Purpose:

Sometimes people dump chemicals that kill bacteria onto the soil and waters of an area and change the way bacteria break down organic substances.  The purpose of this laboratory is to test whether or not a chemical may influence how quickly bacteria break down decaying matter by using an instrument to measure the BOD without a chemical added against how much is being used with the chemical added.  How can we do this?

Hint:  Oxygen is a gas, gases are matter, and matter takes up space!  So, if some oxygen was used it would leave space for something else!!  Now, nature doesn’t like empty spaces, so something would have to run in that space and fill it,  wouldn’ t it?   

            A Manometer is an instrument that can be used to measure the differences in pressure by showing how a volume of, in our case water, is moved when a gas is depleted.  Before you learn more we need to start with two set-ups of the following things you can find around your school science lab:

Laboratory Set-Up: 

Step #1: Obtain your bacterial specimen from your teacher.  The best type to use is the type that is non-harmful and is soil living.  In our case, we can use Sphingomonas pausimobilis, but any non-harmful air loving or aerobic bacteria will do.

Step #2: Place the bacteria, using a bacterial inoculation loop into approximately 200 milliliter or ml (sometimes ml, which is a volume unit can be changed into weight or mass units and then they are called cubic centimeters or cc) of room temperature tap water inside a 500 ml flask.  (Make sure you have a rubber stopper ready that fits the flask and that it has a hole to exactly fit the plastic tubing).

Step #3:  Swirl the mixture or use a magnetic stirrer until all the bacteria is well mixed.  Add to the flask 10 ml of corn syrup or sugar water and mix again.

Step #4:  Divide the mixture between the two 500 ml flasks using a graduated cylinder for measuring the 200 ml bacterial mixture each.

It is important here to know that the stopper and tubing should not be attached until the entire set-up is complete.  Here, there is something else you have to do!

When bacteria break down glucose or sugar not only is use oxygen used, but carbon dioxide (CO₂) is given off.  Remember, nature doesn’t like empty spaces or vacuums.  If carbon dioxide, which is a gas, were present it would fill in the space.  We do not want the space filled with a CO₂, so we need to design a small apparatus to suspend Lithium Hydroxide (a carbon dioxide loving compound) above the water with the bacteria and glucose in it.  This compound will bind with the CO₂.

Step #5:  Make a small cup to fit inside the opening of the flask out of aluminum foil.  Make the cup small enough that once inserted into the top of the flask that the bottom of the cup is not near the level of the mixture.

Step #6:  Place venting holes on the sides of the cup toward the top.  If Lithium Hydroxide were to fall into the mixture it would affect the outcome of the experiment.

Step #7:  In order to keep the flask air tight, we need to grease the edges of the flask before dropping the cup in.

Step #8:  Place the Lithium Hydroxide into the cup and suspend the cup from the top of the flask.

(The placing of the stopper and the tubing is really part of the second part of this activity, which is the manometer.  Please see making the manometer below….)

  You have now the first part of the experiment complete.  Have you wondered about how we would test a “pollutant” in our bacterial mixture?

Step #9:  Repeat the exact same things for a second flask.  In this flask, however, you have added 3 grams of Copper Sulfate (CuSO₄) to the bacterial mixture.  This is your variable in the experimental set-up.

You now have two experimental set-ups with equal quantities of everything except the copper sulfate.

Part 1:

Measuring requires that we have some type of apparatus to measure with.  It is important that our board and measurements be vertically straight, so you may want to use a level or plumb line to line up your measurements vertically.  You may use a graph paper if you find that easier directly onto a wooden board.   You can place lines to mark off centimeters or a shortcut would be to tape a ruler along your vertical line.

Part 2:

Before we put the flask in place with the board we have to place 100ml of water with dye in it into the tubing and stopper.  There should be a space of gas or air before the stopper and a space of air after the colored water in the tubing.  The opposite end of the tubing from the stopper is open to the air.  Do not let the tubing going into the flask touch the Lithium Hydroxide in the cup.

Part 3:

Place the flask on a flat surface with the wooden plank either behind or on side of it.   An ideal situation would be to place the flask on a warming plate to keep tepid with a magnetic stirrer.  If that is not available place in a warm area and manually swirl several times during the experiment. 

Part 4:

Finally the last part of the set-up:  place the tubing in a U along the vertical line with the top measurement of the colored water at 0.  That means we can measure any rise or fall of the colored water column in the tube against 0.  The water levels of the flask and colored water column in the tubing should be even across.  Then attach to the board using tape or tube fasteners making sure that the tubing is not pinched in any way.

There are two set-ups, one a control without the “pollutant” and the other with the “pollutant”.  

Take readings at uniform intervals during the school day for one week.  You do not have to adjust your data for overnight hours since the daytime measurements at intervals once graphed will show the trend. 

Has the color water in the tubing moved?  Did the column of colored water in the tube rise or fall and by how much?  What conclusions have you drawn?  Did the chemical pollutant affect the break down of glucose by your bacteria?

Record and analyze your data and present to your teacher.  Make sure you use charts and graphs

What has your data and graphs shown you?  Is there a change between the control and the experimental set-up with the variable?  What conclusion do you draw from that?

Now, that you know how to make a Manometer you might want to try other compounds around the house.  For example, how does salt of NaCl (by the way Sodium is a metal) as your variable affect the outcome, or Baking Soda, alcohol, soaps, or oven cleaner (remember to read labels).  You may want to do a science project that demonstrates the many ways we pollute our soils and waterways.  GOOD LUCK!