Biology Lab: Water Testing and Purification In this lab activity, students learn how to collect water samples aseptically and how to use a microscope to look for and identify fresh water invertebrates. They also do some simple chemical testing of their water samples and make and test simple, filter-purification systems. Note: culturing and identification of bacteria in water samples will be done in another lab
Biology Lab: Water Testing and Purification Introduction Water pollution is a major worldwide health problem. According to the World Health Organization (WHO) >9 million deaths annually are attributed to waterborne diseases. 38% of those deaths are of children afflicted with diarrhea. 80% of all worldwide infectious diseases are attributed to unsafe water supplies. Typical waterborne infections are Giardia, Cholera, Schistosomiasis, Hepatitis, Shigellosis, Typhoid and coliform-related diarrhea. Coliforms are bacteria that inhabit all human or animal colons; many bacteria in this group are Gram-negative bacilli (e.g. E.coli or Salmonella) or Gram-positive cocci (e.g. fecal Streptococci). Because they are found in all colons, they are often used as an indicator of seweragecontaminated water. It is impossible to keep water and food completely sterile for long as soon as it is unwrapped (in the case of food), or transported through streams or pipes it is going to come into contact with organisms. All we can do is keep contamination at a minimum by keeping streams, pipes and reservoirs clean and separated from waste or sewerage outlets, by storing food properly, by boiling water before use, by cooking food properly, and by good personal hygiene. Choose several sources of water to sample. Select one that you expect to be contaminated or dirty (e.g. pond water) as well as one that you expect to be clean (drinking water) so you can compare them. In this lab, you will also learn how simple, homemade filtration systems may be used to purify water, and you will do an experiment to determine how efficient they are at producing clean drinking water. Introduction 1
This lab has four objectives: To learn how to collect water samples aseptically for quality testing To analyze water samples for common chemical contaminants and learn what their presence means To use a microscope to look for and identify invertebrates in water samples To make a homemade filtration system and test how well it can remove contaminants from your water samples. You will learn more about culturing and identifying bacteria from water samples in another lab, later (see Biology Lab: Culturing Water Samples and Selective Media). Protocol 1. Discuss with your lab partners (your group) and your teacher, where you would like to collect your water samples from. The class should aim to collect a wide a range of samples, but limit the number to two water samples per group. 2. Take two sterile tubes and two sterile pipets with you to the watersampling site. 3. Carefully open your sterile tube using your littlest finger. Using the thumb and first finger of the hand that is holding the lid, dip the tip of your pipet into the water, fill up the pipet and transfer it to the tube. Repeat this until the tube is almost full then close the lid. When collecting water samples, DO NOT immerse your feet or hands in the water. Stand at the edge of the water source and use a pipet to collect your sample. Use a clean pipet for each sample and throw it away after use. Figure 1. This photograph shows you how to open a tube with one hand while holding an object, such as a pipet, with the other hand using aseptic technique. 4. Take your water samples back to the lab. Glass tubes and pipets can be sterilized just before use by immersing them in a pot of water and boiling it for 10 minutes. Use a pair of tongs to retrieve them! Introduction 2
Phase 1: Chemical testing of water samples You have been provided with a chemical testing kit to test for some chemicals commonly found in water supplies. You may test for all the chemicals in the kit for fun, but we suggest that you pay particular attention to the following four: 1. Chlorine: kills a lot of harmful microorganisms and is often added to water supplies to keep them clean, but can be harmful to humans at high doses. Doses of <90ppm or 2.6mg/L are perfectly safe for humans. Doses higher than 90ppm may cause throat irritation or sickness. 2. Iron: sometimes appears in water that has traveled through rusty pipes or has been stored in metal tanks for long periods. It can give the water a reddish-brown color. Small amounts of iron may also leach out from rocks. Iron is actually good for us; it is used by the body to make hemoglobin. It is only dangerous in very high doses (>500ppm). Repeated exposure to high doses may eventually lead to liver damage. 3. Copper: small doses of copper sometimes enter water from particular, copper containing rocks (ores) or from copper piping. However, high doses of copper in water are rare unless you live near a copper mine. Copper levels above 1.5ppm (1.5 mg/l) are rare, but may be harmful to health. 4. Nitrates: come from chemical fertilizers. When fertilizers are used by farmers, they may drain into rivers and water supplies, particularly after heavy rainfall. Human and animal feces also contain nitrates, so water that is contaminated with animal or human feces may be high in nitrates. Nitrate levels in drinking water above 10ppm (10mg/L) may be harmful to infants because nitrate lowers the ability of red blood cells to transport oxygen, resulting in diarrhea, vomiting, and/or lethargy and difficulty in breathing. Follow the directions provided with the kit to test your water samples, and record your results. You will re-test your water samples after filtration later, and compare those results to these. Hemoglobin is a pigment that makes your blood red. It contains iron, and it helps to transport oxygen around your body. People who don t get enough iron may become anemic. Using rivers or lakes as a toilet can increase the levels of nitrate in the water, which may be toxic to other people who use the rivers and lakes for swimming or drinking water. Introduction 3
Phase 2: Microscopy In phase three you will look at a small drop of each water sample under a microscope, to see if it has any microscopic invertebrates in it. Most water supplies have some invertebrates in them and most are harmless. However, there some harmful invertebrates too (see table 1 and the Fresh Water Invertebrates Field Guide). 1. Transfer a few drops of water to the cavity slide using a pipet. 2. Place the cavity slide on the microscope. Carefully focus on a piece of debris in the water using a low magnification, then look for moving organisms on/around the debris. Ask you teacher to check what you are looking at. Some organisms may have moving parts such as tails, flagella or mouth-parts. 3. See how many you can identify and compare your findings to the rest of the class. 4. Estimate how many organisms you can see in ~5 fields of vision (5 different locations on the slide), then record the total number. Table 1: Fresh water invertebrate examples Schistosomiasis miracidia The larval stage that infects humans and causes schistosomiasis or bilharzia Giardia A protist that can cause diarrhea Found in water contaminated with human urine and fresh water snail hosts Found in water contaminated with human feces NOTE: A lot of harmless protists look similar. If you see something like this don t panic Ameba Some amebas can cause severe diarrhea, but many are harmless Rotifers Are very common in ponds and rivers, and are harmless Nematodes Are very common in ponds and rivers. Most come from the soil and are harmless Often found in water. Those that cause diarrhea or dysentery come from human sewerage Some nematodes may come from cat or dog feces, and can be harmful to humans NOTE: these are really fun to watch and have interesting, rotating mouth parts Introduction 4
You may also see different types of algae, which are more closely related to plants and are harmless (see figure 2 below). A lot of algae may be an indication of high nitrate levels in your water sample. If you see a lot of these, check the results of your nitrate test to see if it was high. Figure 2. Some common, fresh water algae Phase 3: Making and testing a water filtration system. Water treatment plants that provide drinking water to cities use a simple filtration system to purify recycled water and make it safe for drinking. The water is simply filtered through a series of beds that contain different combinations of gravel sand or charcoal. Invertebrates don t pass easily through sand and gravel, and bacteria stick to charcoal. It is easy to make a small scale water filter using sand or gravel or even crushed coal that is effective and removes most debris and many organisms. To be completely safe, the filtered water should be boiled before use. Presence of large amounts of algae in water is often an indication that nitrate levels are high. This may suggest that the water is contaminated with fertilizers or sewerage. You can re-use the cavity slides if you rinse them with water and lay them up-side down on a piece of paper to dry. 1. Make a collection of sand, gravel, crushed coal and/or any other particulate matter that you find lying around, by collecting them from the environment. Dry each completely by baking in an oven for an hour, or spreading them out on plastic or metal sheets in the sun for a few days. Introduction 5
2. Store your gravel, sand etc. in plastic bags, buckets or plastic containers somewhere dry. 3. Take a large syringe, pull out the plunger and push a tiny bit of cotton wool (½ cm across) into the bottom with a pair of tweezers. 4. Make your filter by putting ~ ½ - 1 inch layers of the materials you collected in step 1, into the syringe. Put the finest materials at the bottom (and make these layers a little thinner, or the water will take a long time to filter through it). You can make more than one type of filter by using different combinations of materials, and test them all to see what works best. 5. Hold your filter over a clean tube or clean glass container, and carefully pour about ¾ of your dirtiest water sample through the filter. It may take a long time, but your only need a few drops for testing. 6. Put a few drops of your filtered water sample into a clean cavity slide and look at it under the microscope. Estimate the number or organisms in five fields, as before, and record your results. Are there fewer organisms than before? Use the equation below to calculate the % decrease # organisms before filtration # organisms after filtration X 100% Total (# before + number after) 7. Retest your sample for iron, copper and nitrates to see if filtering reduces the contamination level of these pollutants. 8. Take a culture dish (see Lab: Culturing Bacteria for protocol for making culture dishes) with Tryptic Soy agar on it. Divide the dish into two sides by drawing a line across the underside. Carefully spread a little unfiltered water on one side. Label the outside of the dish so you know which side is the unfiltered water. Then, carefully spread a little of the filtered water on other side of the dish and label it. 9. Incubate your plates in a warm place for a few days then count the colonies of bacteria on both sides. Use a similar equation as the one above to calculate the % decrease in bacteria after filtering your water. You can re-use syringes if you immerse them in boiling water for ~10 minutes, allow them to dry then wrap them in paper and store them in a dry place. Use ~ ¾ of your water sample to test your water filtration system, but KEEP the rest to use as a control later. Use a Q-tip to spread your water on the plates. If they are dry before you use them, they will be clean enough BUT DON T TOUCH THE TIP WITH YOUR HANDS! Introduction 6