Microbiology Week 4 Notes
Microbiology Week 4 Notes MICR 3050
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This 4 page Class Notes was uploaded by Toni Franken on Monday February 1, 2016. The Class Notes belongs to MICR 3050 at Clemson University taught by Dr. Whitehead in Spring 2016. Since its upload, it has received 34 views. For similar materials see General Microbiology in Microbiology at Clemson University.
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Date Created: 02/01/16
MICR 3050 – Notes Set 6, 01/25/2016 Dr. Whitehead, Clemson University Chapter 2: Microscopes and Staining Procedures Heat Fixation: It appears that heat fixation actually works because…it just works. Some suggestions, however, do say that proteins and lipids are denatured, and that this allows them to cling to the glass better. Staining Continued: Endospore Staining: Can tell you size, location, and shape of the endospore, or if there is an endospore present at all. o Heated, double staining technique. Have to use heat to force the dye into the spore in order to overcome the resistance. Resistant to destaining as well. o Bacterial endospore is one color and vegetative cell is a different color. o Endospores: Incredibly stress resistant spore formed inside the bacterium (Bacillus anthracis and clostridium are two types of bacteria that make them). Usually forms when conditions are poor, and the bacteria will most likely die, leaving the endospore behind. They are capable of resisting boiling, disinfectants, and more. There is information about the bacteria in the size, location, and shape of the endospore. Capsule Staining: Negative staining is where you stain the fluid around the object, but not the object itself. Will appear clear or white against a dark background). Negative staining technique – capsules are colorless against a stained background. Presence and type of the capsule is important to tell information of the organism, and may even be absolutely vital for an organism to cause an infection. Capsule size can be important. Flagella Staining: Staining of flagellum o There are bacteria capable of movement, and by multiple methods. However, one movement type is by flagella. The number and location of flagellum on a bacterium are important for identification and information. May have flagella everywhere, on both ends, on one end, and may have singular or grouped flagella. o Under normal light microscopy, flagella CANNOT be seen. In flagella staining, we will coat the flagella with a mordant to make them appear thicker, and help the dye stick to the flagella. This allows them to be thicker, and colored, to see with a normal light microscope. Basics of staining Repeated o Acid fast: Red cells are mycobacteria, blue cells are not mycobacteria. o Endospore staining: Endospores are red, rest of cell is blue. o Capsule stain: Bacteria and capsules are not stained, but the fluid around them is. o Gram stain: Purple cells gram positive, red cells gram negative. Chapter 7.5: Laboratory culture of cellular microbes: Koch’s postulates: According to Koch, you must be able to culture a bacterium to identify it – growing bacteria, however, is important for any part of microbiology. Nutrient preparations are devised to support the growth and reproduction of microorganisms: All different media must contain: o Macronutrients (substance needed by bacteria in fairly large amounts, such as Carbon, Nitrogen, or phosphorous). o Micronutrients (substances required at small levels, possibly even trace element levels, such as specific metals). o Growth factors (help promote the growth of microorganisms – larger molecules like biologically relevant molecules – some bacteria require them. Example: Amino Acids must be given). Characteristics of Media: o Nutrient media can be liquid or solid. Solid media are usually solidified with agar (made with seaweed). We don’t use gelatin for a number of reasons. Some bacteria can break down gelatin, which makes it less than ideal for culturing. However, it also has more difficult melting points to work with. o Chemical composition: Defined Media: Concentrations and chemical compositions are exactly known. Nothing is unknown in defined medium. We know the exact amount of EVERY component. Complex Media: We don’t know everything about the medium. We don’t know exact composition and/or concentration of the media. Know general composition, but not exactly how much. Examples: Nutrient Broth, TSA, TSB, MacConkey Agar. How much carbon is in peptone, tryptone, etc? We have no idea the chemical composition, because it varies from batch to batch. It often doesn’t matter exactly how much is in it. May contain extracts: Aqueous extracts, usually beef or yeast. Lyse the molecules, make it easier for bacteria to use, and allow to dry. o Physical nature: Liquid (such as broths) Semisolid (used to see if bacteria are capable of movement, contains some agar, but only enough to thicken it) Solid – agar plates. Agar: Sulfated polysaccharide sued to solidify liquid media. Ideal solidifying agent because: We can melt agar with boiling water at about 90 °C. It solidifies at about 45 °C. You can pour the agar while still liquid because you can comfortably hold things at about 55 °C. Can use it to culture a wide range of microbes without killing them. In addition, most microbes aren’t capable of degrading agar, making the plat turn to mush. o Function: General Purpose media (supportive) – Supports the growth of many microorganisms. Typically nutrient rich. TSA and TSB (tryptic soy agar/tryptic soy broth) are examples. Something will grow in it regardless of what you culture. Enriched Media: More fastidious (aka, picky) microbes will grow on it – there are extra nutrients added in. It is general purpose media supplemented by blood or other special nutrients (usually use sheep blood). Chocolate agar – contains blood and gives it a brown appearance. Minimal Media: Contains the minimal necessities for growth of the wild type organism. They only contain inorganic salts, a simple carbon source, and water. Ecoli can grow on minimal media. Often use to figure out the metabolic capabilities of an organism. Can it use glucose, maltose, chondroitin sulfate, etc? Selective Media: Favor the growth of some microorganisms and inhibit the growth of others: Example is MacConkey agar – selects for gram negative bacteria, and tends to inhibit grampositive bacteria. Can also add antibiotics to select for those resistant to antibiotics. Can also add a carbon source that many bacteria can’t use (such as cellulose – only some bacteria can use it). Differential Media: Distinguish between groups of microorganisms based on their biological characteristics. You will be able to see the difference. Blood agar is a type of differential medium (as well as enriched): A number of bacteria can lyse red blood cells, and degrade them to varying degrees. This is called hemolysis, and different bacteria have different types of hemolytic activity. Bacteria that lyse red blood cells partially will have a cloudy green color around them. Bacteria that lyse red blood cells completely will have a completely clear area that completely destroys blood cells. Bacteria that do not lyse cells, but will simply grow. MacConkey agar: Lactose fermenters versus nonfermenters – selective for gram negatives, and among those gram negatives, some can ferment lactose, and some cannot. A color change will occur when the bacteria can produce a great deal of acid to ferment lactose. It causes a pH change (and therefore a color change due to the pH indicator in the plate) to varying degrees. Ecoli has a very high ability to ferment lactose = big color change. This makes it selective and differential. Mannitol salt agar: selective and differential. Looks at how well organisms can grow in a specific carbon source, and cause a color change. Know blood agar and MacCokey agar. Also, if she gave a new type of media, described it, can you tell its characteristics. Isolation of Pure Cultures: We can only culture about 1 – 10% of known bacteria purely. We can often get them down to a culture of 2, but can’t get them by themselves. o Pure culture: Population of cells that arises from a single cell. If you’re trying to get a genetically pure colony, in theory, a pure culture is as close as you can get. We used to think that pure cultures are clonal (all identical), but mutations happen, and throw it off. It allows for the study of single types of microorganisms. o We can isolate a pure culture in various ways, but all involve putting a mixture of cells on an agar surface so that individual cells are well separated from each other. Techniques for isolating pure cultures: o Streak Plating: Start with a dense area of bacteria placed on a plate with a wire loop. Streak back and forth in 1 quadrant of the plate to create the Primary Streak. Flame the loop, and DO NOT dip it back into the original sample. Once sterile, drag the loop across the second quadrant, dipping into the first quadrant, creating the Secondary Streak. Flame loop, and repeat for the third and fourth quadrants, until your streak towards the middle will, hopefully, produce single, isolated colonies. o Spread Plate: To create a spread plate, you must take a small volume of diluted culture (100 uL, usually), and transfer it to the center of the agar with a pipette. The culture is spread evenly over the surface with a sterile bent rod, often called a hockey stick. The point is to spread a very small number of cells evenly over the surface of the plate. A countable plate should have between 30 and 300 colonies. o Pour Plate: To create a pour plate, take a small amount of diluted sample, and mix it with liquid agar that has cooled to about 45 – 50 °C, and pour the whole thing into an empty petri dish. You want to start with a diluted culture so that the cells are spread out enough that you can count the colonies. The mixing action of agar and culture is what distributes the cells. A countable plate should have between 30 and 300 colonies. Aseptic techniques: Main purpose is to prevent contamination of culture, environment, and experimenter. You don’t want contamination of culture, and don’t want contamination outside of the culture. Maintain a pure culture For aseptic transfer: Do a lot of disinfecting of surfaces before and after the experiment, work with a flame, you flame the loops, the mouth of the tubes, use some alcohol, etc. Hopefully, with these procedures, you’ll have sterile instruments.