Sex

 Pregnant/Breastfeeding Status

 Weight (pounds)

 Height (feed/inches)

 Approximate level of physical activity.

I. Click calculate food plan to review recommended the number of calories that the USDA recommends you intake in order to achieve and maintain a healthy weight. How do these numbers relate to your metabolic rate calculations?

When you are finished, answer the following questions:

13. State and discuss your actual metabolic rate determination and your USDA MyPlate calorie recommendations. Were these consistent? Were they (either or both) what you expected? Why/why not? 14. Visit https://www.choosemyplate.gov/MyPlatePlan and determine how many calories are recommended by the USDA MyPlate program in order to achieve and maintain a healthy weight for a 27-year-old, genetic female, that weighs 145lbs, is 5 feet 7 inches tall. She is not pregnant or breastfeeding, and she is exercises lightly, walking for 30 minutes 1-3 days a week.

 

 

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15. Calculate the actual metabolic rate for a 50-year-old, genetic male, that weighs 230 lbs, is 6 feet 3 inches tall. He is very active, running or playing basketball for 45 minutes to an hour 6-7 days a week. 16. Based on this figure, approximately how long would it take to burn 1,000 kcal at rest, by walking, and while jogging? Explain your answer; how did you come to this conclusion? 17. Discuss the meaning and long-term (over time) implications of the energy balance we calculated in the metabolic rate experiment. If a person’s calculated energy balance was positive every day long-term, what effect would that have on body weight over time? If a person’s calculated energy balance was negative every day long-term, what effect would that have on body weight over time? If a person’s calculated energy balance was 0 every day long-term, what effect would that have on body weight over time? Explain your answer (why this would happen) for each situation.

18. How did your calculated energy balance change when you updated (went up or down) an activity level? If your goal was to gain weight, what changes could you make in your daily diet to improve your energy balance situation? If your goal was to lose weight, what changes could you make in your daily diet to improve your energy balance situation? 19. Based on calorie considerations alone, which dieting strategy should be more effective for weight loss: a low carb diet or a low fat diet (recall: Carbohydrates and proteins each contain 4 kcal/g and Fats contain 9 kcal/g)? Explain your answer. 20. Based on your metabolic rate data and calculations, explain why athletes often gain weight when they retire from sports.

 
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Writing Analysis Biology Elephant Population In Africa

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There is a decline in the elephant population in a specific area in Africa. You are an activist trying increase the elephant population. You are required to present a proposed solution to this problem. Create a proposal to solve this elephant problem. Please include the following:

Background Information. (Describe the main biome where elephants are located; describe an ecosystem, and describe 4 other types of biomes)

Name and discuss how elephants are threatened in Africa. (Research)

Discuss how biodiversity can affect the increase and decline of the elephant population.

Differentiate between the types of population growth models that can increase or decrease the elephant population.

Discuss how community populations relate to your proposal.

Length 1000 Words

Font12 point , Calibri Font, no more than 1″ margins

Format APA

There is a decline in the elephant population in a specific area in Africa. You are an activist trying increase the elephant population. You are required to present a proposed solution to this problem. Create a proposal to solve this elephant problem. Please include the following:

Background Information. (Describe the main biome where elephants are located; describe an ecosystem, and describe 4 other types of biomes)

Name and discuss how elephants are threatened in Africa. (Research)

Discuss how biodiversity can affect the increase and decline of the elephant population.

Differentiate between the types of population growth models that can increase or decrease the elephant population.

Discuss how community populations relate to your proposal.

Length 1000 Words

Font12 point , Calibri Font, no more than 1″ margins

Format APA

 
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Genetics

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Genetics Practice Problems #1-

1. In horses, hair color is controlled by a gene A. The dominant allele A produces black hair; the recessive a gives brown. A second gene B controls hair length, with long hair (B) dominant to short hair (b). A homozygous black, short-haired female horse is mated to a homozygous brown, long-haired male. What will be the genotypes and phenotypes of the F1 generation? Of the F2 generation?

2. In shorthorn cattle, the gene for coat color is an example of incomplete dominance (a heterozygous animal is a color intermediate between the two pure gene colors). A breeder of these cattle has cows that are white and a bull that is roan (a mixture of red and white). What fraction of the calves produced in his herd will be white? Roan? Red?

3. Starting with a roan bull and white cows, as in problem 2, could the breeder eventually establish a true-breeding red herd? How?

4. In humans, normal skin pigmentation is due to a dominant gene C; its recessive c results in albinism. A normal man marries an albino woman. Their first child is an albino. What are the genotypes of these three people? If the couple have more children, what are they likely to be?

5. Watermelons may be either plain green or striped in color, the fruit may be either long or round in shape. A watermelon plant of a homozygous long, green variety was crossed with one of a homozygous round striped variety. The F1 plants all bore round, green melons. (a) How many genes are involved in this cross? (b) Which alleles are dominant? (c) If two plants are crossed, what fraction of the F2 generation would be round and striped?

6. What results would you expect if one of the F1 plants in problem 5 was crossed with a plant of a long, striped variety?

7. The following problems all concern tomatoes. Red tomato fruit color is dominant to yellow, and round tomato shape is dominant to oval (pear-shaped). Each characteristic is controlled by a single gene, and the genes are on separate chromosomes. Phenotypes of parent and offspring plants are given in succeeding questions. For each question, determine the genotypes of the parents.

a. Red/round crossed with yellow/oval produced one-half red/round and one-half red/oval.

b. Red/oval crossed with red/oval produced three-fourths red/oval and one-fourth yellow/oval.

c. Yellow/round crossed with red/oval produced all red/round.

d. Red/round crossed with red/oval produced three-eighths red/round, three-eighths red/oval, one-eighth yellow/round, and one-eight yellow/oval.

e. Red/round crossed with red/round produced 56 red/round, 18 red/oval, 19 yellow/round, and 6 yellow/oval.

 
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BIOLOGY LAB REPORT

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hello

i have a biology experiment attached can write for me the introduction, Materials &

Methods and the conclusion

 

Introduction: Defines the subject, the purpose of doing the experiment; includes some background information (usually can be gathered from the experiment’s intro); reader should be able to understand why the experiment was done, what you already know about the experiment, and the specific purpose of the experiment (usually stated in the title). Do not plagiarize your introduction or you will lose points!!! Also, citing is more than copying the work and listing the source! You must cite within the text as well. Your entire introduction cannot be cited!!! You will lose points for intros that don’t have your original thoughts.

 

Materials & Methods: List the main materials used and write in your own words how the experiment is conducted; reader should understand what materials were used, how the materials were used, and what was actually done during the experiment. If the protocol was altered some way in class, you must make the changes in your lab report. Make sure you underline/italicize all microorganisms!!! Methods should be listed step by step vs. paragraph format.

Conclusion:  answer questions at the end of the experiment, then discuss/conclude what your results mean in terms of the experiment and whether you were successful or not. The conclusion should not just state the experiment was successful, but definitive results should be explained here! Your conclusion should explain how the microbes used in the experiment were affected. For example, if the experiment is on gram staining, then your conclusion should state the gram reaction for each organism used. Ex: E.coli was determined to be gram negative, because it did not retain the purple of the primary stain, but retained the pink of the counter stain.

EXPERIMENT

Microscopic Examination

Of Stained Cell Preparations

Learning Objectives

Once you have completed this experiment, you should be familiar with the

1. Theoretical principles of brightfleld microscopy.

2. Component parts of the compound microscope.

3. Use and care of the compound microscope.

4. Practical use of the compound microscope for visualization of cellular morphology from stained slide preparations.

Principle

Microbiology is a science that studies living or​ganisms that are too small to be seen with the naked eye. Needless to say, such a study must in​volve the use of a good compound microscope. Although there are many types and variations, they all fundamentally consist of a two-lens sys​tem, a variable but controllable light source, and mechanical adjustable parts for determining focal length between the lenses and specimen (Figure 4.1).

Components of the Microscope

Stage A fixed platform with an opening in the center allows the passage of light from an illumi​nating source below to the lens system above the stage. This platform provides a surface for the placement of a slide with its specimen over the central opening. In addition to the fixed stage, most microscopes have a mechanical stage that can be moved vertically or horizontally by means of adjustment controls. Less sophisticated micro​scopes have clips on the fixed stage, and the slide must be positioned manually over the central opening.

Illumination The light source is positioned in the base of the instrument. Some microscopes are equipped with a built-in light source to provide direct illumination. Others are provided with a

 

reversible mirror that has one side flat and the other concave. An external light source, such as a lamp, is placed in front of the mirror to direct the light upward into the lens system. The flat side of the mirror is used for artificial light, and the con​cave side for sunlight.

Abbe Condenser This component is found di​rectly under the stage and contains two sets of lenses that collect and concentrate light as it passes upward from the light source into the lens systems. The condenser is equipped with an iris diaphragm, a shutter controlled by a lever that is used to regulate the amount of light entering the lens system.

Body Tube Above the stage and attached to the arm of the microscope is the body tube. This structure houses the lens system that magnifies the specimen. The upper end of the tube contains the ocular or eyepiece lens. The lower portion consists of a movable nosepiece containing the objective lenses. Rotation of the nosepiece po​sitions objectives above the stage opening. The body tube may be raised or lowered with the aid of coarse-adjustment and flue-adjustment knobs that are located above or below the stage-depending on the type and make of the instrument.

Theoretical Principles of Microscopy

To use the microscope efficiently and with mini​mal frustration, you should understand the basic principles of microscopy: magnification, resolu​tion, numerical aperture, illumination, and focusing.

Magnification Enlargement, or magnification, of a specimen is the function of a two-lens system; the ocular lens is found in the eyepiece, and the objective lens is situated in a revolving nose​piece. These lenses are separated by the body tube. The objective lens is nearer the specimen and magnifies it, producing the real image that is projected up into the focal plane and then magni​fied by the ocular lens to produce the final image.

image1.png

Ocular

(eyepiece)

lenses

Body tube lock screw

Head-

Arm

Mechanical stage

Coarse-

adjustment knob

Fine-

adjustment knob

Objective lenses

Diaphragm lever Condenser

Iris diaphragm lever

Substage light

Condenser adjustment knob

Light control

Base

Power switch

Figure 4.1 Leica ATC 2000 compound microscope

The most commonly used microscopes are equipped with a revolving nosepiece containing four objective lenses, each possessing a different degree of magnification. When these are com​bined with the magnification of the ocular lens, the total or overall linear magnification of the specimen is obtained. This is shown in Table 4.1.

Resolving Power or Resolution Although mag​nification is important, you must be aware that unlimited enlargement is not possible by merely increasing the magnifying power of the lenses or by using additional lenses, because lenses are limited by a property called resolving power. By definition, resolving power is how far apart two adjacent objects must be before a given lens shows them as discrete entities. When a lens

cannot discriminate, that is, when the two ob​jects appear as one, it has lost resolution. In​creased magnification will not rectify the loss and will, in fact, blur the object. The resolving power of a lens is dependent on the wavelength of light used and the numerical aperture, which is a characteristic of each lens and imprinted on each objective. The numerical aperture is defined as a function of the diameter of the objective lens in relation to its focal length. It is doubled by use of the substage condenser, which illuminates the ob​ject with rays of light that pass through the speci​men obliquely as well as directly. Thus, resolving power is expressed mathematically as follows:

resolving power =

wavelength of light

2 x numerical aperture

Overall Linear Magnification

MAGNIFICATION

TOTAL MAGNIFICATION

OCULAR LENS

OBJECTIVE LENSES

OBJECTIVE MULTIPLIED BY OCULAR

40x 100x 400x 1000X

Scanning 4x Low-power 10x High-power 40X Oil-immersion 100x

10x 10X 10X 10X

Based on this formula, the shorter the wave​length, the greater the resolving power of the lens. Thus, for the same numerical aperture, short wavelengths of the electromagnetic spec​trum are better suited for higher resolution than are longer wavelengths.

However, as with magnification, resolving power also has limits. You might rationalize that merely decreasing the wavelength will automati​cally increase the resolving power of a lens. Such is not the case, because the visible portion of the electromagnetic spectrum is very narrow and borders on the very short wavelengths found in the ultraviolet portion of the spectrum.

The relationship between wavelength and numerical aperture is valid only for increased re​solving power when light rays are parallel. There​fore, the resolving power is also dependent on another factor, the refractive index. This is the bending power of light passing through air from the glass slide to the objective lens. The refrac​tive index of air is lower than that of glass; as light rays pass from the glass slide into the air, they are bent or refracted so that they do not pass into the objective lens. This would cause a loss of light, which would reduce the numerical aperture and diminish the resolving power of the objective lens. Loss of refracted light can be compensated for by interposing mineral oil, which has the same refractive index as glass, be​tween the slide and the objective lens. In this way, decreased light refraction occurs and more light rays enter directly into the objective lens, producing a vivid image with high resolution (Figure 4.2).

Illumination Effective illumination is required for efficient magnification and resolving power. Since the intensity of daylight is an uncontrolled variable, artificial light from a tungsten lamp is

the most commonly used light source in mi​croscopy. The light is passed through the con​denser located beneath the stage. The condenser contains two lenses that are necessary to produce a maximum numerical aperture. The height of the condenser can be adjusted with the condenser knob. Always keep the condenser close to the stage, especially when using the oil-immersion objective.

image2.png

Objective lens

Refracted (lost)

Slide —[

Condenser

Light source

Figure 4.2 Refractive index in air and in mineral oil

Between the light source and the condenser is the iris diaphragm, which can be opened and closed by means of a lever, thereby regulating the

amount of light entering the condenser. Exces-

sive illumination may actually obscure the speci​men because of lack of contrast. The amount of light entering the microscope differs with each objective lens used. A rule of thumb is that as the magnification of the lens increases, the distance between the objective lens and slide, called working distance, decreases, whereas the nu​merical aperture of the objective lens increases

(Figure 4.3).

Use and Care of the Microscope

You will be responsible for the proper care and use of microscopes. Since microscopes are ex​pensive, you must observe the following regula​tions and procedures.

The instruments are housed in special cabi​nets and must be moved by users to their laboratory benches. The correct and only acceptable way to do this is to grip the microscope arm firmly with the right hand and the base with the left hand, and lift the instrument from the cabinet shelf. Carry it close to the body and gently place it on the laboratory bench. This will prevent colli​sion with furniture or coworkers and will protect the instrument against damage.

Once the microscope is placed on the labora​tory bench, observe the following rules:

1. Remove all unnecessary materials (such as books, papers, purses, and hats) from the lab​oratory bench.

2. Uncoil the microscope’s electric cord and plug it into an electrical outlet.

3. Clean all lens systems; the smallest bit of dust, oil, lint, or eyelash will decrease the efficiency of the microscope. The ocular, scanning, low-power, and high-power lenses may be cleaned by wiping several times with acceptable lens tissue. Never use paper toweling or cloth on a lens surface. If the oil-immersion lens is gummy or tacky, a piece of lens paper moistened with xylol is used to wipe it clean. The xylol is immedi​ately removed with a tissue moistened with 95% alcohol, and the lens is wiped dry with lens paper. Note: This xylol cleansing pro​cedure should be performed only by the in​structor and only if necessary; consistent use of xylol may loosen the lens.

The following routine procedures must be followed to ensure correct and efficient use of the microscope.

1. Place the microscope slide with the specimen within the stage clips on the fixed stage. Move the slide to center the specimen over the opening in the stage directly over the light source.

2. Raise the microscope stage up as far as it will go. Rotate the scanning lens or low-power lens into position. Lower the body tube with the coarse-adjustment knob to its lowest po​sition. Note: Never lower the body tube while looking through the ocular lens.

3. While looking through the ocular lens, use the fine-adjustment knob, rotating it back and forth slightly, to bring the specimen into sharp focus.

4. Adjust the substage condenser to achieve op​timal focus.

5. Routinely adjust the light source by means of the light-source transformer setting, and/or the iris diaphragm, for optimum illumination for each new slide and for each change in magnification.

6. Most microscopes are parfocal, which means that when one lens is in focus, other lenses will also have the same focal length and can be rotated into position without fur​ther major adjustment. In practice, however, usually a half-turn of the fine-adjustment knob in either direction is necessary for sharp focus.

7. Once you have brought the specimen into sharp focus with a low-powered lens, prepa​ration may be made for visualizing the speci​men under oil immersion. Place a drop of oil on the slide directly over the area to be viewed. Rotate the nosepiece until the oil-immersion objective locks into position. Note: Care should be taken not to allow the high-power objective to touch the drop of oil. The slide is observed from the side as the objective is ro​tated slowly into position. This will ensure that the objective will be properly immersed in the oil. The fine-adjustment knob is read​justed to bring the image into sharp focus.

8. During microscopic examination of microbial organisms, it is always necessary to observe several areas of the preparation. This is ac​complished by scanning the slide without the application of additional immersion oil. Note: This will require continuous, very fine ad​justments by the slow, back-and-forth rota​tion of the fine-adjustment knob only.

Diaphragm Opening

Objective

Working Distance

image3.png

image4.png

image5.png

Scanning 4x

4x

Reduced

9-10 mm

Slide

image6.png

image7.png

image8.png

Low power 10x

10x

Not fully opened

5-8 mm

Slide

image9.png

image10.png

image11.png

High power 40x

40x

Not fully

opened

0.5-0.7 mm

J

Slide

image12.png

image13.png

image14.png

Oil immersion 100x

100x

Fully opened

0.13-0.18 mm

Slide

Figure 4.3 Relationship between working distance, objective, and

Diaphragm opening

On completion of the laboratory exercise, re​turn the microscope to its cabinet in its original condition. The following steps are recommended:

1. Clean all lenses with dry, clean lens paper. Note: Use xylol to remove oil from the stage only.

2. Place the low-power objective in position and lower the body tube completely.

3. Center the mechanical stage.

4. Coil the electric cord around the body tube and the stage.

5. Carry the microscope to its position in its cabinet in the manner previously described.

AT THE BENCH

Materials

Slides

Commercially prepared slides of Staphylococcus aureus, Bacillus subtilis, Aquaspirillum iter-sonii, Saccharomyces cerevisiae, and a human blood smear.

Equipment

Compound microscope, lens paper, and immer​sion oil.

Procedure

1. Review the parts of the microscope, making sure you know the names and understand the function of each of these components.

2. Review instructions for the use of the micro​scope, giving special attention to the use of the oil-immersion objective.

3. Examine the prepared slides, noting the shapes and the relative sizes of the cells un​der the high-power (also called high-dry, be​cause it is the highest power that does not use oil) and oil-immersion objectives.

4. Record your observations in the Lab Report.

Observations and Results

Draw several cells from a typical microscopic field as viewed under each mag​nification, and give the total magnification for each objective.

Lab Report

High Power

Oil Immersion

S. aureus

image15.png

image16.png

Magnification

B. subtilis

image17.png

image18.png

Magnification

A. itersonii

image19.png

image20.png

Magnification

S. cerevisiae

image21.png

image22.png

Magnification

Blood smear

image23.png

image24.png

Magnification

Review Questions

1. Explain why the body tube of the microscope should not be lowered while you are looking through the ocular lens.

2. For what purpose would you adjust each of the following microscope components during a microscopy exercise?

a. Iris diaphragm:

b. Coarse-adjustment knob:

c. Fine-adjustment knob:

d. Condenser:

e. Mechanical stage control:

3. As a beginning student in the microbiology laboratory, you experience some difficulties in using the oil-immersion lens. Describe the steps you would take to correct the following problems:

a. Inability to bring the specimen into sharp focus.

b. Insufficient light while viewing the specimen.

c. Artifacts in the microscopic field.

 
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