WS0210 Practical 1.Introducing fungi and experimental design Introduction What is biology? It is the study of organisms and of life. It includes investigations of tiny bacteria, kangaroos and gum trees, and whole landscapes. It includes investigations of biochemical processes that occur in a fraction of a second and of ancient fossils that may be unchanged for a billion years. It is the discovery of principles and processes that are fundamental to living organisms and being able to predict responses in organisms to changes in conditions. It is being able to reliably grow food, to maintain healthy ecosystems, and to manage the conservation of our diverse flora and fauna. We achieve this by using scientific principles of discovery and understanding that have dramatically increased human ability to understand and manipulate the world around us over the last several hundred years. The process of science Science involves making observations. These can be descriptions, measurements or complex analyses. Science also involves developing and testing hypotheses. A hypothesis is a statement about how nature or life works or operates. A hypothesis is a statement that predicts what will happen and it is a statement that can be tested. Experiments test hypotheses. An experiment is carefully set up so that the statement within the hypothesis can be tested. Any factors that may caste doubt on the result are carefully eliminated. The experimental results show whether or not the hypothesis is supported under the carefully defined test conditions. Science also involves the publication of experiments and their results. This ensures science is carried out efficiently, as other researchers can build on the results of previous experiments. Other researchers can also examine the results and provide criticism to ensure that the conclusions reached by the researchers are justified. If desired other researchers can also repeat the description from the published information and ensure the experiment was carried out accurately and that it did indeed substantiate a hypothesis. Recently two scientists asserted that they had achieved a process to generate energy by cold fusion, which has enormous implications for energy production. However other scientists were able to recreate the experiment using the published methodology and showed that the original research was in error.
Observations and data When living things are observed and studied, it is necessary to keep records of observations. These observations are not "one offs" but generally are repeated many times so that gradually the biologist amasses large amounts of what is described as "data". Observations or data have been traditionally collected as: sketches, drawings or images; photographs; sound and video recordings; written words (descriptions); and measurements and figures. Irrespective of how observations are collected, they must make sense when reviewed months or years down the track, so there are certain conventions for data collection and presentation. When masses of data are collected, organisation and presentation become most important, and eventually an analysis of the data is necessary to test for trends or patterns. This exercise that you will carry out introduces some of the more traditional methods of collecting data and some basic techniques of presenting it. Hypotheses and experiments Set up treatments Experiments test hypotheses by creating different, relevant sets of conditions and determining the response in some condition to those different conditions. Thus processes or organisms are exposed to differing treatments. Are the results those predicted by the hypothesis? Results in these different conditions are compared to see if they are in agreement with the hypothesis. Eliminate influences that are not part of the treatment There are many conditions that could possibly affect the outcome of an experiment and some of these can not be eliminated. Therefore experiments usually need to include a control. A control includes all the conditions that are not being tested but which may affect the experiment. For example: A hypothesis is that when you blow on a feather it will move. To test this you blow on the feather and it moves but perhaps a faint breeze acted on the feather at that time. Therefore you need a second feather which is being acted on by all the same factors that are affecting your test feather, except for the factors that you are testing. Then if you blow on the first feather and only the first feather moves then you know it was only your blowing on it that caused it to move. The second feather in this case is the control.
Practical exercise aims Exercise 1 Exercise 1 introduces you to the process of designing an experiment to test a hypothesis. It also introduces you to descriptive techniques for recording data. Exercise 2 Exercise 2 introduces you to the management of variation through the use of replication. It introduces you to quantitative techniques for recording data. The aim of the report is also to develop an appropriate report writing style for presenting the data that you have recorded. Exercise 1 Media effects on fungal growth Introduction Have you seen the fine grey threads and rough green patches that grow on bread and cheese. These are fungi. The one with fine grey thread that produces pin head sized black fruiting bodies on erect hyphae is often Rhizopus, the green one is often Penicillium. Fungi grow over the bread and excrete enzymes which break down the organic compounds in the bread and then they absorb the carbohydrates and nutrients. Figure 1. Fungus affected bread after incubation. A hypothesis What are the effects of treating the bread on fungal growth? Will fungi still consume the bread if it is covered in sugar or salt? Let us test the hypothesis: Sugar and salt do not affect fungal growth on bread. In this case the hypothesis is expressed as a null hypothesis, that there will be no effect. This is the form in which a hypothesis is generally expressed. How can we test it? Well we can treat some bread with sugar and we can treat some bread with salt and we can examine the outcome. We also need a control to compare it to, otherwise there may be effects on fungal growth due to the weather, temperature or number of fungal spores. We can t control all of these so we set up a treatment without any sugar or salt and use it to see what happens to the bread. This is a control as it is set up identically to the sugar and salt treatments but just without the sugar or salt.
Media effects on fungal growth WARNING Whenever dealing with biological material prevent contact between the material and food. Don't ingest the material and always wash your hands carefully before eating and avoid contact with your mouth. Discard any materials used in the rubbish or wash and disinfect them after use. The fungi involved in this experiment are normal contaminants of food but care should always be taken. Read all parts of exercise 1 before starting this practical. Materials - 3 slices of bread - 1 teaspoon of salt - 1 teaspoon of bread - Plastic bags - Paper towelling Treatments Moisten the bread with a few drops of water and place each slice on a piece of paper towelling (or newspaper would do). The end of the paper should extend beyond the bread and on one slice write 'Control', on the second 'Sugar' and on the third 'Salt'. Sprinkle one teaspoon of sugar on the slice of bread marked sugar. Sprinkle one teaspoon of salt on the slice of bread marked salt. The third slice remains untreated. Leave all slices exposed for 1 hour (kitchen bench is fine as they are not fungal affected yet). Figure 2. Bread after salt treatment and ready for incubation. Place each slice of bread into a plastic bag and tie the ends. Put the slices of bread away from the kitchen or living areas of your house, a balcony or verandah would be fine. Leave the bread for four days. Watch out for ants, horses and all other bread loving creatures! You may need to suspend the plastic bags to prevent ants from getting into the bags.
Observations After four days, record the following information for each slice of bread. If nothing has happened yet on the control treatment leave the slices of bread for another two days. If nothing has happened within 10 days contact the lecturer. AVOID GETTING TOO CLOSE AND BREATHING THE FUNGAL SPORES WHEN RECORDING THE DATA. For each slice, record what proportion of the bread is covered by fungal growth. Is the fungal growth sparse, moderate or dense? What colour are the fungi? Describe the fungi. Draw each slice of bread in pencil, showing the locations of the different fungi. Outline the areas of fungal growth with a sharp dark lead pencil (2B is good), using shading to show where the different types of fungi are growing. Include a legend beside the bread drawing. Your drawing needs a title. A convention in biology is that figure titles are numbered and go below the figure. i.e. Figure 1. Fungal growth on bread coated with sugar. You also need to have a scale on your drawing. Draw a bar 1 cm long and write ' 1 cm ' next to the bar (or if you drew the bread twice as large as it was in reality you would draw a bar 2 cm long and write 1 cm next to it). See the example drawing on the next page. Another common way of presenting data is to summarise the data in a table. A table always has a title describing what is being presented in the table and this is above the table. It also has column or row titles that describe what is presented in that particular column or row. The row or column title also includes a description of the measurement units. Enter the data you have collected into a table like that below. Table 1. Comparison of fungal growth on bread coated with sugar and salt and uncoated bread. Treatment Proportion of bread affected by fungal growth (%) Density of fungal growth Colour and description of the fungi Control Sugar Salt
Examples of figures. Yellow Black Light grey Pink 2 cm Figure 3. Fungal infection on a mango leaf with the different coloured fungal infection areas described in the legend. Figure 4. Final exam results of ENV102 students who completed all five quizzes compared to students who completed fewer than four quizzes. [Note the figure title with figure number, axis values, and axis titles with measurement unit in brackets.]
Experiments and chance Let s look at yet another critical issue for experimental science variation. Variation always needs to be considered when carrying out experiments. Variation occurs due to many different conditions. Say you have four plants and put fertiliser on two of them. A week later the two fertilised plants are larger. One plant may be able to grow faster than the other. Thus there is variation in the experimental response. Note you need the control (the plants without fertiliser) to test how much the plants would grow without fertiliser. One of the plants may have been slightly larger than the other when you started the experiment. Thus there is variation in the experimental material. Say both you and a friend measure the height of one of the plants, one of you records 12.9 cm and the other 12.7 cm. Thus there is measurement variation as well. Or if you use two different rulers, one may show the height as 12.8 and the other 12.7 cm. All of this variation combined is termed experimental variation or experimental error. When we look at the experiment we have some differences between the control plants and the treatment plants but we also have variation between the control plants. How do we know if the treatment has had any effect on plant growth? We do this by having at least three plants that are treated identically and comparing them to three untreated plants. Then we can see if the differences between the control and treated plants are greater than the experimental variation within the control plants. Having identically treated plants to test for variation is called replication. Exercise 2 Fungal effects on banana growth Introduction Now let s look at a local example of fungal growth that has a huge impact on the Northern Territory. In 2000, bananas were the third most important crop in the NT after mangoes and table grapes, with a value of $13 million (NT Department of Business, Industry and Resource Development Technical Bulletin 2002 No. 299). In 1997, Panama disease was discovered in one of the local banana plantations. This disease is caused by the soil borne fungus, Fusarium oxysporum f.sp. cubense. It has now spread to most banana growing areas in the Darwin region and is a threat to the industry. Researchers at CDU, CSIRO and NT Primary Industries have been investigating how to manage and control the disease. Approaches include looking at soil effects on the disease and breeding new resistant cultivars. The fungus may be more virulent in some soil conditions compared to others and different cultivars of bananas show varying levels of resistance to the disease.
A hypothesis Let s design an experiment to see whether the yield of the banana varieties, Cavandish or Gold finger, is affected when they are grown in the presence of the fungus Fusarium oxysporum. How can we test it? We can go to an area of ground infected with Panama disease and grow say 20 plants of each variety in a small plot and measure the amount of fruit produced. We also need a control to compare it to, otherwise we don t know whether any affects that we observe are due to the disease or due to some other factor that may be affecting banana production. The control in this case could include establishing some plots of both banana cultivars in an area that does not contain Panama disease. Treatments Now we have two treatments: a control (without fungal infection) and secondly the plots containing the disease. We will look at both of the cultivars so we will need separate control and disease plots for each of the cultivars. We need to take experimental error into account so each treatment will need three replicates - so we will establish 3 plots of each banana cultivar in the diseased area and three plots of each in the control area. Observations After managing the plots for two years we will return to harvest the bananas. We need separate weights for the bananas harvested from each plot to determine the plot to plot variation, so we need to take care to keep the bananas from each plot separate. Here are the data from the field notebook that records the yield of bananas per plot (these data are fictitious so don t rely on them if you happen to grow bananas!) Cavendish Control plot 1 820 kg Diseased plot 1 456 kg Control plot 2 722 kg Diseased plot 2 296 kg Control plot 3 840 kg Diseased plot 3 351 kg Gold finger Control plot 1 580 kg Diseased plot 1 640 kg Control plot 2 640 kg Diseased plot 2 551 kg Control plot 3 646 kg Diseased plot 3 600 kg Record the data in a table. Remember to include a title and that column headings should contain the measurement units. See Table 1 for an example.
Also present the data for the Cavendish bananas as a column graph. The data for the three control plots should be adjacent and the data for the three treatment plots should also be together. Include a figure title and remember to label the axes. See Figure 4 for an example. Practical report write up Introduction Write a null hypotheses for experiment 1 and also for experiment 2. Results Experiment 1 Include the figure of the fungi growing on the bread. This is figure 1, the three slices can be figure 1a, 1b and 1c. Include Table 1. Experiment 2 Include table 2 with all the banana yield results. Include the graph of the Cavendish results, figure 2. Note that normally you would not present results in both tabular and graphical form. Discussion 1. Why is a control important in the fungi experiment? What is important to consider when treating the control with respect to experimental factors that are being treated and other variable factors that may affect the treatments? 2. How did sugar and salt affect fungal growth on bread? Why is the lack of replication important to consider when discussing the fungi on bread experiment? 3. Why is a control important for assessing whether Panama disease affects banana yield? 4. Why was replication important when assessing the banana yield results?