The Epipelagic Is Divided Into Two Components: The Oceanic Waters And The

The Epipelagic Is Divided Into Two Components: The Oceanic Waters And The. Question

1 of 25

The epipelagic is divided into two components: the oceanic waters and the

photic zone.

neritic zone.

pelagic realm.

upper photic zone.

subtidal zone.

Question

2 of 25

Most of the primary production carried out in the open ocean is performed by

seaweeds.

kelps.

phytoplankton.

seagrasses.

zooxanthellae.

Question

3 of 25

Net phytoplankton consist mostly of

copepods.

diatoms and dinoflagellates.

nanoplankton.

cyanobacteria (blue-green algae).

nanoplankton and diatoms.

Question

4 of 25

Typically the most abundant group in the zooplankton are the.

larvaceans.

krill.

fish larvae.

nanoplankton.

copepods.

Question

5 of 25

Which of these is least likely to be seen in the epipelagic?

Suspension feeders

Deposit feeders

Primary production

First-level carnivores

Second-level carnivores

Question

6 of 25

Which of these groups builds a mucus “house?”

Copepods

Arrow worms

Larvaceans

Planktonic snails such as pteropods

Snail larvae

Question

7 of 25

Which of the following accounts for about 50% of the primary production in epipelagic waters?

Diatoms

Cyanobacteria

Dinoflagellates

Coccolithophorids

Silicoflagellates

Question

8 of 25

The following are an adaptation to the planktonic way of life except

spines.

small size.

decrease in drag.

substitution of heavy ions by light ones.

gas-filled bladders.

Question

9 of 25

The storage of lipids within the body is an adaptation in plankton since lipids

make cells heavier.

increase body density.

contain air pockets so they help in buoyancy.

contain a larger amount of energy.

are less dense than water.

Question

10 of 25

The neuston consists of animals that

swim against currents.

sink to the bottom portion of the water column.

are top carnivores in the pelagic realm.

spend their entire lives in the plankton.

live at the surface, but remain underwater.

Question

11 of 25

Counter shading is a form of

shading with bioluminescence.

warning coloration.

structural coloration.

protective coloration.

cryptic coloration.

Question

12 of 25

The rete mirabile found in some fishes is involved in.

increasing speed.

decreasing buoyancy.

digesting food.

increasing buoyancy.

conserving body heat.

Question

13 of 25

Zooplankton that migrate vertically

hibernate at night and feed during the day.

feed at the surface during the day, and migrate below the photic zone at night.

feed in the photic zone during the day, and migrate to the surface at night.

stay below the photic zone during the day, and feed at the surface at night.

migrate up and down but always stay below the photic zone.

Question

14 of 25

Most animals in the epipelagic are omnivores. This means that they eat

producers and consumers.

part of the neuston.

zooplankton.

detritus.

phytoplankton.

Question

15 of 25

What is the relationship between dissolved organic matter (DOM) and bacteria in the epipelagic?

Bacteria feed on the DOM, making it available to other animals in the food chain that feed on bacteria.

Bacteria supply most of the DOM.

Bacteria feed on DOM and thus it is unavailable to other animals.

Bacteria cannot utilize DOM and thus feed on detritus, depleting it through most of the epipelagic.

Bacteria cannot utilize DOM, making it available to animals.

Question

16 of 25

The most common limiting nutrient in the ocean is

silicon.

oxygen.

nitrogen.

carbonate.

phosphorus.

Question

17 of 25

The fall bloom in temperate waters is caused when

primary production decreases as nutrients increase.

primary production decreases due to light limitation.

primary production decreases as nutrients decrease.

primary production increases as nutrients increase.

primary production increases as the number of zooplankton increases.

Question

18 of 25

Equatorial upwelling occurs as a result of

temperature changes at the Equator.

the divergence of equatorial surface currents.

the convergence of equatorial surface currents.

winds causing the Ekman transport of surface water offshore.

El NiƱo conditions north and south of the Equator.

Question

19 of 25

The Southern Oscillation can be best described as

relative changes between two pressure systems.

variation in wind speed over the Pacific Ocean.

relationship between sea-surface and high-altitude pressures.

tidal differences between the Indian and Pacific Oceans.

wind-speed differences along the Equator.

Question

20 of 25

The long spines and projections seen in many epipelagic plankton are used for

feeding.

reproduction.

increasing drag.

increasing buoyancy.

gathering nutrients.

Question

21 of 25

Most epipelagic fish have a tail that is

short and wide.

short and narrow.

short and thin.

high and wide.

high and narrow.

Question

22 of 25

Which of the following adaptations is least likely to be seen in epipelagic fish?

Stiff fins

A smooth, scaleless body

Increased white muscle

Grove in body for fins

Eyes flush with body

Question

23 of 25

The largest source of dissolved organic material (DOM) in the epipelagic is

viruses.

bacteria.

phytoplankton.

zooplankton.

nekton.

Question

24 of 25

The lateral line system in fishes functions in ________________.

sensing vibrations in the water

detecting magnetic lines of force in water

sensing light in aphotic zones

sweeping surrounding water for plankton

bioluminescence

Question

25 of 25

The remote sensing system found in dolphins and some other cetaceans is ___________.

communal mutualistic behavior

extrasensory perception

echolocation

underwater acoustic sensitivity

heightened smell

 

 

 

The Epipelagic Is Divided Into Two Components: The Oceanic Waters And The

 
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Prologue Autumn Aroma

Prologue Autumn Aroma. Prologue Autumn Aroma

Takamato ridge, crowded with expanding caps, filling up, thrivingā€” the wonder of autumn aroma.

ā€” From the eighth- century Japanese poetry collection Man- nyo Shu

WhAt do you do when your world StArtS to FAll apart? I go for a walk, and if Iā€™m really lucky, I find mushrooms. Mush- rooms pull me back into my senses, not justā€” like flowersā€” through their riotous colors and smells but because they pop up unexpectedly, reminding me of the good fortune of just happening to be there. Then I know that there are still pleasures amidst the terrors of indeterminacy.

Terrors, of course, there are, and not just for me. The worldā€™s climate is going haywire, and industrial progress has proved much more deadly to life on earth than anyone imagined a century ago. The economy is

Elusive life, Oregon. Matsutake caps emerge

in the ruin of an industrial forest.

2 Prologue

no longer a source of growth or optimism; any of our jobs could disap- pear with the next economic crisis. And itā€™s not just that I might fear a spurt of new disasters: I find myself without the handrails of stories that tell where everyone is going and, also, why. Precarity once seemed the fate of the less fortunate. Now it seems that all our lives are precariousā€” even when, for the moment, our pockets are lined. In contrast to the mid- twentieth century, when poets and philosophers of the global north felt caged by too much stability, now many of us, north and south, confront the condition of trouble without end.

This book tells of my travels with mushrooms to explore indetermi- nacy and the conditions of precarity, that is, life without the promise of stability. Iā€™ve read that when the Soviet Union collapsed in 1991, thou- sands of Siberians, suddenly deprived of state guarantees, ran to the woods to collect mushrooms.1 These are not the mushrooms I follow, but they make my point: the uncontrolled lives of mushrooms are a giftā€” and a guideā€” when the controlled world we thought we had fails.

While I canā€™t offer you mushrooms, I hope you will follow me to savor the ā€œautumn aromaā€ praised in the poem that begins my pro- logue. This is the smell of matsutake, a group of aromatic wild mush- rooms much valued in Japan. Matsutake is loved as a marker of the au- tumn season. The smell evokes sadness in the loss of summerā€™s easy riches, but it also calls up the sharp intensity and heightened sensibili- ties of autumn. Such sensibilities will be needed for the end of global progressā€™s easy summer: the autumn aroma leads me into common life without guarantees. This book is not a critique of the dreams of mod- ernization and progress that offered a vision of stability in the twentieth century; many analysts before me have dissected those dreams. Instead, I address the imaginative challenge of living without those handrails, which once made us think we knew, collectively, where we were going. If we open ourselves to their fungal attractions, matsutake can catapult us into the curiosity that seems to me the first requirement of collabora- tive survival in precarious times.

Hereā€™s how a radical pamphlet put the challenge:

The spectre that many try not to see is a simple realisationā€” the world will not be ā€œsaved.ā€ . . . If we donā€™t believe in a global revolutionary fu- ture, we must live (as we in fact always had to) in the present.2

autumn aroma 3

When Hiroshima was destroyed by an atomic bomb in 1945, it is said, the first living thing to emerge from the blasted landscape was a mat- sutake mushroom.3

Grasping the atom was the culmination of human dreams of con- trolling nature. It was also the beginning of those dreamsā€™ undoing. The bomb at Hiroshima changed things. Suddenly, we became aware that humans could destroy the livability of the planetā€” whether intention- ally or otherwise. This awareness only increased as we learned about pol- lution, mass extinction, and climate change. One half of current precar- ity is the fate of the earth: what kinds of human disturbances can we live with? Despite talk of sustainability, how much chance do we have for passing a habitable environment to our multispecies descendants?

Hiroshimaā€™s bomb also opened the door to the other half of todayā€™s precarity: the surprising contradictions of postwar development. After the war, the promises of modernization, backed by American bombs, seemed bright. Everyone was to benefit. The direction of the future was well known; but is it now? On the one hand, no place in the world is untouched by that global political economy built from the postwar development ap- paratus. On the other, even as the promises of development still beckon, we seem to have lost the means. Modernization was supposed to fill the worldā€” both communist and capitalistā€” with jobs, and not just any jobs but ā€œstandard employmentā€ with stable wages and benefits. Such jobs are now quite rare; most people depend on much more irregular livelihoods. The irony of our times, then, is that everyone depends on capitalism but almost no one has what we used to call a ā€œregular job.ā€

To live with precarity requires more than railing at those who put us here (although that seems useful too, and Iā€™m not against it). We might look around to notice this strange new world, and we might stretch our imaginations to grasp its contours. This is where mushrooms help. Mat- sutakeā€™s willingness to emerge in blasted landscapes allows us to ex- plore the ruin that has become our collective home.

Matsutake are wild mushrooms that live in human- disturbed forests. Like rats, raccoons, and cockroaches, they are willing to put up with

4 Prologue

some of the environmental messes humans have made. Yet they are not pests; they are valuable gourmet treatsā€” at least in Japan, where high prices sometimes make matsutake the most valuable mushroom on earth. Through their ability to nurture trees, matsutake help forests grow in daunting places. To follow matsutake guides us to possibilities of coexis- tence within environmental disturbance. This is not an excuse for further damage. Still, matsutake show one kind of collaborative survival.

Matsutake also illuminate the cracks in the global political econ- omy. For the past thirty years, matsutake have become a global com- modity, foraged in forests across the northern hemisphere and shipped fresh to Japan. Many matsutake foragers are displaced and disenfran- chised cultural minorities. In the U.S. Pacific Northwest, for example, most commercial matsutake foragers are refugees from Laos and Cam- bodia. Because of high prices, matsutake make a substantial contribu- tion to livelihood wherever they are picked, and even encourage cul- tural revitalizations.

Matsutake commerce, however, hardly leads to twentieth- century development dreams. Most of the mushroom foragers I spoke with have terrible stories to tell of displacement and loss. Commercial foraging is a better than usual way of getting by for those with no other way to make a living. But what kind of economy is this anyway? Mushroom foragers work for themselves; no companies hire them. There are no wages and no benefits; pickers merely sell the mushrooms they find. Some years there are no mushrooms, and pickers are left with their ex- penses. Commercial wild- mushroom picking is an exemplification of precarious livelihood, without security.

This book takes up the story of precarious livelihoods and precari- ous environments through tracking matsutake commerce and ecology. In each case, I find myself surrounded by patchiness, that is, a mosaic of open- ended assemblages of entangled ways of life, with each further opening into a mosaic of temporal rhythms and spatial arcs. I argue that only an appreciation of current precarity as an earthwide condition allows us to notice thisā€” the situation of our world. As long as authori- tative analysis requires assumptions of growth, experts donā€™t see the het- erogeneity of space and time, even where it is obvious to ordinary par- ticipants and observers. Yet theories of heterogeneity are still in their

autumn aroma 5

infancy. To appreciate the patchy unpredictability associated with our current condition, we need to reopen our imaginations. The point of this book is to help that process alongā€” with mushrooms.

About commerce: Contemporary commerce works within the con- straints and possibilities of capitalism. Yet, following in the footsteps of Marx, twentieth- century students of capitalism internalized progress to see only one powerful current at a time, ignoring the rest. This book shows how it is possible to study capitalism without this crippling as- sumptionā€” by combining close attention to the world, in all its precar- ity, with questions about how wealth is amassed. How might capitalism look without assuming progress? It might look patchy: the concentration of wealth is possible because value produced in unplanned patches is appro- priated for capital.

About ecology: For humanists, assumptions of progressive human mastery have encouraged a view of nature as a romantic space of anti- modernity.4 Yet for twentieth- century scientists, progress also unself- consciously framed the study of landscapes. Assumptions about expansion slipped into the formulation of population biology. New developments in ecology make it possible to think quite differently by introducing cross- species interactions and disturbance histories. In this time of di- minished expectations, I look for disturbance- based ecologies in which many species sometimes live together without either harmony or conquest.

While I refuse to reduce either economy or ecology to the other, there is one connection between economy and environment that seems important to introduce up front: the history of the human concentra- tion of wealth through making both humans and nonhumans into re- sources for investment. This history has inspired investors to imbue both people and things with alienation, that is, the ability to stand alone, as if the entanglements of living did not matter.5 Through alienation, people and things become mobile assets; they can be removed from their life worlds in distance- defying transport to be exchanged with other assets from other life worlds, elsewhere.6 This is quite different from merely using others as part of a life worldā€” for example, in eating and being eaten. In that case, multispecies living spaces remain in place. Alienation obviates living- space entanglement. The dream of alienation inspires landscape modification in which only one stand- alone asset matters;

6 Prologue

everything else becomes weeds or waste. Here, attending to living- space entanglements seems inefficient, and perhaps archaic. When its singular asset can no longer be produced, a place can be abandoned. The timber has been cut; the oil has run out; the plantation soil no longer supports crops. The search for assets resumes elsewhere. Thus, simplification for alienation produces ruins, spaces of abandonment for asset production.

Global landscapes today are strewn with this kind of ruin. Still, these places can be lively despite announcements of their death; aban- doned asset fields sometimes yield new multispecies and multicultural life. In a global state of precarity, we donā€™t have choices other than look- ing for life in this ruin.

Our first step is to bring back curiosity. Unencumbered by the sim- plifications of progress narratives, the knots and pulses of patchiness are there to explore. Matsutake are a place to begin: However much I learn, they take me by surprise.

This is not a book about Japan, but the reader needs to know something about matsutake in Japan to proceed.7 Matsutake first appears in Japanā€™s written record in the eighth- century poem that starts this prologue. Al- ready then, the mushroom is praised for its aromatic marking of the autumn season. The mushroom became common around Nara and Kyoto, where people had deforested the mountains for wood to build temples and to fuel iron forges. Indeed, human disturbance allowed Tricholoma matsutake to emerge in Japan. This is because its most com- mon host is red pine (Pinus densiflora), which germinates in the sunlight and mineral soils left by human deforestation. When forests in Japan are allowed to grow back, without human disturbance, broadleaf trees shade out pines, preventing their further germination.

As red pine spread with deforestation across Japan, matsutake be- came a valued gift, presented beautifully in a box of ferns. Aristocrats were honored by it. By the Edo period (1603ā€“ 1868), well- to- do common- ers, such as urban merchants, also enjoyed matsutake. The mushroom joined the celebration of the four seasons as a marker of autumn. Out- ings to pick matsutake in the fall were an equivalent of cherry- blossom

autumn aroma 7

viewing parties in the spring. Matsutake became a popular subject for poetry.

The sound of a temple bell is heard in the cedar forest at dusk, The autumn aroma drifts on the roads below.

ā€” AkemI TAchIbAnA (1812ā€“ 1868)8

As in other Japanese nature poetry, seasonal referents helped build a mood. Matsutake joined older signs of the fall season, such as the sound of deer crying or the harvest moon. The coming bareness of winter touched autumn with an incipient loneliness, at the edge of nostalgia, and the poem above offers that mood. Matsutake was an elite pleasure, a sign of the privilege to live within the artful reconstruction of nature for refined tastes.9 For this reason, when peasants preparing for elite outings sometimes ā€œplantedā€ matsutake (i.e., stuck mushrooms artfully in the ground because naturally occurring matsutake were not avail- able), no one objected. Matsutake had become an element of an ideal seasonality, appreciated not only in poetry but also in all the arts, from tea ceremony to theater.

The moving cloud fades away, and I smell the aroma of the mushroom.

ā€” KoI NAgAtA (1900ā€“ 1997)10

The Edo period was ended by the Meiji Restorationā€” and Japanā€™s rapid modernization. Deforestation proceeded apace, privileging pine and matsutake. In the Kyoto area, matsutake became a generic term for ā€œmushroom.ā€ In the early twentieth century, matsutake were particu- larly common. In the mid- 1950s, however, the situation began to change. Peasant woodlands were cut down for timber plantations, paved for sub- urban development, or abandoned by peasants moving to the city. Fossil fuel replaced firewood and charcoal; farmers no longer used the remain- ing woodlands, which grew up in dense thickets of broadleaf trees. Hill- sides that had once been covered by matsutake were now too shady for pine ecologies. Shade- stressed pines were killed by an invasive nematode. By the mid- 1970s, matsutake had become rare across Japan.

8 Prologue

This was the time, however, of Japanā€™s rapid economic development, and matsutake were in demand as exquisitely expensive gifts, perks, and bribes. The price of matsutake skyrocketed. The knowledge that mat- sutake grew in other parts of the world suddenly became relevant. Jap- anese travelers and residents abroad began to send matsutake to Japan; as importers emerged to funnel the international matsutake trade, non- Japanese pickers rushed in. At first it seemed that there were a plethora of colors and kinds that might appropriately be considered matsutakeā€” because they had the smell. Scientific names proliferated as matsutake in forests across the northern hemisphere suddenly rose from neglect. In the past twenty years, names have been consolidated. All across Eur- asia, most matsutake are now Tricholoma matsutake.11 In North America, T. matsutake seems to be found only in the east, and in the mountains of Mexico. In western North America, the local matsutake is considered another species, T. magnivelare.12 Some scientists, however, think the ge- neric term ā€œmatsutakeā€ is the best way to identify these aromatic mush- rooms, since the dynamics of speciation are still unclear.13 I follow that practice except where I am discussing questions of classification.

Japanese have figured out ways of ranking matsutake from different parts of the world, and ranks are reflected in prices. My eyes were first opened to such rankings when one Japanese importer explained: ā€œMat- sutake are like people. American mushrooms are white because the people are white. Chinese mushrooms are black, because the people are black. Japanese people and mushrooms are nicely in between.ā€ Not ev- eryone has the same rankings, but this stark example can stand in for the many forms of classification and valuation that structure the global trade.

Meanwhile, people in Japan worry about the loss of the peasant wood- lands that have been the source of so much seasonal beauty, from spring blossoms to bright autumn leaves. Starting in the 1970s, volunteer groups mobilized to restore these woodlands. Wanting their work to matter beyond passive aesthetics, the groups looked for ways restored wood- lands might benefit human livelihood. The high price of matsutake made it an ideal product of woodland restoration.

And so I return to precarity and living in our messes. But living seems to have gotten more crowded, not only with Japanese aesthetics and eco-

autumn aroma 9

logical histories, but also with international relations and capitalist trad- ing practices. This is the stuff for stories in the book that follows. For the moment, it seems important to appreciate the mushroom.

Oh, matsutake: The excitement before finding them.

ā€” YAmAguchI Sodo (1642ā€“ 1716)14

Part I Whatā€™s Left?

Conjuring time, Yunnan. Watching

the boss gamble.

It wAS A StIll- brIght evenIng when I reAlIzed I was lost and empty- handed in an unknown forest. I was on my first search for matsutakeā€” and matsutake pickersā€” in Oregonā€™s Cascade Mountains. Earlier that afternoon, I had found the Forest Serviceā€™s ā€œbig campā€ for mushroom pickers, but all the pickers were out foraging. I had decided to look for mushrooms myself while I waited for their return.

I couldnā€™t have imagined a more unpromising- looking forest. The ground was dry and rocky, and nothing grew except thin sticks of lodgepole pine. There were hardly any plants growing near the ground, not even grass, and when I touched the soil, sharp pumice shards cut my fingers. As the afternoon wore on, I found one or two ā€œcopper tops,ā€ dingy mushrooms with a splash of orange and a mealy smell.1 Nothing else. Worse yet, I was disoriented. Every way I turned, the forest looked the same. I had no idea which direction to go to find my car. Thinking I would be out there just briefly, I had brought nothing, and I knew I would soon be thirsty, hungryā€” and cold.

I stumbled around and eventually found a dirt road. But which way should I go? The sun was getting lower as I trudged along. I had walked less than a mile when a pickup truck drew up. A bright- faced young

14 Part i

man and a wizened old man were inside, and they offered me a ride. The young man introduced himself as Kao. Like his uncle, he said, he was a Mien from the hills of Laos who had come to the United States from a refugee camp in Thailand in the 1980s. They were neighbors in Sacramento, California, and here to pick mushrooms together. They brought me to their camp. The young man went to get water, driving his plastic jugs to a water storage container some ways away. The older man did not know English, but it turned out he knew a little Mandarin Chinese, as did I. As we awkwardly exchanged phrases, he pulled out a smoking bong handcrafted from PVC pipe and lit up his tobacco.

It was dusk when Kao came back with the water. But he beckoned me to go picking with him: There were mushrooms nearby. In the gath- ering dark, we scrambled up a rocky hillside not far from his camp. I saw nothing but dirt and some scrawny pine trees. But here was Kao with his bucket and stick, poking deep into clearly empty ground and pulling up a fat button. How could this be possible? There had been nothing thereā€” and then there it was.

Kao handed me the mushroom. Thatā€™s when I first experienced the smell. Itā€™s not an easy smell. Itā€™s not like a flower or a mouth- watering food. Itā€™s disturbing. Many people never learn to love it. Itā€™s hard to de- scribe. Some people liken it to rotting things and some to clear beautyā€” the autumn aroma. At my first whiff, I was just . . . astonished.

My surprise was not just for the smell. What were Mien tribesmen, Japanese gourmet mushrooms, and I doing in a ruined Oregon indus- trial forest? I had lived in the United States for a long time without ever hearing about any of these things. The Mien camp pulled me back to my earlier fieldwork in Southeast Asia; the mushroom tickled my inter- est in Japanese aesthetics and cuisine. The broken forest, in contrast, seemed like a science fiction nightmare. To my faulty common sense, we all seemed miraculously out of time and out of placeā€” like some- thing that might jump out of a fairy tale. I was startled and intrigued; I couldnā€™t stop exploring. This book is my attempt to pull you into the maze I found.

1 Arts of Noticing

I am not proposing a return to the Stone Age. My intent is not reactionary, nor even conservative, but simply subversive. It seems that the utopian imagination is trapped, like capitalism and industrialism and the human population, in a one- way future consisting only of growth. All Iā€™m trying to do is figure out how to put a pig on the tracks.

ā€” Ursula K. Le Guin

In 1908 And 1909 two rAIlroAd entrePreneurS raced each other to build track along Oregonā€™s Deschutes River.1 The goal of each was to be the first to create an industrial connection be- tween the towering ponderosas of the eastern Cascades and the stacked lumberyards of Portland. In 1910, the thrill of competition yielded to an agreement for joint service. Pine logs poured out of the region, bound for distant markets. Lumber mills brought new settlers; towns sprung

Conjuring time, Kyoto Prefecture.

Mr. Imotoā€™s map of revitalizing. This is his matsutake mountain:

a time machine of multiple seasons,

histories, and hopes.

18 ChaPter 1

up as millworkers multiplied. By the 1930s, Oregon had become the na- tionā€™s largest producer of timber.

This is a story we know. It is the story of pioneers, progress, and the trans- formation of ā€œemptyā€ spaces into industrial resource fields.

In 1989, a plastic spotted owl was hung in effigy on an Oregon log- ging truck.2 Environmentalists had shown that unsustainable logging was destroying Pacific Northwest forests. ā€œThe spotted owl was like the canary in the coal mine,ā€ explained one advocate. ā€œIt was . . . symbolic of an ecosystem on the verge of collapse.ā€3 When a federal judge blocked old- growth logging to save owl habitat, loggers were furious; but how many loggers were there? Logging jobs had dwindled as timber compa- nies mechanizedā€” and as prime timber disappeared. By 1989, many mills had already closed; logging companies were moving to other re- gions.4 The eastern Cascades, once a hub of timber wealth, were now cutover forests and former mill towns overgrown by brush.

This is a story we need to know. Industrial transformation turned out to be a bubble of promise followed by lost livelihoods and damaged landscapes. And yet: such documents are not enough. If we end the story with decay, we abandon all hopeā€” or turn our attention to other sites of promise and ruin, promise and ruin.

What emerges in damaged landscapes, beyond the call of industrial promise and ruin? By 1989, something else had begun in Oregonā€™s cut- over forests: the wild mushroom trade. From the first it was linked to worldwide ruination: The 1986 Chernobyl disaster had contaminated Europeā€™s mushrooms, and traders had come to the Pacific Northwest for supplies. When Japan began importing matsutake at high pricesā€” just as jobless Indochinese refugees were settling in Californiaā€” the trade went wild. Thousands rushed to Pacific Northwest forests for the new ā€œwhite gold.ā€ This was in the middle of a ā€œjobs versus the environ- mentā€ battle over the forests, yet neither side noticed the mushroomers. Job advocates imagined only wage contracts for healthy white men; the foragersā€” disabled white veterans, Asian refugees, Native Americans, and undocumented Latinosā€” were invisible interlopers. Conservation- ists were fighting to keep human disturbance out of the forests; the entry of thousands of people, had it been noticed, would hardly have been welcome. But the mushroom hunters were mainly not noticed. At

arts of notiCing 19

most, the Asian presence sparked local fears of invasion: journalists wor- ried about violence.5

A few years into the new century, the idea of a trade- off between jobs and the environment seemed less convincing. With or without conservation, there were fewer ā€œjobsā€ in the twentieth- century sense in the United States; besides, it seemed much more likely that environ- mental damage would kill all of us off, jobs or no jobs. We are stuck with the problem of living despite economic and ecological ruination. Neither tales of progress nor of ruin tell us how to think about collabo- rative survival. It is time to pay attention to mushroom picking. Not that this will save usā€” but it might open our imaginations.

Geologists have begun to call our time the Anthropocene, the epoch in which human disturbance outranks other geological forces. As I write, the term is still newā€” and still full of promising contradictions. Thus, although some interpreters see the name as implying the triumph of humans, the opposite seems more accurate: without planning or inten- tion, humans have made a mess of our planet.6 Furthermore, despite the prefix ā€œanthropo- ,ā€ that is, human, the mess is not a result of our species biology. The most convincing Anthropocene time line begins not with our species but rather with the advent of modern capitalism, which has directed long- distance destruction of landscapes and ecologies. This time line, however, makes the ā€œanthropo- ā€ even more of a problem. Imagin- ing the human since the rise of capitalism entangles us with ideas of progress and with the spread of techniques of alienation that turn both humans and other beings into resources. Such techniques have segre- gated humans and policed identities, obscuring collaborative survival. The concept of the Anthropocene both evokes this bundle of aspira- tions, which one might call the modern human conceit, and raises the hope that we might muddle beyond it. Can we live inside this regime of the human and still exceed it?

This is the predicament that makes me pause before offering a de- scription of mushrooms and mushroom pickers. The modern human conceit wonā€™t let a description be anything more than a decorative

20 ChaPter 1

footnote. This ā€œanthropo- ā€ blocks attention to patchy landscapes, mul- tiple temporalities, and shifting assemblages of humans and nonhu- mans: the very stuff of collaborative survival. In order to make mush- room picking a worthwhile tale, then, I must first chart the work of this ā€œanthropo- ā€ and explore the terrain it refuses to acknowledge.

Consider, indeed, the question of whatā€™s left. Given the effectiveness of state and capitalist devastation of natural landscapes, we might ask why anything outside their plans is alive today. To address this, we will need to watch unruly edges. What brings Mien and matsutake together in Oregon? Such seemingly trivial queries might turn everything around to put unpredictable encounters at the center of things.

We hear about precarity in the news every day. People lose their jobs or get angry because they never had them. Gorillas and river porpoises hover at the edge of extinction. Rising seas swamp whole Pacific islands. But most of the time we imagine such precarity to be an exception to how the world works. Itā€™s what ā€œdrops outā€ from the system. What if, as Iā€™m suggesting, precarity is the condition of our timeā€” or, to put it an- other way, what if our time is ripe for sensing precarity? What if precar- ity, indeterminacy, and what we imagine as trivial are the center of the systematicity we seek?

Precarity is the condition of being vulnerable to others. Unpredict- able encounters transform us; we are not in control, even of ourselves. Unable to rely on a stable structure of community, we are thrown into shifting assemblages, which remake us as well as our others. We canā€™t rely on the status quo; everything is in flux, including our ability to sur- vive. Thinking through precarity changes social analysis. A precarious world is a world without teleology. Indeterminacy, the unplanned na- ture of time, is frightening, but thinking through precarity makes it evident that indeterminacy also makes life possible.

The only reason all this sounds odd is that most of us were raised on dreams of modernization and progress. These frames sort out those parts of the present that might lead to the future. The rest are trivial; they ā€œdrop outā€ of history. I imagine you talking back: ā€œProgress? Thatā€™s an idea from the nineteenth century.ā€ The term ā€œprogress,ā€ referring to a general state, has become rare; even twentieth- century modernization has begun to feel archaic. But their categories and assumptions of im- provement are with us everywhere. We imagine their objects every day:

arts of notiCing 21

democracy, growth, science, hope. Why would we expect economies to grow and sciences to advance? Even without explicit reference to devel- opment, our theories of history are embroiled in these categories. So, too, are our personal dreams. Iā€™ll admit itā€™s hard for me to even say this: there might not be a collective happy ending. Then why bother getting up in the morning?

Progress is embedded, too, in widely accepted assumptions about what it means to be human. Even when disguised through other terms, such as ā€œagency,ā€ ā€œconsciousness,ā€ and ā€œintention,ā€ we learn over and over that humans are different from the rest of the living world because we look forwardā€” while other species, which live day to day, are thus dependent on us. As long as we imagine that humans are made through progress, nonhumans are stuck within this imaginative framework too.

Progress is a forward march, drawing other kinds of time into its rhythms. Without that driving beat, we might notice other temporal patterns. Each living thing remakes the world through seasonal pulses of growth, lifetime reproductive patterns, and geographies of expan- sion. Within a given species, too, there are multiple time- making projects, as organisms enlist each other and coordinate in making landscapes. (The regrowth of the cutover Cascades and Hiroshimaā€™s radioecology each show us multispecies time making.) The curiosity I advocate fol- lows such multiple temporalities, revitalizing description and imagina- tion. This is not a simple empiricism, in which the world invents its own categories. Instead, agnostic about where we are going, we might look for what has been ignored because it never fit the time line of progress.

Consider again the snippets of Oregon history with which I began this chapter. The first, about railroads, tells of progress. It led to the fu- ture: railroads reshaped our destiny. The second is already an interrup- tion, a history in which the destruction of forests matters. What it shares with the first, however, is the assumption that the trope of progress is sufficient to know the world, both in success and failure. The story of decline offers no leftovers, no excess, nothing that escapes progress. Progress still controls us even in tales of ruination.

Yet the modern human conceit is not the only plan for making worlds: we are surrounded by many world- making projects, human and not human.7 World- making projects emerge from practical activities of

22 ChaPter 1

making lives; in the process these projects alter our planet. To see them, in the shadow of the Anthropoceneā€™s ā€œanthropo- ,ā€ we must reorient our attention. Many preindustrial livelihoods, from foraging to stealing, persist today, and new ones (including commercial mushroom picking) emerge, but we neglect them because they are not a part of progress. These livelihoods make worlds tooā€” and they show us how to look around rather than ahead.

Making worlds is not limited to humans. We know that beavers re- shape streams as they make dams, canals, and lodges; in fact, all organ- isms make ecological living places, altering earth, air, and water. With- out the ability to make workable living arrangements, species would die out. In the process, each organism changes everyoneā€™s world. Bacteria made our oxygen atmosphere, and plants help maintain it. Plants live on land because fungi made soil by digesting rocks. As these examples sug- gest, world- making projects can overlap, allowing room for more than one species. Humans, too, have always been involved in multispecies world making. Fire was a tool for early humans not just to cook but also to burn the landscape, encouraging edible bulbs and grasses that at- tracted animals for hunting. Humans shape multispecies worlds when our living arrangements make room for other species. This is not just a matter of crops, livestock, and pets. Pines, with their associated fungal partners, often flourish in landscapes burned by humans; pines and fungi work together to take advantage of bright open spaces and exposed mineral soils. Humans, pines, and fungi make living arrangements si- multaneously for themselves and for others: multispecies worlds.

Twentieth- century scholarship, advancing the modern human con- ceit, conspired against our ability to notice the divergent, layered, and conjoined projects that make up worlds. Entranced by the expansion of certain ways of life over others, scholars ignored questions of what else was going on. As progress tales lose traction, however, it becomes possi- ble to look differently.

The concept of assemblage is helpful. Ecologists turned to assem- blages to get around the sometimes fixed and bounded connotations of ecological ā€œcommunity.ā€ The question of how the varied species in a species assemblage influence each otherā€” if at allā€” is never settled: some thwart (or eat) each other; others work together to make life pos- sible; still others just happen to find themselves in the same place. As-

arts of notiCing 23

semblages are open- ended gatherings. They allow us to ask about com- munal effects without assuming them. They show us potential histories in the making. For my purposes, however, I need something other than organisms as the elements that gather. I need to see lifewaysā€” and non- living ways of being as wellā€” coming together. Nonhuman ways of being, like human ones, shift historically. For living things, species identities are a place to begin, but they are not enough: ways of being are emergent effects of encounters. Thinking about humans makes this clear. Foraging for mushrooms is a way of lifeā€” but not a common characteristic of all humans. The issue is the same for other species. Pines find mushrooms to help them use human- made open spaces. As- semblages donā€™t just gather lifeways; they make them. Thinking through assemblage urges us to ask: How do gatherings sometimes become ā€œhappenings,ā€ that is, greater than the sum of their parts? If history without progress is indeterminate and multidirectional, might assem- blages show us its possibilities?

Patterns of unintentional coordination develop in assemblages. To notice such patterns means watching the interplay of temporal rhythms and scales in the divergent lifeways that gather. Surprisingly, this turns out to be a method that might revitalize political economy as well as environmental studies. Assemblages drag political economy inside them, and not just for humans. Plantation crops have lives different from those of their free- living siblings; cart horses and hunter steeds share species but not lifeways. Assemblages cannot hide from capital and the state; they are sites for watching how political economy works. If capi- talism has no teleology, we need to see what comes togetherā€” not just by prefabrication, but also by juxtaposition.

Other authors use ā€œassemblageā€ with other meanings.8 The qualifier ā€œpolyphonicā€ may help explain my variant. Polyphony is music in which autonomous melodies intertwine. In Western music, the madrigal and the fugue are examples of polyphony. These forms seem archaic and strange to many modern listeners because they were superseded by music in which a unified rhythm and melody holds the composition together. In the classical music that displaced baroque, unity was the goal; this was ā€œprogressā€ in just the meaning I have been discussing: a unified coordination of time. In twentieth- century rock- and- roll, this unity takes the form of a strong beat, suggestive of the listenerā€™s heart;

24 ChaPter 1

we are used to hearing music with a single perspective. When I first learned polyphony, it was a revelation in listening; I was forced to pick out separate, simultaneous melodies and to listen for the moments of harmony and dissonance they created together. This kind of noticing is just what is needed to appreciate the multiple temporal rhythms and trajectories of the assemblage.

For those not musically inclined, it may be useful to imagine the polyphonic assemblage in relation to agriculture. Since the time of the plantation, commercial agriculture has aimed to segregate a single crop and work toward its simultaneous ripening for a coordinated harvest. But other kinds of farming have multiple rhythms. In the shifting culti- vation I studied in Indonesian Borneo, many crops grew together in the same field, and they had quite different schedules. Rice, bananas, taro, sweet potatoes, sugarcane, palms, and fruit trees mingled; farmers needed to attend to the varied schedules of maturation of each of these crops. These rhythms were their relation to human harvests; if we add other relations, for example, to pollinators or other plants, rhythms multiply. The polyphonic assemblage is the gathering of these rhythms, as they result from world- making projects, human and not human.

The polyphonic assemblage also moves us into the unexplored terri- tory of the modern political economy. Factory labor is an exemplar of coordinated progress time. Yet the supply chain is infused with poly- phonic rhythms. Consider the tiny Chinese garment factory studied by Nellie Chu; like its many competitors, it served multiple supply lines, constantly switching among orders for local boutique brands, knock- off international brands, and generic to- be- branded- later production.9 Each required different standards, materials, and kinds of labor. The factoryā€™s job was to match industrial coordination to the complex rhythms of supply chains. Rhythms further multiply when we move out of facto- ries to watch foraging for an unpredictable wild product. The farther we stray into the peripheries of capitalist production, the more coordi- nation between polyphonic assemblages and industrial processes be- comes central to making a profit.

As the last examples suggest, abandoning progress rhythms to watch polyphonic assemblages is not a matter of virtuous desire. Progress felt great; there was always something better ahead. Progress gave us the ā€œprogressiveā€ political causes with which I grew up. I hardly know how

arts of notiCing 25

to think about justice without progress. The problem is that progress stopped making sense. More and more of us looked up one day and re- alized that the emperor had no clothes. It is in this dilemma that new tools for noticing seem so important.10 Indeed, life on earth seems at stake. Chapter 2 turns to dilemmas of collaborative survival.

Interlude Tracking

MuShroom trAckS Are eluSIve And enIgmAtIc; following them takes me on a wild rideā€” trespassing every boundary. Things get even stranger when I move out of commerce into Darwinā€™s ā€œentangled bankā€ of multiple life forms.1 Here, the biology we thought we knew stands on its head. Entanglement bursts categories and upends identities.

Mushrooms are the fruiting bodies of fungi. Fungi are diverse and often flexible, and they live in many places, ranging from ocean cur- rents to toenails. But many fungi live in the soil, where their thread- like filaments, called hyphae, spread into fans and tangle into cords through the dirt. If you could make the soil liquid and transparent and walk into the ground, you would find yourself surrounded by nets of fungal hy- phae. Follow fungi into that underground city, and you will find the strange and varied pleasures of interspecies life.2

Many people think fungi are plants, but they are actually closer to animals. Fungi do not make their food from sunlight, as plants do. Like animals, fungi must find something to eat. Yet fungal eating is often gen- erous: It makes worlds for others. This is because fungi have extracellular

Elusive life, Oregon. The spoor of deer and elk lead pickers to matsutake

patches. There, cracks signal a deep- seated

mushroom rising through the ground. Tracking

means following worldly entanglements.

138 interlude

digestion. They excrete digestive acids outside their bodies to break down their food into nutrients. Itā€™s as if they had everted stomachs, di- gesting food outside instead of inside their bodies. Nutrients are then absorbed into their cells, allowing the fungal body to growā€” but also other speciesā€™ bodies. The reason there are plants growing on dry land (rather than just in water) is that over the course of the earthā€™s history fungi have digested rocks, making nutrients available for plants. Fungi (together with bacteria) made the soil in which plants grow. Fungi also digest wood. Otherwise, dead trees would stack up in the forest forever. Fungi break them down into nutrients that can be recycled into new life. Fungi are thus world builders, shaping environments for themselves and others.

Some fungi have learned to live in intimate associations with plants, and given enough time to adjust to the interspecies relations of a place, most plants enter into associations with fungi. ā€œEndophyticā€ and ā€œendo- mycorrhizalā€ fungi live inside plants. Many do not have fruiting bodies; they gave up sex millions of years ago. We are likely never to see these fungi unless we peer inside plants with microscopes, yet most plants are thick with them. ā€œEctomycorrhizalā€ fungi wrap themselves around the outsides of roots as well as penetrating between their cells. Many of the favorite mushrooms of people around the worldā€” porcini, chanterelles, truffles, and, indeed, matsutakeā€” are the fruiting bodies of ectomycor- rhizal plant associates. They are so delicious, and so difficult for humans to manipulate, because they thrive together with host trees. They come into being only through interspecies relations.

The term ā€œmycorrhizaā€ is assembled from Greek words for ā€œfungusā€ and ā€œrootā€; fungi and plant roots become intimately entangled in my- corrhizal relations. Neither the fungus nor the plant can flourish with- out the activity of the other. From the fungal perspective, the goal is to get a good meal. The fungus extends its body into the hostā€™s roots to siphon off some of the plantā€™s carbohydrates through specialized inter- face structures, made in the encounter. The fungus depends on this food, yet it is not entirely selfish. Fungi stimulate plant growth, first, by get- ting plants more water, and, second, by making the nutrients of extra- cellular digestion available to plants. Plants get calcium, nitrogen, po- tassium, phosphorus, and other minerals through mycorrhiza. Forests, according to researcher Lisa Curran, occur only because of ectomycor-

traCking 139

rhizal fungi.3 By leaning on fungal companions, trees grow strong and numerous, making forests.

Mutual benefits do not lead to perfect harmony. Sometimes the fun- gus parasitizes the root in one phase of its life cycle. Or, if the plant has lots of nutrients, it may reject the fungus. A mycorrhizal fungus with- out a plant collaborator will die. But many ectomycorrhizas are not lim- ited to one collaboration; the fungus forms a network across plants. In a forest, fungi connect not just trees of the same species, but often many species. If you cover a tree in the forest, depriving its leaves of light and thus food, its mycorrhizal associates may feed it from the carbohydrates of other trees in the network.4 Some commentators compare mycorrhi- zal networks to the Internet, writing of the ā€œwoodwide web.ā€ Mycorrhi- zas form an infrastructure of interspecies interconnection, carrying in- formation across the forest. They also have some of the characteristics of a highway system. Soil microbes that would otherwise stay in the same place are able to travel in the channels and linkages of mycorrhi- zal interconnection. Some of these microbes are important for environ- mental remediation.5 Mycorrhizal networks allow forests to respond to threats.

Why has the world- building work of fungi received so little appreci- ation? Partly, this is because people canā€™t venture underground to see the amazing architecture of the underground city. But it is also because until quite recently many peopleā€” perhaps especially scientistsā€”imag- ined life as a matter of species- by- species reproduction. The most im- portant interspecies interactions, in this worldview, were predator- prey relations in which interaction meant wiping each other out. Mutualistic relations were interesting anomalies, but not really necessary to under- stand life. Life emerged from the self- replication of each species, which faced evolutionary and environmental challenges on its own. No spe- cies needed another for its continuing vitality; it organized itself. This self- creation marching band drowned out the stories of the under- ground city. To recover those underground stories, we might reconsider the species- by- species worldview, and the new evidence that has begun to transform it.

When Charles Darwin proposed a theory of evolution through nat- ural selection in the nineteenth century, he had no explanation for her- itability. Only the recovery in 1900 of Gregor Mendelā€™s work on genetics

140 interlude

suggested a mechanism by which natural selection could produce its effects. In the twentieth century, biologists combined genetics and evo- lution and created the ā€œmodern synthesis,ā€ a powerful story about how species come into being through genetic differentiation. The early- twentieth- century discovery of chromosomes, structures within cells that carry genetic information, gave palpability to the story. Units of heredityā€”genesā€” were located on chromosomes. In sexually reproduc- ing vertebrates, a special line of ā€œgerm cellsā€ was found to conserve the chromosomes that give rise to the next generation. (Human sperm and eggs are germ cells.) Changes in the rest of the bodyā€” even genetic changesā€”should not be transmitted to offspring as long as they do not affect the germ cellsā€™ chromosomes. Thus the self- replication of the spe- cies would be protected from the vicissitudes of ecological encounter and history. As long as the germ cells were unaffected, the organism would remake itself, extending species continuity.

This is the heart of the species self- creation story: Species reproduc- tion is self- contained, self- organized, and removed from history. To call this the ā€œmodern synthesisā€ is quite right in relation to the questions of modernity that I discussed in terms of scalability. Self- replicating things are models of the kind of nature that technical prowess can control: they are modern things. They are interchangeable with each other, because their variability is contained by their self- creation. Thus, they are also scalable. Inheritable traits are expressed at multiple scales: cells, organs, organisms, populations of interbreeding individuals, and, of course, the species itself. Each of these scales is another expression of self- enclosed genetic inheritance, and thus they are neatly nested and scalable. As long as they are all expressions of the same traits, research can move back and forth across these scales without friction. Some hint of coming problems appeared in this paradigmā€™s excesses: when researchers took scalability literally, they produced bizarre new stories of the gene in charge of ev- erything. Genes for criminality and creativity were proposed, sliding freely across scales from chromosome to social world. ā€œThe selfish gene,ā€ in charge of evolution, required no collaborators. Scalable life, in these versions, captured genetic inheritance in a self- enclosed and self- replicating modernity, indeed, Max Weberā€™s iron cage.

The discovery of the stability and self- replicating properties of DNA in the 1950s was the jewel in the crown of the modern synthesisā€” but

traCking 141

also the opening to its undoing. DNA, with associated proteins, is the material of chromosomes. The chemical structure of its double helix strands is both stable and, amazingly, able to replicate exactly on a newly built strand. What a model for self- contained replication! The replication of DNA was mesmerizing; it formed an icon for modern sci- ence itself, which requires the replication of results, and thus research objects that are stable and interchangeable across experimental itera- tions, that is, without history. The results of the replication of DNA can be tracked at every biological scale (protein, cell, organ, organism, pop- ulation, species). Biological scalability was given a mechanism, strength- ening the story of thoroughly modern lifeā€” life ruled by gene expres- sion and isolated from history.

Yet DNA research has led in unexpected directions. Consider the trajectory of evolutionary developmental biology. This field was one of the many that emerged from the DNA revolution; it studies genetic mu- tation and expression in the development of organisms, and the impli- cations of this for speciation. In studying development, however, re- searchers could not avoid the history of encounters between an organism and its environment. They found themselves in conversation with ecol- ogists, and suddenly they realized they had evidence for a type of evolu- tion that had not been expected by the modern synthesis. In contrast to the modern orthodoxy, they found that many kinds of environmental effects could be passed on to offspring, through a variety of mecha- nisms, some affecting gene expression and others influencing the fre- quency of mutations or the dominance of varietal forms.6

One of their most surprising findings was that many organisms de- velop only through interactions with other species. A tiny Hawaiian squid, Euprymna scolopes, has become a model for thinking about this process.7 The ā€œbob- tailed squidā€ is known for its light organ, through which it mimics moonlight, hiding its shadow from predators. But juve- nile squid do not develop this organ unless they come into contact with one particular species of bacteria, Vibrio fischeri. The squid are not born with these bacteria; they must encounter them in the seawater. Without them, the light organ never develops. But perhaps you think light organs are superfluous. Consider the parasitic wasp Asobara tabida. Females are completely unable to produce eggs without bacteria of the genus Wolba- chia.8 Meanwhile, larvae of the Large Blue butterfly Maculinea arion are

142 interlude

unable to survive without being taken in by an ant colony.9 Even we proudly independent humans are unable to digest our food without helpful bacteria, first gained as we slide out of the birth canal. Ninety percent of the cells in a human body are bacteria. We canā€™t do without them.10

As biologist Scott Gilbert and his colleagues write, ā€œAlmost all devel- opment may be codevelopment. By codevelopment we refer to the abil- ity of the cells of one species to assist the normal construction of the body of another species.ā€11 This insight changes the unit of evolution. Some biologists have begun to speak of the ā€œhologenome theory of evo- lution,ā€ referring to the complex of organisms and their symbionts as an evolutionary unit: the ā€œholobiont.ā€12 They find, for example, that associ- ations between particular bacteria and fruit flies influence fruit fly mat- ing choice, thus shaping the road to the development of a new species.13 To add the importance of development, Gilbert and his colleagues use the term ā€œsymbiopoiesis,ā€ the codevelopment of the holobiont. The term contrasts their findings with an earlier focus on life as internally self- organizing systems, self- formed through ā€œautopoiesis.ā€ ā€œMore and more,ā€ they write, ā€œsymbiosis appears to be the ā€˜rule,ā€™ not the excep- tion. . . . Nature may be selecting ā€˜relationshipsā€™ rather than individuals or genomes.ā€14

Interspecies relations draw evolution back into history because they depend on the contingencies of encounter. They do not form an inter- nally self- replicating system. Instead, interspecies encounters are always events, ā€œthings that happen,ā€ the units of history. Events can lead to rel- atively stable situations, but they cannot be counted on in the way self- replicating units can; they are always framed by contingency and time. History plays havoc with scalability. The only way to create scalability is to repress change and encounter. If they canā€™t be repressed, the whole relation across scales must be rethought. When British conservationists tried to save the Large Blue butterfly, mentioned above, they could not assume that a mating population could by itself reproduce the species, although, according to the modern synthesis, populations are formed from individuals formed by genes. They could not leave out the ants without which the larvae cannot survive.15 Large Blue butterfly popula- tions are thus not a scalable effect of the butterfliesā€™ DNA. They are non- scalable sites of interspecies encounter. This is a problem for the mod-

traCking 143

ern synthesis, because population genetics was from the early twentieth century at the core of evolution- without- history. Might population sci- ence need to step aside for an emergent multispecies historical ecology? Might the arts of noticing I discuss be at its core?16

Reintroducing history into evolutionary thinking has already begun at other biological scales. The cell, once an emblem of replicable units, turns out to be the historical product of symbiosis among free- living bacteria.17 Even DNA turns out to have more history in its amino- acid sequences than once thought. Human DNA is part virus; viral encoun- ters mark historical moments in making us human.18 Genome research has taken up the challenge of identifying encounter in the making of DNA. Population science cannot avoid history for much longer.19

Fungi are ideal guides. Fungi have always been recalcitrant to the iron cage of self- replication. Like bacteria, some are given to exchanging genes in nonreproductive encounters (ā€œhorizontal gene transferā€); many also seem averse to keeping their genetic material sorted out as ā€œindivid- ualsā€ and ā€œspecies,ā€ not to speak of ā€œpopulations.ā€ When researchers studied the fruiting bodies of what they thought of as a species, the ex- pensive Tibetan ā€œcaterpillar fungus,ā€ they found many species entan- gled together.20 When they looked into the filaments of Armillaria root rot, they found genetic mosaics that confused the identification of an individual.21 Meanwhile, fungi are famous for their symbiotic attach- ments. Lichen are fungi living together with algae and cyanobacteria. I have been discussing fungal collaborations with plants, but fungi live with animals as well. For example, Macrotermes termites digest their food only through the help of fungi. The termites chew up wood, but they cannot digest it. Instead, they build ā€œfungus gardensā€ in which the chewed- up wood is digested by Termitomyces fungi, producing edible nutrients. Researcher Scott Turner points out that, while you might say that the termites farm the fungus, you could equally say that the fungus farms the termites. Termitomyces uses the environment of the termite mound to outcompete other fungi; meanwhile, the fungus regulates the mound, keeping it open, by throwing up mushrooms annually, cre- ating a colony- saving disturbance in termite mound- building.22

Our metaphorical language (here termite ā€œfarmingā€) sometimes gets in the way and sometimes throws up unexpected insights. One of the most common metaphors in talk of symbiosis is ā€œoutsourcing.ā€ You

144 interlude

could say the termites outsource their digestion to fungi, or, alterna- tively, that the fungi outsource food gathering and niche building to termites. There are lots of things wrong with comparing biological pro- cesses to contemporary business arrangements, too many, indeed, to catalogue. But perhaps there is one insight here. As in capitalist supply chains, these chains of engagement are not scalable. Their components cannot be reduced to self- replicating interchangeable objects, whether firms or species. Instead, they require attention to the histories of en- counter that maintain the chain. Natural history description, rather than mathematical modeling, is the necessary first stepā€” as in the econ- omy. Radical curiosity beckons. Perhaps an anthropologist, trained in one of the few remaining sciences that values observation and descrip- tion, might come in handy.

Prologue Autumn Aroma

 
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Practical manual Immunology

Practical manual Immunology.

Immunology

From Immunology 5e, Goldsby et al.

Practical Program 2016

Prof. Peter Smooker

 

 

Practical manual Immunology

2

Coordinator:

Prof. Peter Smooker School of Applied Sciences Biotechnology and Environmental Biology RMIT-University Plenty Road, Bundoora Melbourne VIC 3083 (: 9925 7129 Fax: 9925 7110 :: peter.smooker@rmit.edu.au Practical Class Times: Friday: 12:00 ā€“ 5:00pm Practical Class Outline: Practical 1: Day 1 Preparation of Antigen ā€“ Sonication 19 August Protein Determination ā€“ Bradford method

Preparation for ELISA ā€“ coating antigen onto microtitre plate

Day 2 ELISA 26 August

SEMESTER BREAK (29/08/2016 ā€“ 02/09/2016)

Practical 2: Day 1 Invasion & Adherence Assay 09 September Day 2 Analysis of results 16 September

Practical 3: 23 September Immuno-bioinformatics

Depending on time constraints, some methods may be demonstrated to

you. Your demonstrators will advise.

 

 

Practical manual Immunology

3

Laboratory Safety:

1. The teaching laboratories in Building 223 are PC2 laboratories and are subject to Australian Standards for Laboratories AS/NZS 2243.3:2010.

2. Long-sleeved laboratory gowns/coats are to be worn at all times in the laboratory. If you do not have a laboratory gown/coat one can be hired from the APS Preparation room after the hiring fee has been paid. These are stored in the plastic bag in a designated place in the laboratory.

3. Suitable footwear must be worn; open sandal or thongs are not acceptable. Long hair must be tied back securely as a protection from Bunsen burners and interference with work being carried out. When working with infectious material wear gloves to ensure no contamination of hands.

4. Keep your bench free of non-essential material at all times. Bags are not permitted near the laboratory benches and therefore must be stored in a designated storage area. Regard all bench tops and other surfaces as potential sources of contamination.

5. When working at the benches avoid all hand-to-mouth operations. Never smoke, eat/drink or put anything in your mouth while in the laboratory.

6. Report any accidents involving cuts, burns, broken glass or spilled cultures immediately to a demonstrator. Tissues and Sodium Hypochlorite disinfectant are provided in the event of a spill.

7. Never place contaminated pipette tips on the bench.

8. Where to discard contaminated and non-contaminated waste:

 

 

 

 

 

 

 

 

 

 

Practical manual Immunology

4

9. Use of the Fume hood: All procedures involving dangerous chemicals will be performed in the fume hood. Students will be instructed in the session.

10. Procedures before leaving the laboratory: a. Replace all empty sterile tip boxes used. b. Empty out non-sterile tips and eppendorf tubes into their snap-lock bags

and return racks to designated trolley. c. Return all plastic racks, pipettes and bottles of reagents to the designated

trolley. d. Ensure all used contaminated material is stored/disposed of appropriately. e. Clean your bench area. f. Close lids of all Biohazard Sharps containers and place them in the

designated storage area. g. Put chairs neatly under your bench. h. Wash hands thoroughly with soap and water.

 

Important Safety Considerations:

ā€¢ In immunology practical classes you are handling potentially dangerous bacterial cultures, please take care and adhere to good aseptic technique at all times.

ā€¢ Any student who may be at increased risk of infection is urged to discuss the matter confidentially with their demonstrator.

 

 

 

 

 

 

Practical manual Immunology

5

GUIDELINES FOR WRITTEN WORK IN ADVANCED IMMUNOLOGY AND CELL TECHNOLOGY:

For each of the three practicals within this subject there is a written component. Practical 1 (ELISA) and Practical 2 (Infection &Invasion) require a scientific report and Practical 3 (Bioinformatics) has an assignment sheet. All of the formal scientific reports must follow this set of guidelines in order to be passed. Please take note of the marking scheme for each report for detailed allocation of

marks for each section within the report. Word Count Limit: The upper limit for these reports is 750 words. Turnitin: All reports are to be submitted to Turnitin for similarities to other studentā€™s reports and already published works. All reports must be written in Third Person/passive text (this means no personal pronouns- No; I saw/ we saw/ we did/ we showed). A correct example of passive writing is ā€œThe data shown representsā€¦.ā€. All sections are to be written in PAST TENSE, except when mentioning a known fact, e.g. ā€œIt was shown that some cells stainedā€¦ā€

HEADINGS FOR THESE REPORTS INCLUDE:

1. Introduction & Aims

The introduction should include the necessary background information required to understand the topic (for this you will need to read current literature) and an aim/purpose of the practical. Be sure to correctly reference any information provided from a reliable source. This section is usually allocated the second most marks in a marking scheme.

2. Materials & Methods

The materials and methods section will contain all of the materials used in the practical and the procedures followed. As these are provided for you in detail you do not need to re-write these, however you must mention any changes made to a protocol and reference them accordingly.

 

3. Results

The results section should have your data organized and presented as clearly and precisely as possible. Avoid repeating results. Data presented in the results section must be the final result (either a graph or table); any raw data (All OD readings, individual group colony counts, calculations etc.) should be placed in an appendix and referred to in text.

 

 

 

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In some cases you may be required to provide results from a calculation. In this case you may show written calculations for one example within the results section and provide the answers from the rest. All other calculations should be in an appendix and referred to in text. All presented data must have a legend (a statement describing what the data represents). For tables the legend is above the table (see Example 1) for figures the legend is below the figure (see Example 2). Example 1: How to include a table in your data

Table 1) Class average of colony counts of E.coli and Salmonella on LB agar E. coli

(Adherence) Salmonella (Adherence)

E. coli (Invasion)

Salmonella (Invasion)

QC Controls

10-2 10-3 10-2 10-3 10-1 10-2 10-1 10-2 10-1 10-2

132 14 85 7 0 0 22 2 0 0

Example 2: How to include a figure in your data

Figure 2) ELISA Detection of Salmonella Typhymurium 82/6915 H-antigen using anti-Salmonella H-antigen antibody and anti-Shigella antibody (negative control). 4. Discussion & Final Conclusions

The discussion section is where you ā€œdiscussā€ the results you saw and how it applies to current scientific research.

Any unexpected result should be discussed, i.e. if something gave a positive response and it was supposed to be negative. However donā€™t spend the entire discussion on the reasons why the experiment did/didnā€™t respond as expected. Any references mentioned in the introduction or in literature can be used as a

0

0.5

1

1.5

2

2.5

3

1 4 16 64 256 1024

O D 4 50

Titre

ELISA for Detection of Salmonella H- antigen

1/400 anti-Salmonella

1/400 Anti-Shigella

 

Anti-Salmonella antibody

Anti-Shigella antibody

 

 

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comparison to our experiments. Use this real knowledge to explain the relevancy of our model practical experiment. Finally there should be a sentence or two concluding statement that summarizing the findings of this report.

This section is usually allocated the most marks in a marking scheme. 5. References

The Referencing section is important part of any scientific communication. Any comment or result that is not your own must be referenced, unless it is widely accepted or known information (e.g., ā€œEscherichia coli is a gram negative bacillusā€ does not need to be referenced).

Any information retrieved from a journal article or website on the Internet must be written in your own words and properly referenced. It is only acceptable to take material word-for-word from another source if you place in within a parenthesis. Direct quotations such as these are generally boring to read and should only be used if there is a special reason.

All reports must have in-text referencing as well as a reference list.

WIKIPEDIA and its constituent websites (anything with a Wiki attached or forum related references) are NOT suitable referencing material!!!

You also cannot reference spoken sources (like lecturers or demonstrators).

It is highly recommended that you use a Referencing Manager program (like Endnote/Procite/Refworks/Mendeley, etc.) to keep references properly managed. They will generate a reference list and will ensure the formatting of references automatically.

Failure to include references will result in a zero mark for the report.

6. Appendices

The appendix section includes any supplementary data or material that does not belong within the report. All raw data and calculations go in this section numbered separately.

Example:

Appendix 1: Raw calculations for the CFU/mL of E. coli and Salmonella for infection and

invasion assay.

OD600 (E.coli) = 2.65 (2.65 * 7.65) – 0.3 = 19.97 X 109 CFU/mL

 

 

 

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BEFORE SUBMITTING:

ā€¢ Keep your report concise. DONā€™T WAFFLE.

ā€¢ To get the highest possible marks, follow the individual marking scheme and these guidelines for each practical.

ā€¢ Make sure the in text citations match the references list.

ā€¢ We understand that you all come from different backgrounds, so to ensure your report follows correct spelling, language and formatting it would be beneficial if you proof-read your assignment and perhaps get someone else to proof-read it to make sure any spelling/grammatical errors are not overlooked.

ā€¢ Ensure all bacterial names are italicised and written out in full once before you start using abbreviations, e.g. ā€œEscherichia coli (E. coli) is a gram-negative rod shaped bacteria. E. coli is a commonly studied organism.ā€

ā€¢ Ensure all Chemical names are written out in full once before you start using abbreviations, e.g. Bovine Serum Albumin (BSA)

ā€¢ Any report submitted without one of abovementioned sections (with the possible exception of an appendix) will be marked down.

ā€¢ Any report submitted after the assigned due date (without prior extension or confirmation from Prof. Peter Smooker) will receive a penalty as per the course guide.

ā€¢ Reports submitted to Turnitin must be the same report as the one submitted for marking. Any report that has is found to be plagiarised from another student or published works will receive a mark of zero

 

 

 

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PRACTICAL 1

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) Background (ELISA) Salmonella enterica subsp. enterica serovar Typhimurium is a Gram-negative, rod-shaped, flagellated, facultative anaerobic bacterium. It is a member of the genus Salmonella. Many of the pathogenic serovars of the S. enterica species are in this subspecies. Salmonella are found worldwide in cold and warm-blooded animals (including humans), and in the environment. They are commonly the cause illnesses such as typhoid fever, paratyphoid fever, and foodborne illness(1). Serotyping is the process by which the Salmonella genus is classified into further serovar subtypes is due to immunogenic surface marker variation in the O-polysaccharide (O-Antigen) and the flagellin protein (H-antigen). Fritz Kauffmann and P. Bruce White initially proposed serotyping in 1934 as a classification scheme for Salmonella (2). In this practical, we will determine the presence of a specific antigen in lysates of Salmonella and E. coli. We will be using the Bradford Assay for the determination of protein concentration, and an Enzyme-Linked-Immunosorbent Assay (ELISA) in order to determine the presence of H-antigen of Salmonella Typhimurium 82/6915. E. coli DH5Ī± will be used as a negative control as it does not express the same H-antigen as Salmonella. Day 1: A. Isolation of antigen by sonication B. Determination of protein concentration using the Bradford assay C. Coating antigen onto microtitre plate Day 2: D. Indirect ELISA References: 1. HERIKSTAD, H., Y. MOTARJEMI, R. TAUXE, nbsp, and V. 2002. Salmonella

surveillance: a global survey of public health serotyping. Epidemiology & Infection 129:1-8.

2. McQuiston, J. R., R. J. Waters, B. A. Dinsmore, M. L. Mikoleit, and P. I. Fields. 2011. Molecular Determination of H Antigens of Salmonella by Use of a Microsphere- Based Liquid Array. Journal of Clinical Microbiology 49:565-573.

 

 

 

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Day 1 A. ISOLATION OF ANTIGEN BY SONICATION Background Sonication can be defined as the disruption of cells by high frequency sound waves. This technique is commonly used to isolate bacterial proteins and involves harvesting and washing of the bacterial cells, followed by sonication on ice (see method below). The cell lysate is then centrifuged at high speed to recover the bacterial proteins, which are found in the supernatant. These proteins can then be used as soluble antigens in the Enzyme Linked Immunosorbent Assay (ELISA). In this practical we are using a strain of Salmonella Typhimurium, which expresses flagella protein (H-antigen). When a lysate is made, it will contain this antigen, in addition to all the other bacterial proteins. As a (negative) control, we use a strain that does not express the antigen (E coli DH5Ī±). Reagents and Equipment – E coli DH5Ī± / Salmonella Typhimurium 82/6915 cultures – 10 mM Tris-HCl, pH 7.4 – 10 ml centrifuge tubes – Benchtop centrifuge – Sonicator – Esky + ice – Pipettes and tips (non sterile) – Eppendorf tubes (non sterile)

 

 

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Procedure 1. A 10 ml overnight culture of E. coli DH5Ī± or Salmonella Typhimurium

82/6915 is used to inoculate 150 ml LB and is grown to an OD of 0.3- 0.6. The bacteria from ten millilitre samples of these are collected by centrifugation at 5,500 rpm, and the pellets stored frozen.

This step has been done for you. Students start here: Label your tubes with your group initials 2. Re-suspend the cell pellet in 1 ml of 10 mM Tris-HCl, pH 7.4 and

centrifuge at 5,500 rpm for 2 minutes. Remove the supernatant and then repeat the re-suspension and centrifugation steps.

3. Resuspend cells in a final volume of 2 ml of 10mM Tris-HCl, pH 7.4 and place tubes on ice for sonication.

The demonstrators will combine the cultures of E. coli DH5Ī± and Salmonella Typhimurium 82/6915, as a larger volume of culture is required to assist in

sonication. N.B Ear muffs must be worn at all times during sonication of the cells 4. Sonicate cells (as demonstrated) using the following programme:

 

Pulse ā€“ 30 secs.

6 times Rest ā€“ 30 secs.

Demonstrators will aliquot sonicated samples back into Eppendorf tubes and return to students. Label your tubes. 5. Transfer the cell lysate to Eppendorf tubes and centrifuge at 14,000 rpm

for 5 minutes. Transfer the supernatant to a clean Eppendorf tube.

This part will be demonstrated to you. Your demonstrators will advise.

 

 

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B. PROTEIN DETERMINATION: BRADFORD METHOD Background The Bradford method utilises the ability of a dye, for example Bio-Rad Protein Assay Dye Reagent, to bind to proteins (specifically arginine, histidine and the aromatic amino acids). Binding of the dye to different amounts of a standard protein, usually Bovine Serum Albumin (BSA) is quantitated by measuring the absorbance at 600 nm and used to generate a standard curve. This can then be used to quantify the unknown protein(s). Reagents and Equipment

– Eppendorf tubes (non sterile) – 96-well microtitre plates – Pipettes and tips

– Bio-Rad Protein Assay Dye Binding Reagent:

This reagent is commercially purchased as a concentrate o Dilute 1 part Dye Binding Reagent Concentrate in 4 parts Distilled,

Deionised (DDI) Water. Then filter through 0.45 Āµm filter, store at 4Ā°C, filter required amount again before use.

– BSA standard: Standard Albumin solution: 1 mg/ml – 0.15 M NaCl

 

 

 

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Procedure 1. Label tubes as follows:

Blank S1 S2 S3 S4 S5 S6 S7 S8

2. Set up the standards as follows:

ĀµL Blank S1 S2 S3 S4 S5 S6 S7 S8

BSA (1 mg/ml) 0 0 3 6 9 12 15 18 21

0.15 M NaCl 100 100 97 94 91 88 85 82 79

Final protein amount (Āµg) 0 0 3 —- —- —- —- —- —-

Fill in the blanks as how much protein is in each tube (Āµg)

3. For test samples, aliquot 10 ĀµL into an Eppendorf tube, then add 90 ĀµL of 0.15 M NaCl (This makes a 1/10 dilution).

4. Add 900 ĀµL Dye Binding Reagent to each tube, mix thoroughly, stand for 2

minutes at room temperature.

5. Aliquot 2 x 200 ĀµL to 96-well microtitre plates as shown below and read on ELISA reader at 595 nm. Pipette standards and test samples as follows:

Make sure that there are no air bubbles present as this can alter the reading.

1 2 3 4 5 6 7 8 9 10 11 12 A B B B B B S1 S2 S3 S4 S5 S6 S7 S8 C S1 S2 S3 S4 S5 S6 S7 S8 D T1 T2 E T1 T2 F G H

B ā€“ Blank S1 to S8 ā€“ Standards T1 & T2 ā€“ Samples

KEEP YOUR SAMPLE as it will be used in the next step!

Sample

 

 

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Calculations to determine your protein sample: Students will be provided an excel spreadsheet containing Raw data (OD595 readings) from the Bradford Assay. From these results students will create a standard curve showing the absorbance versus protein amount (Āµg) for the standards and this can be used to determine the protein content of your cell lysate. Creating the Standard Curve In Excel:

Use these instructions in conjunction with Bradford calculations tutorial given in class.

1. The first thing to be done is to average all of your blank wells, e.g., Average Blank =Average(A1:A4).

2. Then you must average all of your standards in duplicates E.g., S1 (average) =Average(C1:D1), S2 (average) =Average(C2:D2) and so on.

3. Finally you must do the same for all of your samples. 4. Once you have done this, you must then normalise against background. In order to do

this you take all OD readings and minus the Average Blank. 5. Once all of the readings are normalised, you can create the standard curve using the BSA

standard protein concentration (calculated page 13, step 2) as the X-axis, and the Standard OD readings (minus blank) as the Y-axis. To do this fill in the standard concentrations in a column next to standard OD readings and highlight both columns, then go to Chart and select Scatter plot (Marked Scatter).

6. Once you have a graph on the page (ensuring Protein Concentration is X-axis and OD readings is Y-axis) Right click on one of the points and select ā€œAdd trend lineā€. Once this opens up select ā€œLinearā€ and ensure the intercept = 0, and make the equation and R2 value visible on the graph.

7. From the equation you can determine your total protein concentration. Calculating your protein concentration: Once you have your standard curve you can calculate the protein concentration (Āµg/Āµl) of your original sample (obtained in part A of this practical). You will need to take into account the dilution factor and the volume of your diluted sample (from step 3 of this procedure).

This is an example of the calculations required for determining your protein

concentration. Use these to assist your own calculations

but do not use the values included.

If your equation is

y=0.0246x and your Average OD (minus blank) is 0.13338, substitute y for

your OD value: y=0.0246x

0.13338=0.0246x

1. Then you solve for x (your protein concentration) y=0.0246x 0.13338=0.0246x

x=0.13338/0.0246 x= 5.422Āµg (in 10 ĀµL)

= 5.422Āµg/10

= 0.5422 Āµg/ĀµL this is your final concentration!!!

(This is the volume we added to the assay, so to determine Āµg/ĀµL (final)

concentration you must divide by 10).

 

 

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B. COATING ANTIGEN ONTO MICROTITRE PLATE Reagents and Equipment – Antigen (from last weekā€™s practical) – Coating buffer (0.016 M Na2CO3, 0.034 M NaHCO3, pH 9.6) – 96-well flat bottom ELISA plate – Yellow tips ProcedureS Note that some wells are treated differently to others- carefully follow the protocol below (particularly note the underlined points). 1. Use Bradford assay results to estimate total protein content in the

samples, and dilute protein samples to 0.005 Āµg/ĀµL in Coating buffer (which has been provided).

 

 

 

 

Use C1V1=C2V2 calculation to determine dilution of antigen:

Example:

If my lysate concentration is 0.5422 Āµg/ĀµL then the volume I need to add is:

C1V1=C2V2 V1= C2 x V2 = 0.005 Āµg/ĀµL X 3000 ĀµL = 27.665 ĀµL

C1 0.5422 Āµg/ĀµL

Now try it for yourself: If your lysate concentration is Āµg/ĀµL then the volume needed is: C1V1=C2V2 V1= C2 x V2 = 0.005 Āµg/ĀµL X 3000 ĀµL = ĀµL

C1 Āµg/ĀµL

2. Coat the wells with 100 ĀµL of diluted samples (equivalent to 0.5Āµg of antigen per well) to all wells.

3. Incubate the plate at 40C overnight.

C1= your sample lysate concentration (Āµg/ĀµL) V1= what we are trying to find out (ĀµL) C2=0.005 Āµg/ĀµL (final concentration) V2= 3 mL= 3000ĀµL (total volume required to coat a 96-well plate)

 

 

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Day 2

B. INDIRECT ELISA Background The indirect ELISA is used for the screening of antisera for specific antibodies and utilises semi-purified or purified antigen. Q: What does a direct ELISA identify? Antibodies are detected by coating the wells of microtitre plates with antigen, incubating the coated plates with test solutions containing specific antibodies, and washing away unbound antibodies. A solution containing a secondary antibody (against the test antibodies) conjugated to an enzyme such as horseradish peroxidase is then added to the plate. After incubation, unbound conjugate is washed away and substrate solution is added. After incubation, the amount of substrate hydrolysed is assessed by measuring the absorbance at 450 nm, using an ELISA plate reader. The measured amount is proportional to the amount of specific antibody in the test solution. Figure 3. Indirect ELISA to detect specific antibodies. Ag = antigen, Ab =

antibody, E = enzyme.

 

 

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Reagents and Equipment – 96-well flat bottom ELISA plate coated with antigen (prepared previously) – Yellow tips – Wash bottles – 1x PBS – PBS/Tween (1x PBS with 0.05% Tween 20) – Distilled water – Blotto (5% skim milk in PBS with 0.05% Tween 20) – Diluent (1% skim milk in PBS with 0.05% Tween 20) – Anti-Salmonella H-antigen polyclonal antibody – Anti-Shigella polyclonal antibody – Goat anti rabbit IgG ā€“ HRP conjugate – TMB substrate – 2 M H2SO4 – ELISA plate reader

Note that some wells are treated differently to others- carefully follow the protocol below (particularly note the underlined points).

1-3 done by staff)

Table 1) Template for ELISA assay.

 

 

 

 

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Procedure 1. Wash plate with 1X PBS/Tween once, and add 200 ĀµL of Blotto solution

(to all wells except A3, add 100 ĀµL PBS to A3) incubate for 1 hour at 370C with gentle shaking.

2. Discard Blotto solution, and wash wells with 3 times with PBS/Tween.

3. Add 100 ĀµL of diluent into well A1& A3, C2-12 and E2-12.

4. Add 200 ĀµL of Salmonella antiserum into well C1, and 200 ĀµL of Shigella antiserum into well E1. In well A2 & A3 add 100 ĀµL of Salmonella antiserum.

5. Make a serial dilution from C1 to C12 as following:

 

 

 

 

6. Repeat this process for Row E exactly as you have done for C.

 

 

 

7. Incubate the plate at 37oC for 1 hour with gentle shaking.

8. Discard primary antibody and wash wells 3 times with PBS/Tween.

9. Add 100 ĀµL of 1/5000 diluted Goat anti-rabbit IgG-HRP conjugate to all wells, except A2. Add 100 ĀµL of diluent in well A2.

10. Incubate the plate at 37oC for 1 hour with gentle shaking.

11. Discard antibody solution and wash 3 times with PBS/Tween, and once with distilled water.

Perform Step 12 & 13 to be completed in the Fume hood:

12. Add 100 ĀµL of TMB (tetramethylbenzidine) substrate to all wells (Blue colour will develop). Incubate the plates in dark for up to 30min (your demonstrator will define the length of incubation required).

13. Stop the reaction by addition of 100 ĀµL of 2M H2SO4 per well (TMB substrate will turn yellow).

14. The ELISA plates will be read at 450nm.

 

 

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PRACTICAL 2

ADHERENCE and INVASION ASSAYS

Background Most pathogenic bacteria causing infection require virulence determinants that enhance their ability to adhere to sites of infection and invade through the membrane. Humans possess several physical and chemical barriers to infectious agents, which are the same in all individuals. The similarity of these barriers has enabled microbial evolution that use mechanisms based on a similar theme. Adherence is the first and most important microbial mechanism for initiating the infectious disease process. Attachment or adhesion requires the involvement of a receptor on the host and a molecule on the surface of the microbe called an adhesin. Generally host receptors are carbohydrates while adhesins are usually proteins. For example, strains of E. coli possess one type of pilus, referred to as type 1 pili that bind to receptors containing the sugar mannose. Pili are adhesins found in many Gram-negative species. Gram-positive organisms, such as Streptococcus pyogenes, adhere to epithelial cells of the skin and nasopharynx. Epithelial cells are covered with the plasma glycoprotein fibronectin. Fibronectin acts as a receptor for the lipoteichoic acid adhesin of S. pyogenes. Some microbes do not remain on the epithelial surface but instead penetrate to subepithelial layers. The ability to penetrate below the epithelium is referred to as invasiveness. Salmonella Typhimurium is capable of invading the human intestinal epithelium. Salmonella, and some enteropathogenic E. coli strains, use a terminally differentiated epithelial cell, the M cell of the Peyerā€™s patch in the terminal ileum and in other gut-associated lymphoid tissue, as a portal of entry into the host. The M cells found in Peyerā€™s patches are thought to internalise luminal contents for delivery to underlying antigen-presenting cells. M cells possess fewer lysosomes and a sparse mucus layer that makes them a prime target for invasive bacterial pathogens. In this experiment, the invasiveness of Salmonella Typhimurium 82/6915 will be determined. E. coli DH5Ī± is used as a control (this strain of E. coli is not invasive). References:

1. Boyd, Robert F., Basic medical microbiology. 5th ed. 1995. Little Brown & Company (Inc.)

2. Neidhardt, Frederick C., Escherichia coli and Salmonella. 2nd ed. 1996.

ASM Press.

 

 

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Figure 4. Electron Micrograph of E. coli adhering to the intestinal epithelium (From H. W. Moon, B. Naggy, and R. E. Isaacson, J. Infect. Dis. 136[Suppl.]:124, 1977)

Cancer Cells in Culture Both normal cells and cancer cells can be cultured in vitro in the laboratory. However, they behave quite differently. Normal cells pass through a limited number of cell divisions (50 is about the limit) before they decline in vigor and die. This is probably caused by their inability to synthesize telomerase. Cancer cells may be immortal; that is, proliferate indefinitely in culture. For example INT407 cells are cultured in laboratories around the world. They are all descended from cells removed from the human embryonic intestinal epithelium. Cancer cells in culture produce telomerase.

 

 

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Day 1 Materials

– DMEM (Dulbeccoā€™s Modified Eagle Medium) – Salmonella Typhimurium 82/6915 culture – E. coli DH5Ī± culture – 24-well plate (12 wells seeded with INT407 cells) – PBS (phosphate buffered saline) – Gentamycin (200 Āµg/mL) – Triton X-100 (0.1%) – LB agar plates – Sterile spreaders – Sterile Eppendorf tubes – Plastic dropper – Incubator (37Ā°C, 5% CO2)

Procedure Note: Salmonella Typhimurium 82/6915 is a non-attenuated wild type strain and hence, is virulent and infectious. Wear gloves at all times during the experiment. 1. Each pair is provided with Salmonella Typhimurium 82/6915 and

Escherichia coli DH5Ī±. Determine the cell count for both bacterial cultures. You are given the optical density reading of the cell growth at 600 nm. Using the following formula, dilute your bacteria to 5 x 107 CFU in a total volume of 1 mL in 1 x DMEM.

(OD600 x 7.65) ā€“ 0.3 =? X 109 CFU/mL

2. You are supplied with a 24 well plate, which has 12 wells containing a

monolayer of INT407 cells. The cells have been seeded out at 105 cells per well. Wash the monolayer in each well gently with PBS 3 times (500 Āµl of PBS each time).

Growing INT407 cells are adherent ā€“ that means, they stick to the bottom of the well and multiply. Hence, you can discard growth medium/PBS by carefully tipping the medium off.

 

 

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3. Label the plate with columns 1-3 and rows a-d (Figure 5).

 

 

Blank = 1 x DMEM Column 1 ā€“ Control (blank ā€“ DO NOT ADD ANY BACTERIA) Column 2 ā€“ Adhesion and invasion (Stripes) Column 3 ā€“ Invasion only (Spots) 4. Add 200 ĀµL of 1 x DMEM to all wells in column 1. 5. Add 200 ĀµL of 5 x 107/mL bacteria (in DMEM) according to the diagram

shown above. This will equate to 1 x 107 bacteria per well.

6. Incubate at 37 Ā°C in CO2 incubator for 1.5 hour.

During this incubation both E. coli and Salmonella will adhere to the INT407 cells. Only Salmonella will invade the INT407 cells, as E. coli DH5Ī± is incapable of invading host cells. During this incubation period, label the tubes you will need for subsequent steps as outlined on page 23.

7. Wash the monolayer gently with PBS three times (500 Āµl of PBS each time).

8. To columns 1 and 3 add 200 ĀµL of 200 Āµg/mL gentamycin. To column 2 add 400 ĀµL/well of 0.1% Triton X-100.

A

B

C

D

3 1 2

Blank SALMONELLA E. COLI

FIGURE 5

 

 

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Note: Gentamycin kills the bacteria that have adhered but not those that have invaded. Triton X-100 lyses INT407 cells releasing any bacterial cells that have invaded the INT407 cells.

9. Incubate at 37Ā°C in CO2 incubator for 15 minutes.

10. Remove 20 ĀµL from wells A2 – D2 (mix the well with a pipette first).

Immediately return the plate to the incubator.

11. Make serial dilutions (in 1X PBS) of the samples from the 4 wells of

column 2 and plate out 100 ĀµL of 10-2 and 10-3 dilutions onto LB agar. Label these plates adhesion (E. coli or Salmonella).

 

12. Incubate column 1 and 3 for a further 45 minutes at 37Ā°C in CO2 incubator.

13. Wash the monolayer three times with PBS.

14. Add 400 ĀµL of 0.1% Triton X-100 to columns 1 and 3 and incubate at

37Ā°C in CO2 incubator for 15 minutes. 15. Make serial dilutions (in 1X PBS) of the 4 wells of column 3 and only A1

from column 1 plate out 100 ĀµL of 10-1 and 10-2 dilutions onto LB agar. Label plates from column 3 invasion (E. coli or Salmonella) and that from A1 control

 

 

16. Incubate LB agar plates at 37Ā°C overnight.

 

 

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Table 2. Labelling instructions for Eppendorf tubes for dilutions in steps 11-15.

 

Step Well Sample Dilutions req. for plating

Tube labels No.

Tubes req.

11 Adhesion and Invasion

A2 E. coli 10-2, 10-3 A2 E -2 and A2 E -3 3

B2 E. coli ā€œ B2 E -2 and B2 E -3 3

C2 S. Typhimurium ā€œ C2 S -2 and C2 S -3 3

D2 S. Typhimurium ā€œ D2 S -2 and D2 S -3 3

TOTAL (step 9) 12 tubes

 

15 Invasion only

A3 E. coli 10-1, 10-2 A3 E -1 and A3 E -2 2

B3 E. coli ā€œ B3 E -1 and B3 E -2 2

C3 S. Typhimurium ā€œ C3 S -1 and C3 S -2 2

D3 S. Typhimurium ā€œ D3 S -1 and D3 S -2 2

 

A1 Blank 10-1, 10-2 A1 B -1 and A1 B -2 2

TOTAL (step 11) 10 tubes

Total for assay 22 tubes

 

 

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Day 2 Count colonies on plates and record all results as a group. Use following table to record group results. You will have duplicate plates for the same sample at the same dilution. Record your average colony count in the table.

Group

ADHERENCE INVASION QC

BLANK E. coli Salmonella E. coli Salmonella

10-2 10-3 10-2 10-3 10-1 10-2 10-1 10-2 10-1 10-2

1

2

3

4

5

6

7

8

Check for any colonies on control plates and record all results.

Results are expressed as a percentage of the total number of colonies adhered or invaded divided by the inoculum.

Total %

Adhered Bacteria Total %

Invaded Bacteria Further, calculate % of bacterial cells invaded compared to those that adhered

using the total number of cells adhered.

% Invasion compared to

adhesion

 

= Number of colonies adhered X dilution factor X 100 Original inoculum

=

= Number of colonies invaded X dilution factor X 100 Number of colonies adhered =

= Number of colonies invaded X dilution factor X 100 Original inoculum =

 

 

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PRACTICAL 3

Immunology Prac 3. Bioinformatics

For your write-up of this practical, give an introduction on computational methods of epitope prediction (approx half a page), noting both T and B cell, and the differences. Answer the questions posed below in bold, with reasoning. 1. Use the following sequence: >gi|45384056|ref|NP_990483.1| ovalbumin [Gallus gallus] MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRT QINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASR LYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGII RNVLQPSSVDSQTAMVLVNAIVFKGLWEKTFKDEDTQAMPFRVTEQESKPV QMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINF EKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLS GISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFL FCIKHIATNAVLFFGRCVSP Using http://www.syfpeithi.de/home.htm , determine which is the dominant CD8 epitope in mice. Use H2-kb, and search for octomers. Try human HLA-B*08, again octomers. Is the same peptide dominant? Is it predicted that HLA-B*08 can present this peptide? This peptide is widely used in antigen presentation assays in immunology. Does BIMAS http://www-bimas.cit.nih.gov/molbio/hla_bind/ also predict this as the dominant peptide? Using the Hopp-Woods tool at http://www.vivo.colostate.edu/molkit/hydropathy/index.html find the most hydrophilic region of the protein. Where is it? Why might it be important to know this? 2. If you were going to design a peptide vaccine to (A) induce CTLā€™s, or (B) induce antibodies from the following sequence, which regions would you choose? Assume you will test in H2-Db mice. LPKSFDARVEWPHCPSISEIRDQSSCGSCWAFGAVEAMSDRICIKSKGKHK PFLSAENLVSCCSSCGMGCNGGFPHSAWLYWKNQGIVTGDLYNTTNGCQP YEFPPCEHHVIGPLPSCDGDVETPSCKTNCQPGYNIPYEKD 3. Using PAProc, http://www.paproc.de/ predict the proteolytic products from ovalbumin after processing by the human wild-type 1 proteosome.

 

 

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Is the dominant H2-kb epitope in mice available for loading onto human MHC? Around what proportion of the protein would be available for presentation? 4. B cell epitope prediction. These are much more complex, as often epitopes represented by antibody are not linear, and therefore are derived from different regions of the polypeptide chain. If detailed knowledge of the protein structure is known, then prediction may be easier. Generally, prediction revolves around the prediction of surface regions. Go to http://tools.immuneepitope.org/tools/bcell/iedb_input Input the ovalbumin sequence. Use each of the algorithms to see the output. What does each predict for the ovalbumin sequence, and why is this important? Are any of the peptides predicted by more than one method?

 

 

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APPENDIX: Material Safety Data Sheet for Tetramethyl Benzidine (TMB)

EMERGENCY OVERVIEW NON-CARCINOGENIC ANALOG OF BENZEDINE, MILD OXIDIZING AGENT

PRODUCT IDENTIFICATION Form: liquid Colour: Colourless to light yellow Boiling point: approx. 100 Ā°C Solubility in water: miscible: Stability: The product is stable for a minimum of 1 year at 2Ā°-25Ā°C. Protect from direct UV light. Avoid elevated temperatures.

Incompatibility: Strong oxidizing agents and metals.

 

HEALTH HAZARD INFORMATION Primary routes of Exposure: Routes of exposure: may be absorbed by ingestion. Inhalation: Inhalation of vapours is unlikely at normal temperature. Skin: skin contact may cause irritation. Eyes: Splashes may cause irritation. Ingestion: May cause irritation. Accidental exposure/spillage information: Personal precautions: Use personal protection. Ventilate area of leak or spill. Methods for cleaning up/collecting: absorb spillage with an inert material and place spillage in a suitable container for disposal.

 

PRECAUTIONS FOR USE Personal protective equipment: respiratory protection: none with normal use. Skin protection: use gloves of rubber or plastic. Eye protection: wear tight fitting safety goggles when risk of splashing.

 

 

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First Aid: Inhalation: Remove to fresh air. Keep at rest. If needed: get medical attention. Skin contact: Remove contaminated clothing and wash with soap and water. Eye contact: Immediately flush with water or physiological salt water, holding eye lips open, remember to remove contact lenses, if any. If needed: get medical attention.

Ingestion: Rinse mouth and drink plenty of water. Keep under surveillance. If needed get medical attention.

Information: show this Safety Data Sheet to doctor or emergency ward. SAFE HANDLING INFORMATION Flammability: Not flammable. Personal precautions: Use personal protection. Ventilate area of leak or spill. Methods for cleaning up/collecting: absorb spillage with an inert material and place spillage in a suitable container for disposal.

Safe storage: in a well closed container. Storage is recommended at 2 to 25Ā°C. Disposal: Do not empty large amounts into water or drains.

Practical manual Immunology

 
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Biology 121 Lab

Biology 121 Lab. 1. A.Ā  Briefly, outline the main steps of the photosynthetic pathway.Ā  Be sure to indicate how the light-dependent and light-independent reactions are coupled.

B. Why is Chlorophyll-a central to the light-dependent reactions?Ā  AND ā€“ What important role(s) do accessory pigments play in this process?

2. Consider last weekā€™s laboratory exercise concerning carbon-fixation in an aquatic plant.Ā  Recall, we used BTB to monitor pH of the surrounding medium as a proxy for CO2 concentration.Ā Ā Ā  Why might we expect to see a DECREASE in pH in the plant/dark tube?Ā  (i.e., What metabolic process might contribute to this result?)
3.Ā  If a farm pond, stocked with fish and plants, were measured for pH at sunrise and sunset what would be the results?Ā  Why?
4. Describe the redox reaction that was the subject of the ā€œHill reactionā€.Ā  What is normally the final electron acceptor and what did we use as a substitute? Why did we use what we used?

CELLULAR RESPIRATION

 

 

What is CELLULAR respiration?

chemical E (glucose) + O2 ā†’ ā€œbiochemical currencyā€ (ATP)

C6H12O6 + 6 O2 ā†’ 6 CO2 + 6 H2O + E

ļƒ˜ā€Æ Oxygen (O2) is ESSENTIAL for AEROBIC respirationā€¦

ļƒ˜ā€Æ 4 main stepsā€¦1 is common to both aerobic AND anaerobic respiratory pathwaysā€¦

 

 

Aerobic vs. Anaerobic Respiration

ļ¬ā€Æ Aerobic ļƒ¼ā€Æ requires O2 ļƒ¼ā€Æ 4 main steps ļƒ¼ā€Æ yields up to 38 ATP glucose-1 ļƒ¼ā€Æ obligate aerobes, facultative anaerobes

ļ¬ā€Æ Anaerobic ļƒ¼ā€Æ NO O2 required ļƒ¼ā€Æ 1 main step ļƒ¼ā€Æ yields 2 ATP glucose-1 ļƒ¼ā€Æ obligate anaerobes

 

 

The Mitochondrion

ā€¢ā€Æ Glucose is broken down in the cytoplasm ā€¢ā€Æ Krebā€™s Cycle occurs in the matrix ā€¢ā€Æ Electron transport occurs in/on the cristae

(envelope)

 

 

Aerobic Respiration

C6H12O6 + 6 O2 ā†’ 6 CO2 + 6 H2O + E

Step 1: Glycolysis (ā€œglycoā€ ā€œlysisā€; cytoplasm)*

glucose ā†’ 2 pyruvate + 2 ATP + 2 NADH2 (6C) (3C)

cytoplasm

glycolysis

 

 

Step 2: Pyruvate Oxidation (mito matrix)

2 pyruvate ā†’ 2 Acetyl CoA + 2 CO2 + 2 NADH2 (3C) (2C)

pyruvate oxidation

matrix

 

 

Step 3: Krebā€™s Cycle (aka TCA Cycle or Citric Acid Cycle; mito matrix)

2 Acetyl CoA + 2 Oxaloacetic Acid (2C) (4C)

2 Citric Acid + 4 CO2 + 2 ATP + 6 NADH2 + 2 FADH2 (6C)

Krebā€™s Cycle

matrix

 

 

Step 4: Oxidative Phosphorylation (aka e- transport; mito cristae)

ļ¬ā€Æ NADH2 + FADH2 are involved with e – transport

ļƒ¼ā€Æ donate e- to carriers in the transport chain ļƒ¼ā€Æ pumping of H+ ions ā†’ [ ] gradient ļƒ¼ā€Æ generation of ATP

ļ¬ā€Æ O2 is the final e- acceptor oxidative phosphorylation

cristae

 

 

out

in

mito matrix

intermembrane space

NADH2 & FADH2

ATP synthase

 

 

Summary ļ¬ā€Æ 3 ATP per NADH2 (x 10) (= 30; steps 1-3) ļ¬ā€Æ 2 ATP per FADH2 (x 2) (= 4; step 3) ļ¬ā€Æ 4 ā€œsubstrate-levelā€ ATP (= 4; steps 1 & 3)

38 TOTAL glucose-1

ļƒ˜ā€Æ Theoretical maximum = 38 ATPā€¦no system is perfect!

āˆ“ this number is rarely [if ever] achievedā€¦

 

 

Anaerobic Respiration

glucose ā†’ 2 pyruvate + 2 ATP + 2 NADH2 (6C) (3C)

2 lactate 2 ethanol + 2 CO2 (= fermentation) (3C) (2C)

– animals – plants – microbes – microbes

demand > O2 release of metabolic poison

 

 

5.1 Respiration in Peas Protocol 5.1: Germinating vs. Non-germinating

ā‡’ā€Æ Atmospheric/background CO2 level = 350-400 ppm

1.ā€Æ Obtain 25 germinating peas & blot dry 2.ā€Æ Place the peas in the respiration chamber 3.ā€Æ Place the CO2 sensor in the chamber 4.ā€Æ Wait 1 minute ā†’ begin collecting data for 5 minutes 5.ā€Æ Measure & record the weight (g) of the peas 6.ā€Æ Place the germinating peas in a beaker and place on ice for 5 minutes 7.ā€Æ Follow the instructions on pg. 4

ļƒ¼ā€Æ determine the rate of respiration (slope, m = rate; ppm CO2 min-1) ļƒ¼ā€Æ store the data for comparison with other measurements

15.ā€Æ Rinse and dry chamber 16.ā€Æ Place the CO2 sensor in the chamber with non-germinating peas 17.ā€Æ Wait 1 minute ā†’ begin collecting data for 5 minutes 18.ā€Æ Follow the instructions on pg. 4

ļƒ˜ā€Æ Use a notebook to ā€œfanā€ (i.e., clear) the sensor for 1 minute, returning the CO2 level to 300-400 ppm between EACH measurement!

 

 

5.2 Respiration in Peas Protocol 5.2: Room vs. Cold Temperature

1.ā€Æ Empty the chamber by PUTTING THE NON-

GERMINATING PEAS BACK ON THE SIDE BENCH, and ā€œclearā€ itā€¦

2. Rinse and dry chamber

3. Repeat steps 1-7 (Protocol 5.1) using COLD germinating peas

 

 

5.3 Respiration in Crickets Protocol 5.3: Room vs. Cold Temperature

1.ā€Æ Obtain 5-8 crickets & place in the respiration chamber 2.ā€Æ Place the CO2 sensor in the chamber 3.ā€Æ Wait 1 minute ā†’ begin collecting data for 5 minutes 4.ā€Æ Measure & record the weight (g) of the crickets 5.ā€Æ Place the crickets in the chamber on ice for 5 minutes (or until static) 6.ā€Æ Follow the instructions on pg. 4

ļƒ¼ā€Æ determine the rate of respiration (slope, m = rate; ppm CO2 min-1) ļƒ¼ā€Æ store the data for comparison with other measurements

7.ā€Æ Repeat steps 1-6 using COLD crickets 8.ā€Æ Rinse & dry the respiration chamber when finished

ļƒ˜ā€Æ Use a notebook to ā€œfanā€ (i.e., clear) the sensor for 1 minute, returning the CO2 level to 300-400 ppm between EACH measurement!

 

 

Do the results support your predictions? Peas ļ¬ā€Æ Germinating vs. Non-Germinating

ļƒ¼ā€Æ germinating > non-germinating ļƒ¼ā€Æ WHY?

ļ¬ā€Æ Room vs. Cold Temperature ļƒ¼ā€Æ room > cold ļƒ¼ā€Æ WHY?

Germinating

Germinating/COLD

Non-germinating

 

 

Do the results support your predictions?

Crickets ļ¬ā€Æ Room vs. Cold Temperature

ļƒ¼ā€Æ room > cold ļƒ¼ā€Æ WHY?

ļƒ˜ā€Æ What about PEAS vs. CRICKETS?? ļƒ˜ā€Æ Why is it important to ā€œnormalizeā€ by some

biological parameter (= fresh weight) for comparison?

Biology 121 Lab

 
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