MICROORGANISMS are everywhere, from the

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1 Exploring Microbiology MICROORGANISMS are everywhere, from the clouds over Mt. Everest to the bottom of the deepest ocean. They grow in the deserts and the ice. They live in hot acid and solid rock half a mile under the earth s surface. They are on you and in you and every other living thing. Microorganisms have been here on Earth for 3.5 billion years. Their numbers are almost incomprehensible. What is microbiology, and how can you study things so small? Objectives: 1. Describe microbiology and the methods used to study microorganisms. 2. Identify the types of microorganisms and their characteristics. Key Terms: acidophiles agar algae amoebae anaerobic archaea aseptic techniques autotrophic bacilli bacteria bacteriophage capsid capsule chemoautotrophic ciliates cocci colony decomposers destain diatoms dinoflagellates disinfection enzymes euglena eukaryotic fermentation flagella fungi genetic engineering Gram stain halophiles heterotrophs hyperthermoacidophiles hyperthermophiles hyphae incubator inoculating loop media methanogens microbiology microorganism parasitic pathogens peptidoglycan petri dishes pharmaceuticals photosynthetic probiotics prokaryotic protists pseudopods psychrophiles serial dilution soil symbionts spoilage stains sterilization streak plate thermophiles trypanosomes viruses yeasts Page 1

2 Microbiological Importance and Methods Microbiology is the study of microorganisms. A microorganism is a living thing that can be seen only with magnification. Microorganisms are the most numerous organisms in any environment. Their activity is responsible for many beneficial as well as harmful effects. The small size of microorganisms requires the use of specialized methods to study or control their growth. MICROBIOLOGICAL ACTIVITY FIGURE 1. Most microorganisms are studied with some type of microscope. Beneficial Activity Many microorganisms make up a natural beneficial population in and on your body and the bodies of other organisms. Some of these are even introduced into your body as probiotics (beneficial microorganisms) in capsules or in foods such as yogurt. Other microorganisms produce pharmaceuticals (drugs used in medical treatment), such as antibiotics. Some pharmaceuticals, such as insulin, can be made by genetic engineering that is, introducing new genes into a microorganism to change its biological activity. Enzymes (the proteins that catalyze chemical reactions in living things) can be produced in quantity by microorganisms. Some of these are used to convert crop materials into ethanol. Soil symbionts are organisms in the soil that establish a relationship with plants that benefits the organisms and the plants. The rhizobium bacteria that live in the roots of legumes, such as soybeans, are symbionts. While taking nutrient energy from a plant, they change nitrogen from the air into biologically active nitrogen compounds that benefit the plant. Not only do the soybeans grow better, but excess nitrogen compounds are left in the soil after harvest to benefit the next crop. Fermentation is a process for releasing energy from carbohydrates when there is insufficient oxygen for respiration. Though this process can lead to damage of food, when it is controlled it produces desirable food products that are less susceptible to further damage by microorganism activity. The fermentation by yeasts of grapes and grain to wine, beer, and spirits is an example. Bacterial fermentation action is used to produce products such as soy sauce and sauerkraut. Page 2

3 Harmful Activity Pathogens (microorganisms that cause disease) are found in every group except the archaea. There are pathogens responsible for diseases in plants, animals, and other microorganisms. Although many microorganisms are pathogenic, far more are not. Bacteria and viruses cause most of the human diseases with which you are familiar. Some diseases are caused by protists and fungi. Spoilage is the deterioration of food quality from the activity of microorganisms. It is a significant factor, from the farm to the processor to the store and into the home. The microorganisms feed by dumping enzymes out of their cells onto the food and then taking in the digested food. The action of these enzymes often ruins the food s texture, smell, and nutritional value. Some of the chemicals produced by the microorganisms are even toxic. MICROBIOLOGICAL METHODS Controlling Growth FIGURE 2. If food is not properly stored, spoilage can occur from the activity of microorganisms. To study microorganisms accurately, they must be handled using special techniques. The equipment and the environment must be kept free or nearly free of any contaminating organisms. Disinfection is the use of agents or processes such as chemicals, radiation, or heat to clean surfaces of microorganisms. It may not kill every microorganism, but it will greatly reduce the chance of contamination. Sterilization is the use of processes that kill all microorganisms. Dry heat can be used for sterilization on hard-surface materials. Many bacteria can be killed by reaching the boiling point of water. Filtration is the use of filters to purify media for culture. These methods for keeping the microorganisms where you want them are called aseptic techniques. When aseptic techniques are properly applied, you will produce a pure culture, a population of only the microorganism you want. Specially prepared media (the materials used to grow microorganisms) containing water, food, and other nutrients needed by the organism will be used. Sometimes it is desirable to have microorganisms grow in colonies on the surface of a medium. A colony is a small mass of bacteria on an agar medium that resulted from the reproduction of one cell. To get the colony to grow on the surface of a medium, agar is added. Agar is a derivative of algae that is used to produce a relatively solid gel at room temperature. Page 3

4 The agar medium is most often used in tubes or petri dishes. Petri dishes are round glass or plastic dishes with loose, overlapping lids. Isolating Microorganisms To get a pure culture, microorganisms from a single colony must be transferred to a sterile medium. The separate colonies can be obtained from mixed or unknown sources several ways. An inoculating loop (a small ring of wire attached to a handle) is often used for transfer. Such a loop is made of material such as stainless nichrome wire to prevent oxidation from the repeated heating needed for sterilization. Making a streak plate is a way of isolating bacteria by diluting them through successive streaks. The procedure involves taking a loop of bacteria and making a back-and-forth motion across one side of a medium-filled petri dish. In the process, the lid of the petri dish should be raised at an angle. Care should be taken so the loop is not pushed into the medium surface. The loop is heat sterilized, touched to the medium to cool, and then swept across the first streak. The loop carries some bacteria to an area of the medium that is bacteria free. This process is repeated by sweeping the loop across the second streak to make a third streak and across the third streak to make a fourth. Care must be taken not to overlap the original streak. Since the third or fourth streak is taking only a few bacteria from the previous streaks, the number of resulting colonies is reduced. As a result, single isolated colonies are allowed to develop. The plate is sealed and inverted and then placed in an incubator (a device to keep microorganism cultures at the optimal temperature for their growth). Isolating or separating bacteria can also be achieved by serial dilution (the use of repeated measured transfers to separate bacteria). When serial or repeated dilutions are made and transferred to a solid agar medium, separate colonies can be detected, and from the dilution, an approximate count of the original sample can be calculated. Seeing Microorganisms Microorganisms can be seen only when present in large numbers as colonies on agar or individually under a microscope. In both cases, many of them still look very much alike. To be able to differentiate them and make them more distinguishable, stains (chemicals that make microorganisms more visible) are added. Eosin methylene blue is added to nutrient agar, making EMB agar, specifically to detect Escherichia coli bacteria. Most bacteria are divided by a specific staining procedure called the Gram stain. This staining procedure uses two different stains and differences in the bacteria to tell the bacteria apart. The bacteria are stuck to the slide by FIGURE 3. E. coli bacteria on EMB agar. Page 4

5 UNDER INVESTIGATION LAB CONNECTION: What Do Yeasts Eat? Yeasts are one-celled fungi that do not make their own food. They can ferment their food, or break it down to release energy in an oxygen-poor environment. They give off carbon dioxide gas when they are fermenting their food. You will need a resealable clear sandwich bag for each food tested, a packet of dry baker s yeast, a ½-cup or 150-ml measure, a cookie sheet with edges or other large flat container, water, and food sources. Some suggestions for the foods are a teaspoon of sugar, a packet of sugar substitute, a teaspoon of honey, diet soda pop, high-fructose soda pop, soda pop with sugar as the main ingredient, or a sports drink with glucose or dextrose as a major ingredient. The soda should be clear and left open over night or be shaken to dissipate its carbon dioxide bubbles. Mix half the yeast packet in warm water. Fill each bag with 150 ml of liquid. Use water to dissolve the sugar or sugar substitute. Add 1 teaspoon of yeast solution. Lay the bag flat in the container, and remove all air bubbles. Place the container in a warm environment. The amount of gas that collects will tell you the relative amount of food available for the yeast. heating them. They are then stained with crystal violet, rinsed, and treated with iodine. It is then necessary to destain the slide that is, rinse it with an acetone/alcohol mixture to remove the stain. If the bacteria have thick walls of peptidoglycan, a protein/sugar complex organic compound, they will not destain and will appear violet under the microscope. If the bacteria have little peptidoglycan in their cell walls, they will destain and will not be visible. They must, therefore, be counterstained with safranin, a red stain. Violet bacteria are said to be Gram positive, and the red or pink ones Gram negative. Some bacterial structures that do not take other stains well can be visualized by a negative stain, one that attaches to the slide rather than to the bacteria. The capsule, or the sugary slime layer of the bacteria, is damaged by heat fixing and can be visualized by mixing bacteria with nigrosin and making a smear. The slide is air dried, and the cells appear as clear areas against a black background. Microorganisms Are Extremely Diverse DOMAINS OF LIFE Microorganisms have representatives in all three domains of life: Bacteria, Archaea, and Eukarya. Two of those domains Bacteria and Archaea consist of only microorganisms. Though only tens of thousands of species of microorganisms have been cultured and identified, DNA surveys have indicated that hundreds to thousands of times more species exist in Page 5

6 the environment. There are more bacteria on a human or in a scoop of soil than there are humans on Earth. Microorganisms are found in every environment on Earth that has life. In some environments, they are the only life. Bacteria Bacteria are microscopic unicellular prokaryotic organisms. A cell that is prokaryotic has no nuclear membrane. The groups are mainly divided by shapes bacilli (rod shaped), cocci (round or seed shaped), and spiral. The shapes are further divided by their response to Gram staining. Though we are familiar with the pathogenic, or disease-causing, bacteria, most bacteria are not pathogenic. Many bacteria are autotrophic, producing their own food. Some are photosynthetic, using the energy of sunlight for food production, and some are chemoautotrophic, using chemical reactions for food production. The majority of bacteria are heterotrophs, taking in complex food molecules, and most are decomposers, feeding on nonliving organic material. Archaea Archaea are unicellular prokaryotic microorganisms that are significantly different biochemically from bacteria. They are difficult to culture, so they were thought to be unusual bacteria until analysis revealed their biochemical differences. Their DNA indicates a closer relationship to eukaryotes than to bacteria. There are no known archaeal pathogens. Many archaea have unusual biochemistry. Some are chemoautotrophs. One such group is the methanogens, which produce methane from the reduction of carbon dioxide with hydrogen. They are common in swamp mud, sewage sludge, and animal guts, such as those of cows and humans. Other archaea are extremophiles, organisms that can live in environments too extreme for most life. There are halophiles, which can live in high salt concentrations, such as those of the Dead Sea. Thermophiles survive high temperatures, and hyperthermophiles can exist even at temperatures above the boiling point of 100 C, where ocean pressure keeps the water liquid. Psychrophiles live at very cold temperatures, and acidophiles require environments too acidic for other forms of life. There are even hyperthermoacidophiles, which combine the need for high temperatures with the need for low ph. Eukarya A microorganism that is eukaryotic has a membrane-bound nucleus. Protists Protists are primarily unicellular eukaryotic organisms. Some, such as algae, euglena, dinoflagellates, and diatoms are photosynthetic. Algae range from microscopic photosynthetic cells to macroscopic filaments to giant kelp. Some are found as single cells in both aquatic environments and moist terrestrial environments. Unlike photosynthetic plants, euglena and dinoflagellates swim with flagella, long whiplike structures. Diatoms are unusual in Page 6

7 having cell walls of silica that resemble ornate petri dishes. Euglena and some dinoflagellates can function as heterotrophs, eating food but not producing it. Ciliates are mostly free-living heterotrophs that move with many short flagellum-like structures, called cilia. Amoebae are mostly free living, though a few are pathogenic and parasitic, living in or on other living things. Amoebae move by having their cell contents flow into projections of their cell membrane called pseudopods, or false feet. Trypanosomes are all flagellated parasites, the most notable being the cause of African sleeping sickness. Fungi Most of the fungi, such as mushrooms, are not microscopic. However, there are stages in the life of most fungi where they are microscopic. Some, such as yeasts, are always microscopic. The feeding structures of fungi, called the hyphae, are long, but only one cell wide and therefore not readily visible. Yeasts are single-celled fungi. Some species have the ability to break down food readily by fermentation. Humans have used yeasts and their fermentation process to produce many useful products. FIGURE 4. Many types of fungi can be easily seen with the naked eye. VIRUSES Viruses are microorganisms that are not cellular and do not have their own metabolism. They are included in the microorganisms, though by most definitions they are not living organisms. They are all ultimate parasites. Within a host cell, a virus s genetic material takes over and uses the host cell s biological process to reproduce. There are viruses that infect virtually every kind of living thing, from the T4 bacteriophage that infects bacteria to flu viruses that infect you. A virus consists of a protein capsid, or cover with specific proteins that help in identifying the virus, and a genetic core. The genetic material can be in the form of DNA or RNA and can be either single or double stranded, depending on the type of virus. Summary: Microbiology is the study of living things that are too small to be seen without a microscope. Though some microorganisms are pathogenic, many more are benefi- Page 7

8 cial members of every biological environment. Some are important components in human-controlled processes. Examples are soil symbionts in farming and yeast in food production. Microorganisms small size requires the use of specialized methods to study or control their growth. The microorganisms are the most numerous and most diverse forms of life on earth. All members of the domains Bacteria and Archaea and many of the domain Eukarya are microorganisms. Checking Your Knowledge: 1. How are microorganisms beneficial to humans? 2. Other than causing disease, how are microorganisms harmful? 3. Describe how to obtain a pure culture. 4. What are aseptic techniques? 5. What is the most significant biological similarity between bacteria and archaea? Expanding Your Knowledge: Take the position that viruses should be considered alive. Prepare biologically sound arguments for your position to be presented to a panel of biologists. Be sure to include your answers to the anticipated arguments from those who will disagree with you. Web Links: Photos, Animations, and Descriptions of Microbial Life Methods of Detecting Microorganisms Microbiology Science Project Ideas From=Tab Agricultural Career Profiles Page 8