Belle Vernon Area School District



Name ___________________________Microbiology Chapter 3: Tools of the Laboratory Notes3.1 Methods of Culturing Microorganisms: The Five I’sWhat does it mean “to culture” microorganisms? What is a “culture”?It means to “grow” microorganisms. Typically, a microorganism, such as bacteria, is purposely placed in an environment with nutrients so that they may multiply over and over again, until there are enough present that they can be seen and studied.Why is sterile (aseptic) technique so important when culturing microorganisms?When sterile procedures are used, only the microorganism that is intended to multiply and grow will do so. When contamination occurs, the scientist doesn’t know what’s growing.Media:What is media?An environment, either liquid or solid, that is full of microorganism nutrients where they can multiply and grow.What is enriched media? Give an example of how it can be used in the laboratory.This type of media contains more than just microorganism nutrients, but also specialized nutrients such as blood, serum, hemoglobin, or special growth factors that specific microorganisms need to survive and multiply. An example is streptococci (causes strep throat) because this bacterium needs animal elements to survive.What is selective media? Give an example of how it can be used in the laboratory.This media contains specific ingredients that inhibit the growth of certain microbes, but allow the growth of others (its selects for what can grow and what cannot grow!) This is often used for use of identifying the genus and species of an unknown microorganism. An example would be determining what specific microbes are present in water or soil.What is differential media? Give an example of how it can be used in the laboratory.This media allows multiple types of microorganisms to grow but is designed to display visible differences amongst them. For example, each type of microorganism my show up in a different color, allowing scientists to identify their species.What is a reducing media? Give an example of how it can be used in the laboratory.This type of media absorbs oxygen to reduce its availability. This allows only anaerobic (doesn’t need oxygen) microorganisms to survive and grow, killing off the aerobic (must have oxygen) ones. This type of media may be used to determine if an unknown microorganism uses anaerobic or aerobic respiration to make ATP.What is enumeration (means to count) media? Give an example of how it can be used in the laboratory.This type of media is used to count the number of microorganisms in milk, water, food, and soil. It is used to assess the cleanliness and safety of things we consume.What is transport media? Give an example of how it can be used in the laboratory.This type of media typically contains salts and buffers and is used when microorganisms must be preserved for a long period of time, without dying off. This can be used when the microorganism needs to be studied but time and resources prevent the scientists from doing so immediately, thus they must be preserved for later study.Describe the difference between liquid and solid media.Liquid:Solid: Liquid comes in a test tube and the microorganism freely floats in the liquid. The liquid becomes more and more cloudy as the microorganism multiply.Solid comes in the form of agar in a petri dish, where the microorganisms grow as visible colonies, that look like raised bumps on top of the agar.What is agar? How do we typically use agar in microbiology?Agar is “jello” that contains nutrients for microorganisms. It solidifies in a petri dish and then scientists purposely contaminate the gel, which introduces the microorganism so that it can survive and multiply.What is an inoculation loop? Why is sterile technique so important when using an inoculation loop?An inoculation loop is a sterile tool (either plastic and comes sterile or metal and is sterilized by a flame) that is used to collect a sample of microorganisms and place them into media for growth.Describe how to “streak” a plate. Watch the video at purpose of streaking a plate is to spread out the microorganism enough so that individual colonies, or groups will be visible. Cultures:What is a pure culture? Grows one and only one type of microorganism and the scientist knows exactly what he/she is growing.What is a mixed culture?Grows two or more microorganisms and the scientist knows exactly what he/she is growing. It is easy to tell them apart. For example, one may grow red and the other yellow.What is a contaminated culture?When microorganisms make their way onto media that aren’t supposed to be there and grow. Then the scientist doesn’t truly know what is being studied.The Five I’s are used to sample and profile microorganisms. Describe each step:Inoculation: Known sample is placed onto media containing the proper nutrients for its growth. This must be done using sterile technique.Incubation: After inoculation, the sample is placed in an “oven” that provides the correct growth conditions for the microorganisms. The temperature can be adjusted. Most microorganisms grow well in a temperature warmer than room temperature, but if it gets too hot they will die.Isolation: Once the microorganism has grown colonies should be present. A sample of one of the colonies is taken and steps “a-c” are repeated to grow only the type of microorganism that was in that colony; this it has been “isolated.”Inspection: Visual inspection includes looking at the colony with the naked eye for colony color, size and shape, and then taking a sample from that colony and placing it under the microscope to identify details not present to the naked eye, such as shape (cocci, bacillus, spirillum) Identification: Biochemical tests, such as gram staining or DNA analysis, are performed to identify the species and biological characteristics of the microorganism.How are microorganisms disposed of in a sterile manner?Autoclave: An oven that reaches very high temperatures and pressures. Nothing can live under such conditions so it kills off any living microorganisms.Incineration: To burn with fire, killing off the microorganisms.3.2 The Microscope: Window on an Invisible Realm Review your microscope packet for parts and functions before the next test.We know that microorganisms are microscopic. This means their measurements are measured in “micros.” What symbol is used for “micro”?The Greek letter m, which looks like a cursive u. It is .How would you write:Microliters: lMicrometers: mMicrograms: gWhat are fixed, stained smears?The bacteria have been “heat fixed” to the slide, meaning permanently stuck to. The stained portion refers to attempting to stain the bacteria to see if the stain sticks, indicating the presence of a thick cell wall.Where are fixed, stained smears typically used?In gram staining.What is the difference between a positive and a negative stain?Positive means the stain was able to stick to the microorganism, and negative means the stain did not stick to the microorganism and was washed away.What is Gram Staining and what is one major way it can be used in the medical field today?Gram staining “stains” the thick cell wall of some bacteria. By staining the wall it shows the presence of the wall. This is used in the medical field to determine if antibiotics will be effective against a bacterial infection. Antibiotics only work on bacteria with thick cell walls.Take notes as your teacher shows a video on gram staining and presents a power point presentation on the steps and uses of this technique! ................
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