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151 Cards in this Set
- Front
- Back
How are human pathogens categorized taonomically?
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Kingdom, Phylum, Class, Order, Family, Tribe, Genus, Species (Kings play chess on fat, tiny guy's stomach's)
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What is the proper way to write a MO's name?
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The genus should be capitalized and the species should be lower case, If typing you hsould italicize the name, if writing, you should underline it.
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When should alcohol gel not be used to clean your hands?
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When hands are visibly soiled
When dealing with spore forming agents |
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What are some advantages to using alcohol gel?
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-faster
-more efficient -reduces bacterial count for an extended period of time -easier on hands |
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How should nails be kept?
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short and clean, polish is ok but fake nailsare not
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How do microbiologists define sterile
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an environment completely free of all living organisms including vegetative bacteria, bacterial spores, fungi, and viruses
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Which bacteria turn puple in a gram stain?
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Gram positive
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Which bacteria turn pink in a gram stain?
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gram negative
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Describe the proper microscope settings for viewing at low magnification
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-voltage @ 4-6
- condenser in up position - iris diaphragm closed down by 1/2 |
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Describe the proper microscope settings for viewing at 1000x magnification
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-voltage @ 8-10
-conderser in up position -iris diaphragm wide open |
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Describe the gram staining process
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0. Let slide dry then Heat or methanol fix
1. Add crystal violet for 1 min then rinse with tap water 2. Add iodine for 1 min then risnse with tap water 3. Flow a few drops of acetone-alcohol decolorizer then imediately rinse in tap water 4. Counter stain with safranin for 1 min 5. Blot with a paper towel |
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How do Bacillus spp. gram stain?
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gram positive rods (often appear gram -)
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How do Staphylococcus spp. stain?
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gram positive clusters of cocci
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How does E. coli stain?
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gram negative short rods
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How do Listeria spp. stain
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gram + rods
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What should you do before storing your microscope
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-remove the slide and clean the 100x objective by BLOTING with sparkle
-adjust the voltage control to 1 -turn off the power and unplug, wrap the cord - cover |
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What is the difference between Bacillus and bacillus
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Bacillus (capital B) is a species, bacillus is a morphology
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Discuss how crystal violet interacts with bacteria during a gram stain
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The positively charged CV ion interacts with the negativley charged components of the bacterial cell wall and stains the cell purple (both gram + and Gram - stain purple in this initial step_
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Discuss the role of iodine in the gram stain
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The iodine acts as a mordant by complexing with the CV. This keeps the stain in the cell
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Describe how the decolorizing step of the gram stain interacts differently with gram + and gram - organisms
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A gram-negative cell will lose its outer lipopolysaccharide membrane (denature) and the inner peptidoglycan layer is left exposed.Porosity is increased and the CV–I complexes are washed from the gram-negative cell along with the outer membrane
A gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. Porosity decreases and the CV stain is retained |
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Which has more peptidoglycan gram + or -
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Gram positives has tons more peptidoglycan!
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What is unique about the gram - bacteria membrane
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There is an inner membrane and then a periplasm. Next there is an outer LPS membrane
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What is the purpose of the safranin stain in the gram stain process
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It stains the gram negative bacteria so they can be seen. (They become colorless during the decolorization step)
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what is the magnifcation of the ocular lens of the microcope? How does this effect total magnification
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The ocular lens has 10x magnification. To get total magnification you need to multiply the magnification of the objective by 10.
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What is the purpose of the condensor
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focuses and shapes the light entering the objective, should be in the up position for stained material
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What is the purpose of the iris/ diaphragm
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controls stray light, used to increase image definition and contrast, should be partially closed for low mag but open for 100x objective lens (1000x) magnification
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What can cause loss of cell wall integrity and therefore cause a gram + cell to appear gram -?
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-antimicrobial exposure
-age of organism grown in culture (especially gram + bacilli) -exposure to oxygen (obligate anaerobes) |
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What adjustments should you make when using another person's already focused microscope?
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-fine focus
- interpupillary distance - diopter if necessary |
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define pure culture
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a population of cells all derived from a single parent cell
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Define Asepsis and Aseptic technique
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Asepsis- state of being free of microorganisms
Aseptic technique- procedures characterized by precautions for the exclusion of bacteria |
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Why is aseptic technique necessary?
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-maintain pure cultures
-safety of personnel |
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Define aerosol
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microorganisms suspended in air
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What are the three goals of primary isolation from clinical specimens
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1. Culture all bacterial species present and to see if any predominate
2. Differntiate species by characteristic responses to ingreidiants in the medium 3. Selectivley encourage growth of species of interest while supressing indigenous flora |
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What are the two ways to obtain pure cultures?
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1. streak plate
2. spread plate |
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Describe a streak plate
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-mixture of bacteria is spread over the surface of a solid nutrient medium
-Allows study of colonial morphology, hemolytic reaction, pigment of colony or medium color change, etc |
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describe a spread plate (aka pour plate)
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-used to quantitate the number of bacteria in an aqeous sample
-known quantity or dilution is spread over the entire surface of the agar |
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Why should you not mix or spread cultures vigorously?
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it can create aerosols and destroy charateristic arrangemetns of cells such as chains or clusters
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Describe how to prep a smear
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-place small drop of water on slide away fro medge
-transfer tiny bit of MO with sterile wire (not loop) -spread in even film |
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What are two common mistakes during streak plating
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1. using too much innoculum- results in over crowded plate and no isolated colonies
2. using too little surface area- will not produce isolated colonies |
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What is the fundamental difference between a streak plate and a spread plate?
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A spread plate has a measured amount of sample plated and known dilutions are made, this allows for exact quantitation in CFU/ml rather than the aprox. of +1-+4 of streak plates
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what are two common sources of error during spread plate serial dilutions
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1. pipetting errors.
2. failing to mix |
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How can sterilization procedures be monitored for performance
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autoclave tape, spores
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What are some important things to consider when using a disinfectant (in terms of efficacy)?
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-dilution and concentration
-shelf life -contact time -water pH, temperature, physio-chemical concerns -microbial load -hazards associated with the chemical |
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how should a wire loop be introduced into a flame to sterilize
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holding the loop at an angle, introduce the loop at the end closet to the handle then slowly draw downwards and towards you to the tip of the loop
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T/F disinfectants and antiseptics create a sterile environment
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False, They do not necesarily create a sterile environment. The destroy or inactivate most of the organisms present
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define disinfectant
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disinfectants destroy microorganisms on inert surfaces. They can be either bacteriostatic or bacteriocidal. They are influenced by several factors including concentration, pH, and contact time
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define antiseptic
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Antiseptics destroy or inhibit the growth of MOs when applied to living tissue. They must have a high toxicity for MOs but a low toxicity for human cells.
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Describe boiling as a disinfection method
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requires 20 minutes at 100 degrees, consider pressure, temp, and humidity
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Describe Non Ionizing radiation as a disinfection method
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UV light at 260 nm damages nucleic acids, can be used on surfaces, or as air cleaner in sterile hoods
Gamma (ionizing) radiation can be used for plastics and food items |
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Describe pasteurization as a disinfection method
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used to kill food pathogens especially in liquids, recommended 72C for 15 seconds
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Describe filtration as a disinfection method
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used to disinfect gases, liquids and other heat labile fluids, 0.22um pore size filter removes spores and most bacteria but not viruses can small bacteria like mycoplasma
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What MOs can filtration with a 0.22um pore not remove
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viruses and small bacteria like mycoplasma
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How can labs monitor the performance of autoclaves and drying ovens
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expose bacterial endospores to the sterilization method, run through the sterilization cycle then incubate in nutrient medium to check for viability
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What can an autoclave with steam and pressure be used to sterilize
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heat stable media/supplies/ instruments
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What are the usual conditions for autoclaving with steam and pressure
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121C at 15 lbs/sq in, for 15-60 min depending on load size and makeup
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What are some limitations to autoclaving
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cannot use on heat labile materials (sugars, plastics, proteins etc), oil based materials will not be well penetrated
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What can dry heat be used to sterilize
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glassware, oils, instruments, powders
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what conditions is dry heat sterilization carried out under
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oven at 160-200 C for 1-4 hours depending on load size and make up
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What are some drawbacks to dry heat sterilization
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takes longer to kill spores than moist heat
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What can gas vapor (ethylene oxide, plasma gas, formaldehye,peroxide, chlorine oxide) be used to sterilize
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surgical supplies, plastics, instruments, bandages
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How is gas sterilization carried out
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Special sterilizer, 700 mg of Ethylene oxide/liter, chamber space at 60C for 2 hr
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What are some drawbacks to gas sterilization
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residual gas on materials can irritate skin, they are toxic, flammable, and expensive
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What is incineration used to sterilize
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infectious waste
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How is incineration carried out
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special facility, 870-980C
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What are some drawback to incineration
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emissions heavy metals, ash
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what is ionizing/ gamma radiation used to sterilize
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plastics, food
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What are some drawbacks to ionizing radiation as a sterilization technique
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facility expense and public acceptance
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What is UV light/ non ionizing radiation used to sterilize
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inanimate surfaces
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What are some drawbacks to using UV light to sterilize
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poor penetration
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What 5 points should be considered when evaluating a potential pathogen
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1. agent's biological and physical nature
2. sources that are likely to harbor the agent 3. host susceptibility 4. procedures that may disseminate the agent 5. best method to effectively inactivate the agent (Remember No Home Stays Dry Indefinitely, Nature, Harbor, Suceptibility, Dissemination, Inactivate) |
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Describe risk group 1 pathogens
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not associated with disease in healthy adult humans
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Describe risk group 2 pathogens
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associated with human disease that is rarely serious and for which preventative or therapeutic interventions are often available
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Describe risk group 3 pathogens
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associated with serious or lethal human disease for which preventative or therapeutic interventions may be available (high individual but low community risk)
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Describe risk group 4 pathogens
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likely to cause serious or lethal human disease for which preventative or therapeutic interventions are not usually available (high individual and community risk)
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What are the four most common routes of infection
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1. inhalation
2. exposure of mucous membranes 3. ingestion 4. percutaneous self inoculation |
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What is 70-90% alcohol effective against?
What is it not |
vegetative forms of bacteria
not effective against spores or hydrophilic viruses cannot be used if hands are visibly soiled |
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what are the drawbacks to aldehydes even though they have broad germicidal activity
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they are toxic to humans
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how should unstained wet mounts or mycology slides be viewed?
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minimize the amount of light by lowering the condenser and partially closing the diaphragm
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describe dark field microscopy
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-light is directed toward the specimen from the side
-Only light to reach objective is relfected from the subject -specimine structure shows up light against a dark background -used to study very small bacteria |
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describe phase contrast microscopy
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-used to observe living MOs
-light refracted by living cells differently, easily seen -used to view tissue culture cell monolayers and mycology slides |
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describe fluorescence microscopy
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-UV light illuminates object but does not pass into objective
-The light hits dyes and causes them to emit light against a dark background used to show antibodies on bacterial antigens and as a serological diagnostic test |
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T/F streak plates are quantitative
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Sort of true. Streak plates are semi-quantitative they provide a rough estimate of the number of bacteria present in sample by noting the number and position of colonies deposited in each quarter
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T/F Serial dilution/ spread paltes are quantitative
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Ture. The concentration can be accurately be determined by diluting the sample and then counting a plate. The number of organisms is reported as CFU/ml
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How can growth be semiquantitativly described on a streak plate
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Growth can be describe as +1 (few) to +4 (heavy) depending on the number of quarters that show growth
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Describe E. coli (gram - short rod) colony morphology on BAP
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Grey, irregular, umbonate
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Describe Staphylococcus epidermidis (gram positive cocci clusters) colony morphology on BAP
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white, small, slimy, round
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Describe Micrococcus sp.(gram positive cocci in tetrads) colony morphology on BAP
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small, yellow, flat, round
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how are CFU/ml calculated
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# colonies X reciprocal of dilutionX 1/volume plated
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calculate the number of CFU/ml if 45 colonies were counted on the 10^4 plate and 0.5 ml was plated
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45 X 10^4 X 1/0.5= 9.0 x 10^5 CFU/ml
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How many colonies should be on the plate used for a spread plate count
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30-300
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How would a gram stain appear if you didn't heat fix it?
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the bacteria would wash off and you wouldn't see anything
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How would a gram stain appear if you omitted the decolorizing step
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All of the cells would appear purple
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Why is it important to streak plates away from dust and air drafts
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fungal contamination and other bacteria
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What are 3 reasons why we need pure cultures
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1. need isolated colony morphology and gram stain cellular morphology
2. for transfers to make stains and rapid tests 3. to help determine next steps/tests/ media needed for identification |
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Why do we need to do semi-quantitation of growth
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-magnitude of infection
-pathogen vs. indigenous flora -figure out which mo predominates |
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what is the difference between a differential and a seletive media
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differential- a compound in the media allows you to tell the difference between different types of bacteria
selective- has an inhibitory compound against a certain type of bacteria |
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What information can a direct exam provide?
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-information on the host response or inflammatory celluar components
-cellular morphology -single or multiple MOs -predominate organism -gram stain rxn -size, shape arrangement |
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Why are direct exmas fixed in methanol and not heat fixed
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heat fixing destroys the morphology of the host cells
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T/F a direct exam can reveal the genus and species of a specimen
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false, the specimen must be cultured on appropriate media and then subjected to biochemical/ enzyme protocols before an identification can be made
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What are clues that a sputum sample is really just saliva
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-high number of epithelial cells
- a mix of many different types of bacteria (oral flora) |
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What constitutes a good sputum sample
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-low number of epithelial cells
-1 or very few types of bacteria (e.g. gram + diplococci lancet shaped streptococcus pneumonia) |
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What would a normal (non infected) vaginal smear look like
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-epithelial cells
-gram + lactobacilli, non adherent |
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What are clue cells
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adherent MOs on epithelial cells, multiple species, be careful not confuse with stain particles
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What might be a cause of overgrowth of bacteria without inflmamatory cells?
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-improper prep that was heat fixed or an old sample
-fresh wound -immune deficient |
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What might be the cause of WBCs without bacteria?
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-cleared infection
-peripheral sample |
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what is the proper procedure for collecting a sputum sample
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first cleanse the oral cavity, brush teeth and risne to avoid oral epithelial cells, saliva, and oral flora
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What must be done before taking a sample from a wound
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the skin must be cleansed so that skin flora does not contaminate the sample
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What are three different ways to collect a specimin from a wound site
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-swab (but cotton can trap MOs)
-aspirate -biopsy |
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What macroscopic observations should be made from sputum
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-color
-consistency -mucous/ glycoprotein -blood tinged? |
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What are the cut off numbers for cells in a sputum sample
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very few epithelial cells
>25 WBCs for 100x mag |
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T/F epithelial cells are usally present in a wound sample
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false
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What speimen concentration do you need to see 1 mo per field @1000x
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10^5 CFU/ml
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Describe the indole test
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-the bacterial enzyme tryptophanase degrades trytophan into indole
-Kovac's reagent forms a complex with the indole and a red ring forms -this test tests for the presence of the tryptophanase enzyme |
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describe how kovac's reagent works
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The amyl alcohol extracts the indole, the aldehyde complexes with it and forms the red ring
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Describe the phenylalanine deaminase test
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-tests for the prescence of the deaminase enzyme
-addition of 10% FeCl3 will form a green color if phenylpyruvic acid (the by product of phenyalanine degradation) is present |
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describe the lactose fermentation test
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-medium turns from purple to yellow if lactose is fermented to acid because of bromocresol purple pH indicator
-gas production can be tested with durham tube |
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Describe the catalase test
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-tests to see if the bacteria has catalase
-use wooden stick to transfer small amount of isolated colony into H2O2 and look for bubbles -make sure to use positive and negative controls |
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What can give a false positive in the catalse test?
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-picking up RBC
-using a loop with nickel in it |
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Why does having catalse benefit bacteria
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-aerobic and facultative bacteria have catalase to protect themselves from the oxidative end products of aerobic metabolism
-Can be used to protect themselves from the H2O2 in PMNs |
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T/F catalase test should be preformed on gram negative bacteria
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false, it is only done on gram +
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T/F the indole test should be done on gram + bacteria
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false, it should only be done on gram negatives
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T/F the phenylalanine deaminase test should be done on gram + bacteria
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False, the phenylalanine test should only be done on gram - bacteria
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T/F the lactose fermentation test should be done on gram + bacteria
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false lactose fermentation should only be done on gram - bacteria
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Describe the results of the following tests for B. subtilis
gram stain Colony on BAP indole Phenylalanine Lactose fermentation Catalase |
gram stain= + rods
Colony on BAP= grey, puffy, rough, dull, fuzzy indole= N/A Phenylalanine N/A Lactose fermentation N/A Catalase + |
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Describe the results of the following tests for E. faecalis
gram stain Colony on BAP indole Phenylalanine Lactose fermentation Catalase |
gram stain + chains of cocci
Colony on BAP- darker creamy white, flat and round indole- N/A Phenylalanine- N/A Lactose fermentation- N/A Catalase negative |
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Describe the results of the following tests for S. aureus
gram stain Colony on BAP indole Phenylalanine Lactose fermentation Catalase |
gram stain + grape like cocci clusters
Colony on BAP-creamy white, round, slightly convex indole- N/A Phenylalanine- N/A Lactose fermentation N/A Catalase postive |
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Describe the results of the following tests for P. aeruginosa
gram stain Colony on BAP indole Phenylalanine Lactose fermentation Catalase |
gram stain- negative skinny rods in clusters
Colony on BAP-large, dark grey, rough, slimy, smelly indole- negative Phenylalanine-negative Lactose fermentation-negative Catalase n/a |
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Describe the results of the following tests for E. coli
gram stain Colony on BAP indole Phenylalanine Lactose fermentation Catalase |
gram stain- negative short rods
Colony on BAP- yellowy grey umbonate indole- positive Phenylalanine- negative Lactose fermentation- positive with gas Catalase n/a |
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Describe the results of the following tests for P. mirabilis
gram stain Colony on BAP indole Phenylalanine Lactose fermentation Catalase |
gram stain-negative tiny rods in clusters
Colony on BAP-off white wave (swarmer) indole-negative Phenylalanine-positive Lactose fermentation-negative Catalase N/A |
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What are the disadvantages of a dichotomous key
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-must interpret the tests as either positive or negative
-cannot account for strain differences -single error can result in misdiagnosis |
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What are the advantages of using a profile matrix
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-can compare many tests at once
-accomodates strain variants |
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What are the disadvantages of the profile matrixq
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-as more tests are added it gets difficult to keep everything straight
-many do more tests than needed |
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What are the three main reasons for preforming suceptibility testing
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-physicians need to know what the patient's infection is resistant to
-suceptibility patterns can help with identification -set up appropriate treatment |
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What are three common techniques used to test antibiotic suceptibility
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1.disk diffusion
2. broth/ agar dilution 3. E test (gradient diffusion method) |
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Describe the disk diffusion method for suceptibility testing
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A paper disk is infused with an antimicrobial substance and zones of clearance are mesured
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What are the advantages to the Kirby Bauer suceptibility testing method
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cheap, simple, reproducible
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What are the three categories that are used to describe zones of clearance in the kirby bauer test
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1. susceptible-the organisms will be suceptible at normal therapeutic doses
2. resistant-the organism will be resistant at therapeutic doses 3. intermediate- induvidual differences between patients and bacterial strains, drug combinations |
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Describe the broth dilution technique for suceptibility testing
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-determines the MIC
-dilutions of antimicrobials are incorporated in broth or agar and innoculated with standard amount of test organism -The first tube showing inhibition of growth is the MIC |
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What is the diffrence between MIC and MBC
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MIC is determined by finding the tube with no visible growth. This tube can be subcultured to determine wether the organism was inihbited or killed (minimum bactericidal concentration)
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What are the three ways broth dilutions to determine MIC can be carried out
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1. macrodilution in test tubes
2. microdilution in plates 3. agar dilution |
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Describe the E test
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strip with range of concentrations of antimicrobial is placed on lawn, bacteria growth in an elliptical shape, MIC is where elipse intersects strip
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What are the 5 components of suceptibility testing that must be standardized
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1. bacterial inoculum- 10^8 CFU/ml or 0.5 Mcfarland standard
2. Growth medium- Muller Hinton agar, 4mm 3.concentration of antimicrobials 4. incubation environment- 35C 24 hrs, CO2 not used 5. Quality control using specific strains of organisms hint (I'm CEQ, Innoculum, Media, Concentration, Environment, Quality control) |
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T/F the disk diffusion test is quantitative
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False, the test is not truly quantitative, it cannot be directly translated into ug/ml
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Describe how to read a microdiution suceptibility test
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pink wells= alieve
blue wells= dead MIC is recorded as the lowest concentration of antimicrobial that inhibits growth (the lowest blue well) -make sure to have controls with no drugs) |
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Describe the chromogenic beta-lactamase test
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Tests for beta lactamase activity (penicillin resistance), substrate is hydrolyzed to produce pink color
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Which antimicrobials were effective against both gram positive and gram negative
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cephalothin, gentamicin
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Which bacteria that we studied was resistant to many antimicrobials
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Pseudomonas aeruginosa
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What could cause hazy edges in a KB disk diffusion test
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-multiple organisms
-could have resistant colonies |
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Why should a culture being used for suceptibility testing be less than a day old?
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the culture needs to be in the log phase of growth
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Which suceptibiltiy tests give quantitative results? qualitative?
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quantitative- dilutions and E test
qualitative- disk diffusion |
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how can an infection with a beta lactamase producing bacteria be treated
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give a combo of a beta lactam drug and a beta lactamase inhibitor
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