Lab Ex.'s 3,4,5,7
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| What is a smear? | To stain bacteria, a thin film of bacterial cells must be placed on a slide to be viewed through a microscope.
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| Fixing | Denatures bacterial enzymes, preventing them from digesting cell parts, which causes the cell to break(autolysis).
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| Heat fixing | A slide is run through a Bunsen burner flame several times to dry the smear, does not kill the bacteria.
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| Chemically fix | Cover the smear (with bacteria on it) with 95% methanol for 1 minute.
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| What is a chromophore? | The ion that is colored. If chromophone is a (+) ion like methylene blue , the stain is considered a basic stain; if it is a (-) ion it is an acidic stain.
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| Simple stain | Staining procedures that use only one stain. Can be used to determine cell morphology, size, and arrangement.
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| Direct stain | A simple stain that stains the bacteria.
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| Negative stain | A simple stain that stains the background but leaves the bacteria unstained.
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| Of what value is a simple stain? | quick observation of the morphology and arrangement of bacteria.
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| What is the purpose of heat fixing the smear? | To makes the bacteria sticky so they stick to the slide.
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| In heat fixing, what would happen if too much heat were applied? | It would burn and/or kill the bacteria. Cells would be completely destroyed "plasmylosis" and burst and bacteria would not hold stain.
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| What bacterium is a rod? | Baccillus
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| Which bacterium is larger? | B. megaterium
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| Gram stain | A differential stain that allows you to identify and classify bacteria as either gram- positive (+) or gram-negative (-).
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| Primary stain (1st step in gram staining) | Crystal Violet (basic dye stains bacteria purple).
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| Mordant (2nd step in gram staining) | Gram's Iodine (combines with crystal violet in cell to from a crystal violet-iodine complex (CV-I)).
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| Decolorizing agent | Ethanol of ethanol-acetone (primary stain is washed out (decolorized) of some bacteria, whereas others are unaffected.
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| Secondary Stain or Counter Stain | Safranin (a basic dye which stains the decolorized bacteria red).
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| Gram-negative(-) | Those that decolorized easily
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| Gram-positive(+) | Those that decolorize slowly and retain the primary stain.
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| Peptidoglycans | Bacteria cell walls are composed of this complex lattice structure along with other compounds. Gram(+) cell walls contain multiple layers of peptidoglycans, whereas gram(-) bacteria contain a thin layer of peptidoglycan.
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| Why will gram (+) cells more than 24 hours old stain gram (-)? | They may be old cells that have a deteriorated cell wall, as cell age there cell walls degrade which may nor allow bacteria to retain stain and give inaccurate results.
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| Can iodine be added before the primary stain (crystal violet) in a Gram stain? | No
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| Suppose you performed a Gram stain on a sample from a pure culture of bacteria & observed a field of red and purple cocci. Adjacent cells were not always the same color. What do you conclude? | That some were a successful stain & the others may have been dead or just didn't retain the stain.
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| Suppose you are viewing a Gram-stained field of red rods and purple cocci through the microscope. What do you conclude? | A mixed culture OR contamination has occurred.
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| Considering you can't identify bacteria from a Gram stain, why might a physician perform a Gram stain on a sample before prescribing an antibiotic? | Antibiotic sensitivity correlates with the cell wall type of the bacteria.
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| If you performed a Gram stain on human cells, what would happen? | NO cell walls, the cells would not hold the stain, ETOH (alcohol) would destroy the membranes (phospholipid bilayer).
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| Chemically defined medium | A medium whose exact chemical composition is known.
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| Complex media | Media for which the exact chemical composition varies from batch to batch.
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| Nutrient broth | Is a commonly used liquid complex medium.
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| Nutrient agar | When agar is added to the nutrient broth it becomes a solid medium.
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| Agar | An extract from marine red algae, has some unique properties that make it useful in culture media. Few microbes can degrade agar, so it remains solid during microbial growth.
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| Steam sterilization OR autoclaving | Which uses steam under pressure( during this process, material to be sterilized is placed in auto-clave and heated to 120 C at 15 lbs. of pressure for 15 minutes. It's the most common method of sterilizing culture media that are heat stable.
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| Petri plates | Contain solid media which provide a large surface area for examination of colonies.
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| Inoculated | Intentionally introduced, into various forms of culture media to keep them alive and to study their growth-without introducing unwanted microbes.
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| Incubation period | Bacteria that are inoculated into culture media increase in number during this period. A period of usually 24-48 hours were bacteria are heated a 98.6 degrees Fahrenheit to allow microbial growth.
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| Turbid | After a suitable incubation, liquid media become cloudy as a result of bacterial growth.
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| Colony | A population of cells that arise from a single bacterial cell.
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| Colony-forming unit | When a colony arises from a group of the same microbes attached to one another.
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| What is the minimum number of different bacteria present on one of your plates? How do you know? | > or equal to 1.
> or equal to 1 colony type seen.
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| What is the value of Petri plates in microbiology? | Have a larger surface area, easier to subculture so that you can see the isolated colonies of bacteria.
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| What are bacteria using for nutrients in the nutrient agar? | Peptones and beef extract
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| What is the purpose of the agar? | A solidifying agent ( solid/semi-solid surface to grow bacteria).
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| Why is agar preferable to gelatin as a solidifying agent in culture media? | It stays solid at incubator temp of 98.6 degrees Fahrenheit and most bacteria can't eat or digest it.
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| Did all the organisms living in or on the environments sampled grow on your nutrient agar? | No Way! We only provided one food type and one growing temperature.
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| Contaminants | Unwanted microbes
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| Aseptic technique | Used in microbiology to exclude contaminants and is performed under sterile conditions.
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| Sterilized | Rendered free of all life, prior to use, usually accomplished during autoclave.
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| Broth cultures | Provide large numbers of bacteria in a small space and are easily transported.
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| Agar slants | Test tubes containing solid culture media that were left at an angle while the agar solidified. Provide solid growth surface but are easier to store and transport than Petri plates.
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| Inoculating loop and Inoculating needle | When end of wire is bent into a loop AND when it's straight.(Aseptic transfer and inoculation are usually performed with this sterile, heat-resistant, noncorroding Nichrome wire attached to an insulated handle).
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| Agar deep | Solidifies at the bottom of the test tube, often used to grow bacteria that require less oxygen than is present on the surface of the medium.
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| Were any of the bacteria growing in the semisolid agar deeps motile? | Yes, resembled an upside down Christmas/pine tree. (Motile bacteria will move away from the point of inoculation, giving the appearance of an inverted pine tree)
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| What is the primary use of slants? | Solid surface,easier to store than plates and not easily contaminated.
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| What is the primary use of deeps? | Motility and oxygen requirements.
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| What is the primary use of broths? | To maximize growth (the rate/amount)
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| What is the purpose of flaming the inoculating loop before use? After use? | Before for sterilization to minimize contaminants. After for the safety of people within the lab.
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| Why must the loop be cool before you touch it to a culture? Should you set it down to let it cool? | So you don;t burn and or kill the bacteria your trying to obtain. No because you risk contaminating it again and will have to re-heat the loop, always keep in your hands when in use.
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| Why is aseptic technique important? | It minimizes the transfer of bacteria and contaminants from one thing to another.
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