Kinetics of Food Vacuole Formation

Modified from:  J.d. Berger and  S. Pollock, 1981, Kinetics of food Vacuole Accumulation and Loss in Paramecium tetraurelia.  Transactions of the American Microscopical Society, 100 (2): 120 - 133.

  A mathematically more sophisticated version of this experiment is contained in that paper.

Procedure

Equipment and Supplies

(Do this in groups of 3 or 4.  This list is for one group.)

1.  A well populated culture of Paramecium.  You need 40 ml per group. 

2. 
A series of small test tubes.  Label them 0, 1, 5, 10, 15, 30, 45, and 60.

3.  Formalin.  Put 1 ml in each small test tube. 

4.  A tube of artist's water color.  Get this at an art  supply store.  It comes in a container that resembles a small tooth paste tube.  Get lampblack or a red pigment that  is not based on cesium or chromium or other heavy metal. 

5.  A compound microscope and a supply of microscope slides and cover slips.

6.  Glass or plastic pipettes with bulbs.  Be sure that the pipettes used on the live cells never come in contact with formalin.  Even a small droplet of formalin will kill the cells and thus stop feeding.

7.  A clock or stopwatch  for timing.

Procedure

1.  Mix a little water color with spring water or autoclaved tap water to produce a few ml of a distinctly red suspension.  You will add the water color to the 40 ml culture in just a moment.  But first, remove 5 ml and put it in the tube labelled "0" that contains formalin.  Mix it up well.    Now immediately add 0.5 ml of that suspension  to your Paramecium culture.  This is time 0.   You just took the Time 0 sample.

2.  At the designated times remove another 5  ml from your culture of Paramecium containing water color and pipet it into the appropriately labeled tube (1, 5, 10, 15, 30, 45, and 60 minutes).  Mix each well.  The formalin will kill the cells, thus stopping food vacuole formation.  It will also preserve the Paramecia for a little while so you don't have to do the counts (steps 3 & 4 below) on the same day as you do the transferring.  

3.  Let the tubes stand undisturbed for at least 30 minutes after you added the cell suspension to the formalin.   During this time the cells, now dead, will settle to the bottom of the tube.  Anytime from 30 minutes to 24 hours after Step 2, remove the dead cells form a tube.  They will be piled up at the bottom of  the tube.  Put a Pasteur pipette with a bulb so that the tip is right at the bottom.  (Squeeze the bulb BEFORE you stick the pipet in the tube.)  Now release the bulb and the paramecia will be sucked into the tube.

4.  Put the material you sucked up on a microscope slide and add a cover slip.  Now look under low power at the dead paramecia on the slide.  The red water color will be in the medium surrounding the cells, but look more closely and you will see that some of the cells contain food vacuoles in which there is red water color.  This is a "labelled vacuole."  Count the number of labeled vacuoles in at least 10 Paramecia.  Arrange your data this way for each time you sample.

Cells Time 0

Number labeled vacuoles

1

 

2

 

3

 

4

 

5

 

6

 

7

 

8

 

9

 

10

 

Background

Paramecium eat bacteria which they ingest by phagocytosis.  A group of bacteria is swept into a membrane-bound pocket at the end of the cytostome, and then the pocket pinches off, forming a  food vacuole. 

Then the food vacuoles circulate through the cytoplasm where lysosomes empty digestive enzymes into the food vacuole.  When all the digestible material has been transported out of the vacuole and into the cytoplasm, the remaining material is dumped out of the cell by exocytosis at a special place on the cell membrane called  the cytoproct or cytostome.

Study of the process of food vacuole formation and loss can be carried out very easily using artists' water colors to label  the vacuoles.  This can be done with students at many levels of biological and mathematical ability from simply asking what the maximum number of food vacuoles is, or graphing the number of food vacuoles as a function of time, to more sophisticated kinetic studies including the development of equations to describe the process.  Refer to the paper by Berger and Pollock to learn how  to do the mathematically more sophisticated experiments.

Once the basic experiment has been done, lots of inquiry is possible of the sort that asks what the effect of some treatment is on food vacuole formation or loss.  For example, what is the effect of mild osmotic shock on the shape of the graph?