Algae Population Growth

•  Test hypotheses about the influence of light exposure and/or nutrient levels on population growth in algae  

•  Learn how to properly use laboratory equipment such as microscopes and hemacytometers to collect experimental data

• Practice using Excel to calculate mean and SD and make  properly labeled column graphs 

• Perform t-tests to determine significant differences between two experimental groups and write an interpretation of your results

• Write the Lab Title on a new page on the right-hand side of your notebook. *Remember to include the Lab Date!*

• Write the Background, Aim and your Hypothesis for the lab

• Draw the six required Data Tables (See Procedures, Step 3)

• Add an entry for this lab in your Table of Contents.

• Experimental Questions: How do light exposure and nutrient levels influence algal population growth?

• Model Organism: Tetraselmis algae  

• Experimental Groups: Algae populations grown in varying treatments:  high light/high nutrient,  low light/high nutrient,  high light/low nutrient

• Independent Variable: light level or nutrient level 

• Dependent Variables: population counts

1. Light hypothesis: 

I hypothesize that light levels influence algae population growth, and that algae grown in  (low light / high light ) conditions will have significantly larger population sizes. 

OR  

I hypothesize that light conditions will have no significant impact on algae population growth.

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2. Nutrient hypothesis: 

I hypothesize that nutrient levels influence algae population growth, and that algae grown in  (low nutrient / high nutrient ) conditions will have significantly larger population sizes. 

OR  

I hypothesize that nutrient conditions will have no significant impact on algae population growth.

Algae are phytoplankton

Tetraselmis  is a genus of algae that contains both freshwater and marine (salt water) species. Theses algae are classified as phytoplankton, meaning that they are microscopic 'drifter' organisms that perform photosynthesis to make their own food.  In addition to sunlight and CO2 for photosynthesis, phytoplankton require nutrients obtained from the water in order to grow, survive, and reproduce. 

Phytoplankton reproduce asexually and are very abundant in aquatic ecosystems. These photosynthesizing autotrophs serve as the base of the food web in freshwater and marine ecosystems. The are consumed by a variety of zooplankton (heterotrophic microscopic organisms that eat other organisms for food). 

Why study population growth in algae?

Environmental biologists study algae in natural ecosystems because when algae populations grow rapidly (meaning the asexual reproduction rates are high), they can cause harmful algal blooms that release toxins, contaminate water bodies, and cause mass mortalities among marine organisms. Algal blooms can have substantial negative impacts for ecosystems and economies. 

Biologists and industry professionals who perform aquaculture , the production of aquatic organisms for food, routinely grow algal cultures to feed  aquatic animals, such as mussels and clams, that are farmed for human consumption. 

Finally, scientists and engineers are interested in growing large amounts of algae for use as biofuels, which could be environmentally responsible alternative to fossil fuels.

In today's lab, you will learn techniques that biologists use for quantifying algal populations and investigate algal population growth under varied growth conditions.

Introduction to Compound Microscopes

Magnification

Compound microscopes have two sets of lenses that magnify the specimen, the ocular/eyepiece lenses and the objective lenses. The ocular/eyepiece lenses magnify the image ten times (10X). There is a wheel of three objective lenses that can be used that have different magnifications: 4X (scanning), 10X (low power), 40X (high power).

Magnification is how many times larger the microscopic image is than the real specimen. This is determined by multiplying the magnification of the objective lens by the magnification of the ocular lens (Total magnification = ocular x objective magnification). 

For example, if you look at a specimen using the 4X objective, the specimen will be magnified 10 times from the ocular and 4 times from the objective lens. ( Total magnification = 10 x 4 =400X). What will the total magnification be if you use the 10X low power objective lens?  the 40X high power objective lens?

Light

Your microscope has an on/off switch that will turn on the light. The brightness of the light can be changed by moving the sliding knob on the base of the microscope. 

A condenser is located under the stage. By adjusting the diaphragm lever on the condenser, you can regulate the amount of light entering the stage and specimen. In most instances, you do not need to move the condenser itself. Check with your instructor first if you think the condenser is incorrectly positioned.

When you look through both ocular lenses, the circle of light is the field of view of your microscope.

Stage

The stage is where you place your specimen slide. Slide clips hug your slide and keep it in place while you are viewing. Two stage control knobs hang down from the stage. One knob moves the stage forward and back and the other knob moves the stage left and right. 

Focus

On the side of your scope near the base you will see a set of two concentric knobs. The large knob is called the coarse focus, and moves the stage up (toward your objective lens) and down (away from your objective lens). The knob inside of that is the fine focus and helps you to view your specimen really clearly.

Using Hemacytometers for Counting Algal Cells

Determining significant differences between groups: t-test

In order to determine if the observed differences between your experimental groups are significant, you need to run a statistical test. 

If your experimental question examines whether there are significant differences between exactly two groups, you will use an unpaired t-test  (aka two-sample t-test) as a method to statistically compare the two samples. 

To conclude that the difference between your two groups is large enough to be considered statistically significant, the p-value from your statistical test must be less than or equal to 0.05 (p ≤ 0.05). 

You will be performing a t-test on your data data. To understand how t-tests work and what the p-value actually means, you need to know some statistical lingo.  To prep for lab, read more about the t-test here

1. Using a microscope to examine prepared slides or wet mount slides of live algae

Before we perform our algae population counts, we will practice using the compound microscopes to view a prepared slide and/or live wet mount of algae.

PREPARING A WET SLIDE OF LIVE ALGAE (IF USING)

1. Swirl the highly concentrated algae sample flask provided. 

2. Take a clean glass microscope slide and use a transfer pipette to place a few drops of highly concentrated algae sample on your slide.

3. Place a small square coverslip on top of the water.

SETTING UP YOUR MICROSCOPE

1. Use both hands to hold a microscope.  One hand holds it by the arm while the other supports its base.

2. Place the microscope gently on the bench.  Do not drag it around once seated on the bench.

3. Plug in your microscope and turn it on. Adjust the light. Move the ocular lenses closer together or further apart until you see just one circle of light when you look through both ocular lenses. 

4. Place your prepared slide or wet mount on the stage by putting it into the slide holder clip. 

MICROSCOPE FOCUSING INSTRUCTIONS

1. Start with the scan objective (4X) in position over the stage.

2.  Position the prepared slide in the center of the light beam using the stage dials. 

3. Move the stage all the way up using the coarse focus knob  

4. Looking through the ocular, slowly use the coarse adjustment knob to move the stage down until an image comes into view

5. Once your specimen comes into view, stop turning the coarse focus knob. Turn the fine focus knob in one direction and then maybe the other to focus on your specimen as clearly as possible. 

6.  Switch to the 10X low power objective lens.  You DO NOT need to change the coarse focus knob.  Use the fine focus knobs (a quarter of a turn clockwise or counterclockwise) to bring the specimen into focus.

Notice that as you increase the magnification (aka "power"), the field of view decreases (you "zoom in" on one spot) and the object may be seen in greater detail.  

2. Preparing algae for hemocytometer counting

The  lab group as a whole will choose  2 different populations of algae to count.  Each person in lab will perform at least one replicate count on an algae sample. Then all replicate counts will be pooled in a class data set to compare the population size of the two groups.

1. Gently swirl the algae stock flask. Transfer 10 ml of algae from the stock flasks into a 50 ml beaker.

2. Microwave your beaker of algae for 7 seconds. This kills the algal cells so they are able to be counted. 

3. Label the beaker with a piece of tape that contains information about the algae population treatment (e.g. the light and nutrient level) and write the word "Killed."

3. Loading the hemocytometer with algae

1. Gently rinse the hemocytometer with a squirt bottle containing distilled water. Pat dry with a papertowel Kim wipe.

2. Gently swirl your killed algae sample and draw up ~0.25 ml of algae into a sterile transfer pipette. 

3. Gently expel algae in the middle of one grid area (one side) of the hemacytometer.

4. Carefully lower the plastic coverslip on top of the grid area so that the grid area is completely covered with the sample. To minimize bubbles, place one end of the coverslip on the far end of the hemacytometer and gently lower the other size on top of the sample.

5.  Wait ~2 minutes before you begin counting to allow the algal cells to drift and settle onto the grid. 

4. Performing hemocytometer counts for each treatment

1. As you wait for the algae to settle, set your microscope to the 4x objective. Place the hemacytometer into the stage clips and use the stage controls to center the counting grid area that contains your sample.

2. Use the coarse focus knob to bring that stage all the way up. Then look through the oculars and slowly turn the course focus knob until the hemacytometer grid is in view. Then use the inner fine focus knob to make it even clearer/better-focused. You should be able to see the grid and the dead algae.

3. Orient yourself to the hemocytometer grid. Move the stage up and down and left and right to explore the hemocytometer grid. Do you see the 4 large (4x4) corner squares through your scope? Identify the upper left, upper right, lower left, and lower right corner squares.

4.  Move the stage so that the upper left corner is once again in view. Switch to the 10X objective to increase cell size for counting and adjust the fine focus if needed. 

5. Count the algae in the upper left corner (4x4) by counting one small square at a time. Record your count for each small square and repeat until you have completed all 16. *Remember that if algae are on the line, only count them if they are on the top or left side of the square!

6. Complete counts for the remaining three corners (upper right, lower left, and lower right) and record all counts in your notebook. 

5. Performing hemocytometer calculations for each treatment

1. Add up the amount of cells in each of the four corner squares and record it on your data table.

2. Take the average count from your four corners by adding the four totals together and dividing by four. Record this average value in your notebook. 

3. Multiply your average count by 10,000 to get the count per ml of water. Record this  value (# algae / ml of sea water) in your notebook.

6. Putting away your microscope

1. When finished using the microscope, use lens paper to wipe off ocular and objective lenses

2. Rotate the objective lens system so that the 4X scan power lens is facing down.

3. Unplug the scope and neatly wrap the cord.

4. Place the microscope back in its allocated space.

Analysis Step 1: Calculating Mean & SD of Algae Population replicates

You will use Excel to calculate the Mean and Standard Deviation for data from the two algae populations.

Use formulas in Excel to calculate Mean and Standard Deviation separately for the two columns of data on each tab of the Excel file.

Analysis Step 2:  Column Graph to display mean & SD counts for the two algae treatments.

You will use Excel to create a Column Graph to display mean & SD counts for Algae Treatment 1 and Algae Treatment 2. 

Use the mean and SD values calculated in your Excel file to create a properly formatted column graph.

Analysis Step 3:  t-test to compare algae in Treatment 1 and Treatment 2

You will use GraphPad to perform a t-test to determine if there was a significant difference in counts for Algae Treatment 1 and Algae Treatment 2.  You will report your data analysis and  the p-value from the t-test in your written results analysis.

Follow the t-test instructions for how to perform each t-test and what data to record in your notebook.