 Introduction: (Initial Observation)
Microorganisms play an important part in our lives. As decomposers of organic material, they help keep soil fertile, and recycle once-living matter into valuable nutrients for plants. Among large varieties of microorganisms, some including paramecia have also commercial value and are used to feed fish. Because of this special application, many are culturing paramecia and it is important to know how different environmental conditions affect the reproduction and development of paramecia.
In this project you may study the effect of pH, light and temperature on the population (growth and reproduction) of paramecia. If you are doing this as a Science Project, you may choose to study only one of the above factors; however, if your time allows, you may choose to study all three factors.
Information Gathering:
Find out about paramecia, their habitat, their food and reproduction. Read books, magazines or ask professionals who might know in order to learn about the possible effects of pH, light and temperature on paramecia. Also gather information that will help you to design your experiments. For example you need to know how big are paramecia and how can you observe or count them. Keep track of where you got your information from.
Following are samples of information that you may find.
Paramecia are unicellular microorganisms belonging to the phylum Ciliophora. Members of this phylum (ciliates) are characterized by their external covering of continuously beating, hair-like cilia. Under favorable conditions they multiply rapidly by a process called binary fission where they divide in half forming smaller duplicates of themselves. They can also reproduce by conjugation in a similar manner as sexual reproduction in more complex animals.
Paramecia are a cosmopolitan organism and are found in suitable habitats all around the world. Global distribution of Paramecia species is believed to be the result of the break-up of the super-continent Pangaea over 200 million years ago. This continent was home to ancestral paramecia that have subsequently been separated by continental drift. The oldest reported fossil Paramecium was discovered in a piece of amber dating back to the Cretaceous period, over 65 million years ago.
Paramecia are oval flat creatures, and bear a number of tiny cilia that serve to propel it through the water. As they move through the water they collect small particles of food that are swept into the gullet. Most Paramecia are bacteriovorous and feed voraciously on bacteria that accompany decaying organic matter. They are an important link in detritus-based food webs in aquatic ecosystems and are consumed by other small animals, which are in turn preyed upon by larger organisms. Paramecia are an excellent primary food source for newly hatched rainbow fish larvae.
Most aquarium related cultures of paramecia are generally referred to as Infusoria. The term infusoria is a collective name for many microorganisms and can include paramecium, microscopic algae, bacteria, protozoans, desmids, rotifers and a host of other small organisms. Old time methods for obtaining a culture of infusoria was by boiling hay, lettuce, spinach or other vegetable matter and allowing the resultant infusion to stand in the air for a while in the hope that stray infusorians will alight therein. At best this was a hit and miss method and in most cases it simply didn’t result in a successful culture.
The best way to bring about a successful culture is to make a vegetable based infusion similar to the above (banana skin works well) and introduce a pure culture of Paramecia (obtainable from biological and aquaculture supply companies or live food culture dealers). There are a number of different paramecium species available however, Paramecia multimicronucleatum has been found to be one of the best. It is a very large paramecium and promotes rapid growth of fish larvae. The culture is placed in the infusion and before long a pale; ever changing cloud of tiny motes will be seen when the jar containing the infusion is held to the light. Although just visible to the naked eye, to see paramecium properly, a microscope is needed. Once you have started a good culture of your own it is a simple matter to prepare a series of cultures from the original one.
Paramecia multimicronucleatum can be obtained from Carolina Biological Supply or Connecticut Valley Biological. The strain of very large paramecia promotes rapid growth of the fish larvae. The following method of growing the paramecia is simple, quick and very suitable for small, low-budget, fish-raising as well as for a large facility.
Grow seed cultures in 150 x 25 mm plastic petri dishes at 28.5oC. In addition, grow several small batches of seed cultures in other rooms at room temperature as a backup in case of a rather rare “crash” in the paramecia population. These cooler cultures grow slowly and need care only every four or five weeks.
Paramecia Seed Cultures:
1. Add 10-15 grains of boiled wheat to 175 mls of system water.
2. Inoculate with 20 ml from an excellent existing seed culture dish or with a sample from the commercial inoculants.
3. Grow for 7 to 12 days before using.
Paramecia are found in ponds and other quiet waters among the muck and decaying vegetation. A large number of Paramecia in your backyard pond would indicate less than desirable water conditions. This specimen came from my pond in late October, after I had taken the fish indoors, shut down the filter and allowed the decayed vegetation to settle to the bottom for the winter. These two Paramecia came from a sample of the bottom muck.
Paramecia have a weapon defense system that they deploy against their enemies. When attacked, they release dart-like objects from capsules located on their undersides. It usually works, however, it is completely ineffective against the Paramecium’s most deadly enemy – the Didinium. The Didinium simply forges ahead, undeterred, gulping down as many as two Paramecia a minute.
A Brief History of the Infusorian Culture:
by Humphry Axelbearing
In the beginning there was scum! By some divine miracle, a few such bits of organic scum came together to produce the precursors of all life on earth. The existing organisms that best represent our slimy origins, may be collectively referred to as the “Infusorians”. Single-celled, and propelling themselves with cilia, the protozoa are one class of such creatures, and the representative of this group we will discuss in the present column is the king of protozoans, the incredible, the fantastic, just returning from a tour of the greatest swamps in the world, put your cilia together and give it up for the one, the only, the majestic paramecium!
Culturing Paramecia:
The paramecium is near and dear to the hearts of may fish enthusiasts due to several key characteristics – They are large enough to see without magnification, damned easy to culture, and readily eaten by even the smallest of killie-fry! Paramecium can be cultured in any vessel that will hold water, whether it is a 2-liter soda bottle (perhaps my favorite container) or the toilet bowl in a South-American prison cell (definitely my least favorite ;-). Whatever container you choose, in order for paramecia to flourish, you must create an ecosystem that provides the basic needs of the organism (i.e. nutrients, water quality, etc.). I eschew (geschuntite!) the need for sterile lab technique and do not worry about contamination of cultures with other organisms of infusorial heritage. In my experience, a healthy population of paramecia will literally out-eat and out-reproduce any invading organisms that cannot swallow them whole. In fact, if you have a hard time finding a “pure” paramecium starter culture I recommend starting with a pint of water from any aquarium that contains live plants. Siphon up some of the detritus (Latin for rotting brown junk) in the bottom of the tank and treat it as a starter culture. Initially you will probably have a dozen or more different infusorial species, but by the time you have sub-cultured twice, paramecium will be the dominant (if not the only) species remaining! The health and vigor generated by random reproduction, is another advantage to culturing “native” infusoria rather than one of the pure cultures you can get from a biological supply house. Native organisms are already adapted to your water conditions, and have not been weakened by many generations of line breeding from single individuals. In other words, they will be much more likely to thrive and multiply in your fishroom!
My standard method (which up till now was a carefully kept secret, known only to a few hundred of my closest killie keeping colleagues!) is a three step process. The first step is to set-up the food chain. Fill a 2-liter bottle 3-4 full of clean aged tap water, and add one chunk of Purina Dog Chow and 2 square inches of dried iceberg lettuce. Leave this bottle overnight (12-24 hours) uncovered, thus creating a bacterial bloom. I have tried many different sources of organic matter to start paramecium cultures and nothing even comes close to matching dog food for initial population growth. I think it is because dog food contains a complex combination of nutrients (proteins, fats, vitamins and minerals) that are not always present in adequate amounts in traditional culture media (i.e. hay, peas, soy flour, dried lettuce, oatmeal, etc). I am sure other brands of dog foods are probably adequate for these purposes, although you will need to adjust for the size of the pellets (PDC chunks are spherical and about 1-2 inch in diameter). I do not recommend adding dog food to an older culture, however, because it can cause a bacterial bloom so intense that the culture goes anaerobic and the paramecia literally suffocate producing a smell rivaling that of a microworm culture gone bad!
Step two is to add the starter culture (about a cup of a thriving paramecium culture) and cover the bottle loosely – I just set the cap on the bottle. It will take approximately 6 -10 days for the bacterial cloud to clear, but when it does you will have millions of paramecia. I feed my fry tanks directly from the culture bottle with an eyedropper, but only when the culture is clear. I am in the habit of making paramecia available to all my fry for the first week after they hatch. Obviously many killies are large enough to eat other foods and do not absolutely need paramecium as a first food. However, the way I see it, the paramecia are available 1 swim around and eat bacteria in the fry tanks a no-lose situation!
Step three is maintaining the population… At first the paramecia are thick in the container, but a week or so after the bottle has cleared you will notice a decline in the population density. That is because the when there was an abundance of food (bacteria) the paramecia grew and multiplied. The population growth is exponential, and the paramecia rapidly exceed the available food supply. Eventually the paramecia and bacteria will reach a balance point, where the population level can be sustained by the available food. So, if you want to keep the paramecia population up, you have to keep the bacteria up as well i.e. you have to keep feeding the bacteria. I suggest adding 8-12 rolled oats (old fashioned Quaker Oatmeal), about once a week. You can vary the amount, and the frequency of feeding, to maximize population density. Another tip is to add a couple snails to the culture (after it clears initially). Snails eat excess food and their waste products encourage bacterial growth. The bottom line is that cultures with snails will consistently out produce those without snails! Eventually the waste products of metabolism will accumulate to the point of toxicity where the population does not revive upon re-feeding. I can usually keep a culture going strong for 2-3 months before the inevitable decline. The worst case is when the culture “goes bad” (hold your nose as you pour it out) and you have to start over. One way to assure a constant supply of paramecia is to keep at least 3 cultures going all the time – dumping the oldest of the 3 existing cultures and starting a new culture each month. That way if one culture goes bad, or you overfeed and a culture gets cloudy, you will probably have at least one other clear culture from which to feed or start a new bottle. By the way, an old culture need not go down the drain, so to speak – instead dump it into your daphnia culture, or use it to water your house plants!
Sexual and Asexual Reproduction in
Paramecia
The one celled organism called the paramecium (plural: paramecia) is unique in the sense that it can reproduce both sexually and asexually. Identical offspring result from the process of asexual reproduction, as we know from the definition. However, we also know that the genetic information passed on in asexual reproduction is fixed, in the sense, that it only comes from one parent and offers no genetic variety.
Since the paramecium can also reproduce sexually (two parents are needed) this organism can experience genetic variation which could enable it to exist better within its environment. The mating of two paramecia is called conjugation and it allows for the exchange of genetic material. A paramecium is also unique in that it has two nuclei which allows for this phenomenon.
Analysis:
In the environment of the paramecia subtle changes begin to disrupt the delicate balance of the ecosystem. Some of the organisms are able to adapt, most cannot.
In your opinion what are the advantages (or disadvantages) of being able to reproduce both asexually and sexually?
Explain how this applies to their ability or inability to survive.
Question/ Purpose:
What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.
The purpose of this study is to find out how different environmental factors such as light, temperature and pH affect paramecia (population/ reproduction). Our goal is to further develop our understanding of which factors affect paramecium population growth most.
This project guide only focus on the effect of light on the population of paramecia. All variables, hypothesis and the main experiment are suggested for testing the effect of light. You can modify these to adapt this project guide to any other factor that you may choose to study. The main question for this project is:
How does sunlight affect the population of paramecia?
Identify Variables:
When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.
Manipulated variable is the light condition with two possible values. Dark, Light.
Responding variable is the population of paramecia.
Controlled variables are temperature, pH and available food.
Hypothesis:
Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.
Following is a sample hypothesis:
The amount of light a paramecium population gets should have an impact on its growth rate and final population size. Among other things, paramecia eat algae, and light has a profound effect on algae growth; So, paramecia will have more algae to eat.
My prediction is that paramecium population put in an environment with plenty of sunlight would grow much quicker than population put in a very dark environment.
Experiment Design:
Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”
Experiment 1: (Effect of light on paramecia)
Introduction:
Paramecia reproduce by binary fission – each paramecium splits into two paramecia. For this experiment you grow paramecia populations in 40 ml of pond water with 1 ml of liquid food, which is a suspension of ground plant material in water. You can see the fragments of plant material floating around in the water. The paramecia don’t actually eat the “Paramecium Food.” Instead, bacteria and fungi grow on the plant material and the paramecia eat the bacteria and fungi. To test your hypothesis you will grow cultures of Paramecium caudatum (or any other relatively larger species of paramecia) in two environments, one light environment, and one dark environment.
Material:
- stock cultures of Paramecium caudatum
If you don’t have access to a local pond and prefer to purchase paramecia culture, search the internet for paramecium culture and you can find different resellers. A starter culture that comes in a 4 ounce jar can be grown into a quart sized culture in about a week and from there, a gallon in about a month. The price of a 4 ounce culture is about $5.00. - pasturized pond water (It can also be boiled water)
- liquid “Paramecium Food” (Vegetable juice)
- culture vials or test tubes
- dissecting microscopes (a good quality magnifier may also work fine if you are growing large species of paramecia)
- pipettes
- rubber bands to organize culture vials
Procedure/Methods:
Obtain or prepare about 4 ounce paramecium culture that you can use for this experiment. Paramecium culture is a any water containing live paramecia.
Prepare 12 plastic tubes and add 40 ml pasteurized pond water to each tube.
Stir the culture so the paramecium will distribute evenly across the culture.
Get one drop of your culture and under a microscope or strong magnifier, count the number of paramecia in one drop. Repeat this 3 times and then take an average. Now you should have a good estimate of the number of paramecia in each drop.
To each test tube add about 400 healthy paramecium individuals. For example if you have 40 paramecium in each drop of culture, then 10 drops of culture will make up 400 paramecia.
Add one ml of liquefied grass into each test tube.
Paramecia need oxygen. If you are using test tubes with caps, make small holes on the caps of tubes to allow air to enter, or you may leave the test tubes open.
Place six of the tubes near a window, where they would be exposed to a significant amount of light. Wrap other 6 tubes in aluminum foil so they will not get any light at all. Allow your cultures grow for seven days.
Observe samples of cultures every day, and estimate the population size for each tube each day by taking sample and count the number of paramecia under a microscope. Before taking each sample, use a transfer pipette full of air to blow some air in each tube, near the bottom. To do this, simply squeeze the pipette to bubble up the water. This action will spread the paramecia out evenly, and add oxygen to the water. Look at five drops from each tube under a microscope, and count the paramecium from each sample. Then multiply the total from the five drops by 6, (assuming there are 30 drops per ml. Then multiply this number by 41, the number of milliliters of liquid for each tube.
The numbers that we have provided here are just examples. You may change the amounts as you need. For example you may start with smaller test tubes and have only 20 ml water in each tube. Or at the end instead of counting the number of paramecia in 5 drops sample, you may count the number of paramecia in 1 drop of sample and change your calculations accordingly.
Experiment 2: (Effect of pH on paramecia)
Note:
This experiment is identical to the experiment one. Differences are as follows:
- All paramecium cultures will get identical amount of light.
- Grow paramecia in 18 test tubes that can be divided in 3 groups of 6. Label the groups as Low pH (acidic), neutral and High pH (basic). In each tube in Low pH group add 5 drops of vinegar to reduce the pH. In each tube in high pH group add 5 drops of ammonium solution to increase the pH.
- Continue observation, sampling, counting and recording the paramecia as described in the experiment 1.
Experiment 3: (Effect of temperature on paramecia)
Note:
This experiment is identical to the experiment one. Differences are as follows:
- All paramecium cultures will get identical amount of light.
- Grow paramecia in 18 test tubes that can be divided in 3 groups of 6. Label the groups as Cold, room temperature and Warm. Use cold, warm and room temperature water baths to produce different temperatures. You may use ice in cold water bath and use an aquarium heater in the warm water bath.
Materials and Equipment:
Material for experiment 1:
- stock cultures of Paramecium caudatum
If you don’t have access to a local pond and prefer to purchase paramecia culture, search the internet for paramecium culture and you can find different resellers. A starter culture that comes in a 4 ounce jar can be grown into a quart sized culture in about a week and from there, a gallon in about a month. The price of a 4 ounce culture is about $5.00. - pasturized pond water (It can also be boiled water)
- liquid “Paramecium Food” (Vegetable juice)
- culture vials or test tubes
- dissecting microscopes (a good quality magnifier may also work fine if you are growing large species of paramecia)
- pipettes
- rubber bands to organize culture vials
Results of Experiment (Observation):
Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.
Sample Results
The cultures from the light environment reacted very differently than the cultures in the dark environment (Figure 1). By the third day, the populations of the paramecium cultures that were placed next to the window increased much more than the paramecium cultures that were wrapped with tin foil, although their average progress was identical during the first two days. Some of the cultures stored in the same environments were rather inconsistent with each other, but as the days progressed, our data started to develop a trend. On the fourth day, the cultures from the dark environment had a higher average paramecia population per tube. During the next three days, the population of the paramecia from the cultures exposed to light decreased, while the populations of the dark cultures increased. On the final day, the light cultures had about the same population size as when we began the experiment. The average population size of our dark cultures were nearly three times as large as when they began. The population size of our light cultures peaked in the middle of our experiment, then steadily decreased. The population size of our dark cultures increased slowly in the beginning, then started increasing much faster halfway through the experiment.
Average Population Size For Each Environment
| Environment | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| With Light | 420 | 448 | 1148 | 1107 | 644 | 504 | 448 |
| Without Light | 420 | 448 | 779 | 1435 | 1064 | 1260 | 1475 |
Figure 1
Population Size of Cultures With Light
| Culture | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| A | 504 | 504 | 246 | 492 | 504 | 336 | 168 |
| B | 504 | 336 | 984 | 738 | 168 | 336 | 336 |
| C | 168 | 672 | 1230 | 984 | 672 | 504 | 336 |
| D | 336 | 504 | 1968 | 1472 | 168 | 504 | 672 |
| E | 168 | 168 | 492 | 1722 | 1008 | 672 | 336 |
| F | 840 | 504 | 1968 | 1230 | 1344 | 672 | 840 |
Figure 2
Population Size of Cultures Without Light
| Culture | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| G | 840 | 840 | 738 | 2460 | 1344 | 1344 | 2296 |
| H | 672 | 672 | 984 | 1230 | 1176 | 672 | 1680 |
| I | 168 | 168 | 738 | 1476 | 840 | 2016 | 1512 |
| J | 168 | 672 | 984 | 984 | 1008 | 1008 | 1176 |
| K | 504 | 0 | 246 | 1476 | 672 | 1512 | 840 |
| L | 168 | 336 | 984 | 984 | 1344 | 1008 | 1344 |
Figure 3
Calculations:
Write your calculations in this part of your report.
Summary of Results:
Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.
It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.
Conclusion:
Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.
Sample conclusions / Discussion
We concluded that light plays a big role in the population growth of Paramecium caudatum from the results of this study. The light cultures grew faster at first, but after four days, their population size started decreasing until it reached its original population size. The populations of the dark cultures started out growing slower than the light cultures, but eventually started to increase much faster, nearly tripling their original population size. We predicted that the populations set in the light environment would grow much faster than those in the dark. Our prediction was partially correct, as the population of the light cultures grew faster during the first three days. The last four days of the experiment were completely unexpected, and contradicted our prediction.
The light appears to help the paramecium populations get started in a new environment, accelerating the growth for a brief period of time. Judging from our results, absence of light appears to delay the paramecium population growth for a short time, but overall provides a better environment for paramecia to thrive. There is no apparent reason why the populations of the light cultures decreased halfway through the study, leading us to believe that other factors may have played a role.
Although the results of our study did not completely match our prediction, it seems that light does indeed play a big role in the growth rate of paramecium populations, even though it did not happen quite as we had anticipated. Perhaps the tin foil we used to wrap each tube for the dark environment served as insulation, keeping those cultures warmer than the cultures placed near the window. It was starting to get cold outside, which may have affected our light cultures since they were close to the window.
Related Questions & Answers:
What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.
Significance
Applied Significance:
The significance of this experiment is to obtain a greater understanding of how light can affect certain organisms. Paramecium caudatum feed off of things that are affected by light, making more resources available for the population, and encouraging growth. The results of this study, most likely, apply to other organisms that consume food that is affected by light. The results of this experiment may help develop a greater understanding of paramecium population growth, as well as the population growth of other organisms.
Ecological Significance:
Our experiment demonstrates how Paramecium caudatum populations react to different environments. It is very likely that paramecium could have a strong reaction to light, because light is very important to life, in one way or another. Our experiment tests how paramecium adjust to different amounts of light in a previously studied environment where they multiplied steadily.
Evolutionary Significance:
By introducing our paramecia to different environments, we have the opportunity to witness how they adapt to new environments. Our results will tell us how well they are adapted to varying amounts of light, or how well they can adapt. Organisms that can adapt well to a different environment have a higher chance of survival among other species, as opposed to if they are unable to adapt, where they have a smaller chance of surivival.
Possible Errors:
If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.
If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.
References:
Visit your local library and find books related to microorganisms. Find the chapters that discuss paramecia.
Following are some additional web links.
http://zfin.org/zf_info/zfbook/chapt3/3.7.html
http://ebiomedia.com/gall/classics/Paramecium/paramecium1.html
