Case study 1

Student Name

Professor Name

University

Date

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Case study 2

Answer:

Case Study/Application Assignment Number One Spring 2014

General Instructions:

The Case Study beginning on page 2 will involve concepts you learn in Chapters 3, 4, 7, 2

and 8. Use those chapters and related references outside your text to thoroughly and thoughtfully

complete each question you are asked in the case. Read each section carefully. A grading rubric

for this case will be posted by Feb 10th in Shared Files.

Online Students:

Print a copy of the case study first to use as a reference and to give yourself a “full-picture” of

where you’re headed as you work your way through it. As you work your way through this Case,

please type all of your answers directly into the document in the spaces provided. Follow the

required word count. For the poster graphic, mark your suggested changes right on the page. My

expectation is that each student completes their own work. You are welcome to bounce ideas off

one another and work on solving ideas together but the writing must be yours and yours alone.

For online students in particular, all submitted writing goes automatically through “turnitin.com”

and is scored for originality (including if the work is original to other students in the class).

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Case study 3

ELVIS Meltdown!

Microbiology Concepts of Culture, Growth and Metabolism

Adapted from the Department of Cell Biology and Molecular Genetics

University of Maryland - College Park, MD

Part One: Return to Sender

Fresh out of college, with your degree in microbiology, you have landed your first “real job”

as a scientist with DuPont, a company that specializes in the development and production of

polyurethane derivatives (specialized plastics). You (and your boss) are not quite sure why

DuPont has a microbiologist on staff, but you are both about to find out why. Your boss has

called you into his office. “Read this article!” he says, pushing the front page of a national

newspaper across his desk to you.

Stifling your initial reaction to the article, you manage to mumble, “What a tragedy.”

“Yes. Yes. And this could take an ugly turn for DuPont!” your boss says. “I’m not sure what

caused this mess, but I do know a couple of things that didn’t make it into that news article: (1)

the only plastics showing damage in the ELV were polyurethanes; and (2) our company provided

those polyurethane products to NASA at a cost of $15,000,000. We’re in big trouble if we can’t

prove that something from that planet is responsible for destroying ELVIS!”

He continues, “The polyurethane we provided was first-rate. We didn’t cut any corners. Products

from the same batches of polyurethane have been sent into outer space before, and returned with

no damage. There must be some explanation other than our incompetence. This is where you

come in. I need you to find that explanation!”

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“Why me?” you ask.

“Because of the stink!” your boss exclaims.

“What do mean by “the stink”? You ask.

Your boss replies. “Some of the scientists present at the ELVIS disaster said the smell

reminded them of an old fermented or an autoclave. Those are microbiology terms, aren’t they?

Those comments tell me that this whole stinking mess might be caused by microorganisms – you

know, bacteria, fungi, viruses, germs…something like that. Get right to work on this! You and I

will have to work closely on this, you know. I’ll handle all the communications with the press,

and you handle the science. Just make sure that you explain everything clearly to me so that I can

speak about it to the press without making a fool of myself and DuPont!”

Lets Get Started: As a microbiologist for DuPunt you must determine what has happened to the

polyurethane. Here is your hypothesis:

The degradation of polyurethane products was caused by a microorganism or

microorganisms present in the soil samples collected by ELVIS.

Questions:

1. Using the light microscope, you examine all the soil samples and the “goo” from the

degraded polyurethane. Will this approach allow you to observe all the potential microorganisms

in the sample? Why or why not? If not, what are the limitations of this approach? (Minimum

300-word response) There are limitations with using a light microscope. In order for a

microscope to work to the best of its ability it needs to have adequate magnification, resolution

and clarity of the image. The light microscope provides a magnification from 40x to 2000x.

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Magnification is how much bigger a sample appears under microscope than in real life. The

resolution power of the microscope defines the object. It helps distinguish between two points on

an image. The resolution of an image is limited by wavelength because when objects in the

specimen are smaller they don’t interrupt the waves so they go undetected. You can magnify the

image all you want, but if you don’t have the resolution you can’t see detail and the image will

just appear blurry. Next comes the type of light microscope used.

They are described by the nature of their field. There is the bright-field in which it forms its

image as light is transmitted through the specimen. The specimen will appear darker than the

surrounding illuminated field. A bright-field microscope can be changed to a dark-field by

adding a disc to stop all the light from entering the objective lens. This will illuminated

specimens surrounded by a dark field. The next field microscope is phase-contrast microscope.

Phase-contrast microscope contains devices that change the light to make subtle changes as its

passes through the specimen. This is used to detected internal cellular detail. The final light

microscope to mention is the differential interference. The DIC provides a detailed view of

unstained, live specimens. There are bacteria that are too small to be detected and those require

electron microscopy. There are bacteria that are distorted and not be stained by normal methods.

All these factors need to play into finding the right approach to find the microorganism that

caused the degradation of polyurethane.

2. You next use phase contrast microscopy to observe a wet mount of a soil sample (see

picture below) and a “goo” sample (see picture below) from the ELVIS. Based only on the

pictures below, in what ways are the samples you see in both the soil and “goo” similar to

microbes previously characterized on Earth? In what ways are they different? (Minimum 350-

word response) Let’s start by describing the make-up of soil. Soil is made up of minerals,

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organic matter, water, and air and soil organisms. Soil is a big ecosystem that supports complex

relationships between geologic, chemical and biological factors. Organic matter, in soil, is plant

and animal residue at various stages of decomposition, cells and tissues of soil organisms, and

other substances that are synthesized by microorganisms. There are many organisms that find

their home in the soil such as fungi and bacteria. When comparing the slides there are many

things to examine. Microbial sizes, shapes and arrangements are all things that help classify the

sample out into categories as microbes-bacteria, archaic, fungi, algae, protozoa, and viruses

come in all different sizes and shapes. When we are comparing the slides we have to remember

that the images are under a light microscope. Viruses are the smallest of microbes and not

usually detectable with a light microscope. Archie are single-celled simple organisms.

They contain many bacterial characteristics but their ribosomal RNA contains unique

signature sequences. Fungi are very popular in soil. They are single celled or complex

multicellular. Their role is beneficial in cycling carbon and other elements. Algae can also be

single celled or multicellular. They are the eukaryotic protests that photosynthesize with

chlorophyll. Protozoa are unicellular eukaryotic protests. This now leads us to bacteria. Bacteria

have no nucleus. Their genetic information is contained in a single loop of DNA. Bacteria are

classified into five groups based on shape. Spherical (cocci), Rod (bacilli), spiral (spirally),

comma (Vibrio) or corkscrews (spirochetes) are all shapes of bacteria. Bacteria can be then in

single, pairs, chains or clusters. Let’s go back to the samples, the images of the Earth samples

have flagella where the goo does not. The goo sample is actually much simpler as it has much

less organelles. Distant microbes were less complex and did not have flagella to move to new

locations and find nutritional sources. One thing to consider is the complaint of smell when they

returned. This would imply that the microorganisms underwent fermentation versus respiration.

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Figure 1 – Soil Sample Figure 2 – Goo Sample

Your boss has done a little reading about microorganisms but he finds it all pretty

complicated. “It’s like a foreign language!” he complains. “I have to face the press to explain our

idea that microbes might be responsible for all the damage to ELVIS. Clearly, I’m going to need

some visual aids or the press won’t have a clue as to what I am saying. I think I’ll need to explain

what a bacterium looks like and how it might be possible that they can degrade polyurethane. I

really can’t waste time showing them all the potential types of bacteria (archea, mycoplasma,

etc), so I think I’ll just show them what a gram negative bacterium looks like as an example. I’ve

already put together a poster with a diagram of a typical gram-negative bacterium. Will you take

a look at it, to make sure I haven’t made any mistakes? I labeled all the features and also

indicated the major biochemical composition of each feature. I’m sure this figure will wind up in

lots of newspapers and magazines, so it really needs to be scientifically correct. We wouldn’t

want to make DuPunt look stupid, would we? Just proofread it and make any necessary

corrections, okay? Go ahead and mark up the poster as needed.”(see Figure 3 next page)

Make any changes you wish to make to the actual graphic. Highlight those changes.

Part Two – Suspicious Minds

Your direct microscopic observation of microorganisms in the soil samples has sparked your

boss’s interest. He is eager to determine what type of microorganism(s) is/are present. Though he

only presented a model of a bacterium for his public press conference, there is still not enough

evidence to determine if the microorganism is a prokaryote, eukaryote, or even some type of

organism never seen before. He asks you to take a sample of the soil to the electron microscope

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for further analysis. To be the most objective, he asks you to run a sample through the electron

microscope and decides to do the same himself for comparative analysis.

You are just as interested in the nature of the microorganism(s) as your boss, but instead of

directly analyzing the soil, you use both the soil sample and the “goo” sample and, using pure

culture techniques, isolate a similar organism from each for testing.

Question:

1. How would you go about isolating a pure and matched culture from each of your samples?

How would you know that you have isolated the same organism from both the soil and the

“goo” sample? (Be specific) (minimum 450-word response) First thing first…definition of

culture is to cultivate microorganisms. It is a process to take seeds (microbes) and plant

them into an environment (medium) to let them thrive and grow. To do so one would

introduce a sample into a nutrient also known as medium. This provides an environment in

which the sample can grow or multiply. A medium is the foundation of culturing. As all

microbes are different some require a few inorganic compounds for growth versus others

who require a complex list of specific inorganic and organic compounds. Media (plural for

medium) come in over 500 different types. They can be contained in test tubes, flasks, or

Petri dishes. There are three main categories of media. Those categories are based on their

properties: physical state, chemical composition and functional type. Physical state of

media is the consistency of the medium.

It can be liquid, semisolid, solid that converts to a liquid or solid that can’t be liquefied.

The chemical composition is either synthetic or chemically defined or non-synthetic with

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is complex. The functional purpose is the purpose of the medium such as general purpose,

enriched, selective, differential, anaerobic growth, specimen transport, assay or

enumeration. Some media can serve more than one function. Isolation techniques are used

to help separate microbes and spread them apart to create isolated colonies that contain a

single microbe. This method helps define making a pure culture. Proper isolation is

required that a small number of cells be inoculated into a large area of the medium. The

tools needed to perform isolation would be a medium as described earlier, Petri dish or

place media is controlled and inoculating tools. Inoculating tools can be loops, needles,

pipettes and swabs. For a controlled sample, sterile technique needs to be used. Sterile

technique is when you start with a sterile medium, sterile inoculating tools and nothing

non-sterile contaminates the specimen. Things that would be considered non-sterile would

be room air, fingers non-sterile materials.

So now that we have a background on what we are using let’s carry on to that actual

process. We have collected our sample of the goo and of the soil. We placed those samples

into a container of medium that will support the growth. After placing the sample in the media

we place it into an incubator. An incubator is a controlled environment that helps promote

growth. After optimal growth in the medium we use isolation techniques to spread the colonies

apart to grow colonies that contain a single microbe. Those isolation techniques could be

techniques such as the streak plate method or pour plate (loop dilution) method. There is also the

spread plate technique. All these methods use various ways to spread the surface promoting

individual colonies. Once our subculture is separated into a pure culture of the bacteria we

observe for the macroscopic appearance and then under the microscope for basic details such as

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cell type and shape. Those basic details should be seen in the soil and goo samples. This is

reassurance that you don’t have a contaminated culture.

Later, you and your boss compare samples (see table below).

Test Boss’s sample Your sample

80 S ribosomes + -

70S ribosomes+ +

Circular DNA + +

Linear DNA + -

RNA + +

Phospholipid membrane + +

Peptidoglycan + +

Lipotechoic acid + -

Flagellar basal body proteins + +

Cytoskeleton proteins + +

Mitochondria + -

Histone proteins + -

Nuclear pore proteins + -

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“I’m not sure what’s wrong with your sample, but my results prove that we are dealing with a

new kind of life form here…I’m calling it the “preukaryote” because it has components

characteristic of both prokaryotes and eukaryotes. It’s time for a press conference!” boasts your

boss.

Question:

2. If your goal is to isolate this “new microorganism”, which results are more informative:

yours or your boss’s? Why? What do your results indicate about the nature of this

microbe? Does its structure closely resemble that of a prokaryote or a eukaryote? Do you

agree with your boss’s conclusion that this new microorganism is a prokaryotic-eukaryotic

hybrid? Why or why not? (Minimum 450-word response).

As you notice the results vary greatly. This goes back to the pure culture or axenic method

that I used. My boss took the sample and viewed it under an electronic microscope and found

that all the elements were viewed. This is due to the fact that soil contains many organisms and

since they weren’t separated out, he was able to view all sorts of things. He was viewing a

mixture and nothing can be excluded or included as it isn’t about a single microbe. I had gone

through the work of isolating the soil and the goo to find the microorganism that was found and

able to grow in both samples. My boss states that the new organism is a “preukaryote” due to the

fact that the components characterize both prokaryotes and eukaryotes. Let’s work this through.

Due to the fact that my boss’s sample was in my eyes not pure, I am going to look at my positive

viewings to see if his speculation is true. The first thing is the 70S ribosome. Ribosome is made

up of RNA and protein and is the site of protein synthesis. 70S ribosome means that it is heavier

more compact structure that sediment faster. Prokaryotic ribosome is 70S as it actually composed

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of two smaller subunits. The next positive finding is circular DNA. Most bacteria’s hereditary

material is in the form of circular DNA which is designated as the bacterial chromosome.

Moving on to RNA, this is found in both prokaryotes and eukaryotes. It is a single strand

containing ribose sugar instead of deoxyribose and uracil instead of thymine. Phospholipids are

fatty acids plus glycerol plus phosphate that are found in membranes; both prokaryotic and

eukaryotic cells have a cell membrane. Although those cell membranes are made up differently

and have different thicknesses but are present in both. Peptidoglycan is a special class of

compounds in which glycans are lined to peptide fragments, also known as a short chain of

amino acids. Eukaryotic cells do not contain peptidoglycan as of this point we could say that my

boss’s theory has proven false but let’s carry on and see what the last findings show.

I couldn’t help but notice that within my findings I didn’t have a positive lipotechoic acid.

Lipotechoic acid is found on the cell wall of gram-positive cells. Gram-negative cells don’t have

this acid. Flagellar basal body proteins are that provide motility or self-propulsion. They are

found in some not all prokaryotic cells but also some eukaryotic cells as well. They help move

the bacteria and this mobility helps growth to spread. The last positive finding is that of

cytoskeleton proteins. Cytoskeleton was known to the eukaryotic cells as an intracellular

framework of fibers and tubules that bind and support the cell. Prokaryotic cells have more

recently been found to house a cytoskeleton into the fine structure of certain rod and spiral

shaped bacteria. Upon reviewing my findings compared to my boss’s I would still have to say

that I disagree with his theory as the findings are more closely related to a prokaryotic cell.

Later on, as you are getting ready to head home, you hear your boss bellow, “What the H-E

double hockey sticks are going on here!” You ask him what happened. “This morning I put a few

thousand cells from your pure culture onto two slides in water. Because I had to leave for the

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press conference and didn’t want them to dry out, I sealed the cover slips. When I left, they were

clearly distributed evenly on the slides. Now look! On this slide I used a rubber gasket to make

the seal. On this slide, I used a Lycra gasket. Now look at the cell distribution! On the rubber-

sealed slide, the cells are evenly distributed, but on the Lycra-sealed slide all the cells are

congregated around the edge of the cover slip. Look…they are all over the edges; none are left in

the middle part of the slide”.

Question:

3. Come up with at least two possible explanations for the “amazing” redistribution of the

new microorganism on the Lycra-sealed slide. (Minimum 400-word response)

There are a couple possibilities on the explanation of why the redistribution of the unknown

bacteria on the Lycra-sealed slide. Two of those explanations would be motility and taxis.

Motility means the movement of the bacteria by the flagella that are driven by a proton gradient.

Taxis mean a motile response to an environmental stimulus. Bacteria under the microscope

often have appendages noted from their surface. Appendages are divided into two groups,

motility and attachments/channels. If the pure culture was put onto two slides of water and

evenly distributed and sealed and movement happened, motility has occurred. There can be

movement of some cells without it actually making progress. That is why electronic microscopic

examination and sometimes staining of the specimen is necessary to see if the species is motile.

Flagella provide the power of motility or propulsion within bacteria cells. Flagellum is an

appendage that allows a cell to swim freely though an aqueous habitat. There are three parts to

the flagellum; the filament, hook, and basal body. In prokaryotic cells the hook and filament are

free to rotate 360 degrees.

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This is a differential when comparing eukaryotic cells and prokaryotic cells. The flagella are

arranged by two patterns; the polar arrangement and the peritrichous arrangement. In a polar

arrangement the flagella are attached at one or both ends of the cell. In a pertrichous arrangement

the flagella are randomly placed over the cell surface. There is growth spread though out the

entire medium that is an indication of motility as it is widespread and didn’t just stay in the same

spot. The flagella can guide the bacteria in the wanted direction because of the system for

detecting chemicals is linked to the drive of the flagellum. How this works is on the cell surface

molecules that bind specifically with other molecules, receptors, bind specific molecules from

the environment. The attachment of sufficient numbers of these molecules transmits signals to

the flagellum which in return set the flagellum into a rotary motion. As the flagellum rotate

counterclockwise the cell itself moves in a linear motion called a run. Runs are interrupted by

tumbles. Tumbles are when the flagellum reverses direction and rotates clockwise causing a

tumble. When placed in a medium a cell moves randomly in short runs and tumbles until it gets

closer to an attractant and then it spends more time in runs.

So what causes a cell to run and tumble toward or away from something? That would be

called chemo taxis. Chemo taxis is the tendency of organisms to move in response to a chemical

gradient, either towards or away from a chemical stimulus. The chemical stimulus is usually

known as a nutrient or away from a potentially harmful compound. There are other taxis such as

photo taxis which the movement is in response to light rather than chemicals.

Part Three – All Shook Up

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You have found media that support growth of the pure cultures you isolated (now named by your

boss as the Extraterrestrial Polyurethane-Degrading Microbe (EPTUM)). The recipes for these

media are shown below:

Medium 1 Medium 2

5 g yeast extract 10.5 g K2HPO4

20 g tryptone extract 4.5 g KH2PO4

0.5 g NaCl 1 g MgSO4

3.6 g glucose 10 g polyurethane

1 liter H2O 1 liter H2O

Growth Medium 1 Medium 2

EPTUM Growth - aerobic + +

EPTUM Growth - anaerobic + -

E. coli - aerobic + -

E. coli - anaerobic + -

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You are excited because, in Medium 2, EPTUM utilizes polyurethane as its energy source and

its sole source of carbon and nitrogen, a finding that raises the possibility that EPTUM could be a

useful tool for bioremediation of polyurethane-containing wastes (in landfills etc.). You have

also made some progress in characterizing the central metabolic pathways and related

biochemical properties of EPTUM. In particular, you have discovered that:

EPTUM secretes an enzyme (polyurethanase) that catalyzes the degradation of

polyurethane and generates citric acid (citrate) as a product.

The cytoplasmic membrane of EPTUM contains a transport system capable of

transporting citrate across the membrane and into the cell at the expense of 4 ATP molecules

(hydrolyzed to form ADP and phosphate) per molecule of citrate transported.

The cytoplasm of EPTUM contains all the enzymes necessary for glycolysis and the

citric acid (Kreb’s) cycle.

The cytoplasmic membrane of EPTUM contains proteins that form a functional electron-

transport system that utilizes oxygen as the final electron acceptor.

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Questions:

1. Which medium would you consider to be “non-selective” and which “selective”? Is it

possible that one of the media is “differential?” (Minimum 350-word response)

Non-selective and selective are terms used when describing medium. Microbiologists can mix

and match agents to fine-tune a medium for any purpose. Selective medium is described as a

media that contains one or more agents that hinder the growth of certain microbe or microbes.

Selective media allows a certain microbe to grow by itself. This method subdues the unwanted

organisms and allows for growth of the desired ones. A selective medium is one that all the

chemicals used are known and no yeast, animal or plant tissue is present such as in Medium 2.

Some selective media contain strong inhibitory agents to favor the growth of a pathogen that

would otherwise be overlooked because of its low numbers in a specimen. Examples of selective

media are mannitol salt agar with is used for the isolation of staphylococcus aureus from infected

material. Phenylethanol agar which is used for isolation of staphylococci and streptococci.

MacConkey agar (MAC) is used to isolate gram-negative enteric. Sabourad’s agar (SAB) is

media that has an acid that inhibits bacteria so it is used to isolate fungi. The list goes on and on

of the medium, selective agent and what they are used for.

Non-selective media is a medium in which all species will grow. It is a medium that will

contain water, various salts, carbon source and a source of amino acids and nitrogen such as

yeast extract as noted in Medium 1. Differential media grow several types of microorganisms but

are designed to bring out visible differences amongst them. The differences that may appear in

the differential media are such things as colony size or color, formation of gas bubbles and

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precipitates. The changes can also appear as changes in media colors too. The changes come

from the types of chemicals contained in the media and the way the microbes react to them. Dyes

are effective differential agents because of the pH indicators that change color in response to the

production of an acid or base. So since not all bacteria will grow on the same media, the question

is what bacteria grow where? Medium 2 was an environment that grew EPTUM in an aerobic

situation. This would make Medium 2 a selective media.

2. Given that polyurethane is a huge polymer, why is it important that the polyurethanase is

a secreted enzyme? If we assume that polyurethane is the source of energy for the

organism, how can carbon atoms from the polyurethane find their way into the central

metabolic pathways of the microbe? What is the “entry point”? (Minimum 400-word

response)

3. Why does the growth of EPTUM in Medium 2 require oxygen? Address each of the

following questions in your answer: (at least 3 full sentences per question) Would there

be a net gain or loss of ATP in the transport of the citrate across the EPTUM membrane?

Explain.

According to the growthr observations, will glycolysis be useful for generating any ATP

during growth of EPTUM on medium 2? Why or why not?

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Could glycolysis be useful for generating any ATP during growth of EPTUM on medium

1? Why or why not?

How many ATPs can be generated during the citric acid cycle? Where would the citrate

generated in the breakdown of polyurethane on Medium 2 enter the cycle?

What is the relationship between the citric acid cycle and substrate-level phosphorylation

(or the electron transport system)? Can EPTUM generate any ATP via this cycle when grown on

Medium 2? If it can, how many overall ATP is EPTUM capable of generating when grown on

Medium 2? If it can’t, explain why.

How many overall ATP is EPTUM capable of generating when grown on Medium 1?

What is the importance of oxygen as it relates to the ATP tally for EPTUM on either

medium 1 or medium 2?

Part Four – A Little Less Conversation

At a press conference announcing your company’s isolation and characterization of EPTUM, a

reporter raises an important question: “How do you know that this microbe actually came from

the Nearby Previously Invisible Planet (NPIP) and not from Earth? Could this be a microbe be an

Earth microbe that was present in/on ELVIS before it was launched into space?”

Question:

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1. What kinds of experiments would need to be done to determine an answer to the

reporter’s question prior to a new attempt to launch ELVIS into outer space? Briefly

suggest a plan of action. (Minimum 450-word response)

Why that is a very good question, how do we know that the microbe actually came from the

Nearby Previously Invisible Planet and not from Earth? There are many things that would need

to happen before we can attempt a new launch of ELVIS into outer space. We would first have to

find a process of decontamination of the structure prior to launch. That is, if there is such a thing.

The method of microbial control is called decontamination. There are many methods to

decontamination such as heat or radiation, chemical agents like disinfectants and antiseptics.

There are some microbial forms that constantly present in the external environment. So what is

the difference in disinfection versus sterilization versus antisepsis? Disinfection is the destruction

or removal of vegetative pathogens but no end spores. Sterilization is the complete removal and

destruction of all viable microorganisms and antisepsis is chemicals that are applied to body

surfaces to destroy or inhibit vegetative pathogens. To achieve microbial death the cell is

exposed to an agent that promotes cell structures to break down and the entire cell sustains

irreversible damage. The permanent loss of reproductive capability even under optimum growth

conditions is the definition of cell death.

The plan of action to determine this would be to go back to the beginning and sample the

areas of interest. In the specific case those two areas would be Earth and the NPIP. We would

have to inoculate the sample into a container of medium. The inoculated media is then placed in

a controlled environment to grow. After the growth, the known bacteria will be isolated out to

obtain a pure culture. Then information will be gathered to see about finding a suitable way to

promote cell death that is feasible and physically able.

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