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Diatoms

A Test of Water Quality of a Local Watershed(?)

Developed from Slijk

Background

• algae– phytoplankton.

• most diatoms are unicellular, • producers within the food chain• unique cell wall made of silica (hydrated silicon

dioxide-think glass!) called a frustule. – usually consist of two asymmetrical sides (sometimes

called shells or valves) with a split between them

• Fossil evidence suggests that they originated during, or before, the early Jurassic Period

1. Diatoms are a type of _________, which as a group, are responsible for a significant portion of the

oxygen in the earth’s atmosphere.

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1. Bacteria

2. Prokaryote

3. Heterotroph

4. Phytoplankton

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Diatoms are the only known organism to build a cell wall with silica, the same as that found in glass. This unique cell

wall is given a special name called a _____.

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1. Frustule

2. Gram Positive Wall

3. Peptidoglycan

4. Jurassic

What will you see?

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Day 1(see Slijk procedures)

• Collect samples from multiple locations along a continuum within watershed– Scrape sample of yellow-brown algae from top

of rock exposed to sunlight.• Use knife or edge of 50ml centrifuge tube

– Add 25ml of pondwater and mix– Transfer volume to 200ml beaker

Where on the rock should you collect your sample from?

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1. Bottom

2. Top

3. Sides

4. Anywhere, all the same

Day 1

• Cooking organics– Fill beaker to 50ml mark with 30% Hydrogen Peroxide

(H2O2)– Place on hot plate (300 degrees) in fume hood,

enclosed behind glass– Boils fast - watch it!

• Adjust location on hot plate away from center if it is going to bubble over

• Kills organics• Cook for approx. 10-15 min until rxn stops (no foam, only

bubbles)

What is the purpose of the Hydrogen Peroxide and heating the sample?

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1. Speed up cellular replication

2. Kill the living material

3. Create pores in the cell wall to enhance viewing

4. To extract the DNA from the nucleus

Day 1

• Remove from heat

• Pour into new 50ml centrifuge tube

• Leave overnight at table

Day 2

• Washes (removing peroxide)– Perform this procedure 3x– Spin in centrifuge for 15min– Draw of supernatant with syringe and tubing if

needed• Don’t disturb diatom pellet at bottom of tube• Ie. Don’t go below 15ml in your tube

– Add distilled water to 50ml again– repeat

Day 2 - mounting

• Place 4 drops of mixed solution on glass coverslips– 1 per student in group + 1 extra in case of

mistake

• Let coverslip dry at lab station overnight

Day 3 - mounting continued

• Place 1 drop of mounting oil (zrax or karo corn syrup) on slide (not coverslip!)

• Put coverslip diatom side down onto oil and gently press to spread oil a little.

• Place slide now onto hot plate and let mounting oil boil– Once completely boiling, remove slide quickly– Use coffee stirrer to gently press out air bubbles by

pressing on coverslip• Don’t tap, gently press or slip will crack

Day 3 - viewing/counting• Use Compound Light Microscope for viewing

– Get to 400x (blue) and observe first– Place 1 drop of immersion oil on coverslip while slide is on stage

and objective lens is set to low power (red)– Rotate black lens in place (longest) to observe diatoms at 1000x

now.– Complete count

• Do 1 transect per slide– Identify dominant species within transect (anywhere from 1-4)– Draw/diagram/photograph these diatoms– Count # of each dominant species compared to total # of cells

observed and calculate % of population occupied by this diatom species.

• Classify by group/genera and calculate Avg. PTI of sample (see pdf from Texas with PTI values)– 4=sensitive– 1=tolerant

• For all group slides, calculate Avg. PTI thus giving you an Avg. PTI number for the sampled area of the watershed.

• Tryblionella - Avg. PTI = 2• Thalassiosira - Avg. PTI = 2• Synedra - Avg. PTI = 2.71• Surirella - Avg. PTI = 2.4• Etc.• Take average from document of species PTI numbers

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