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151 Cards in this Set

  • Front
  • Back
How are human pathogens categorized taonomically?
Kingdom, Phylum, Class, Order, Family, Tribe, Genus, Species (Kings play chess on fat, tiny guy's stomach's)
What is the proper way to write a MO's name?
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.
When should alcohol gel not be used to clean your hands?
When hands are visibly soiled
When dealing with spore forming agents
What are some advantages to using alcohol gel?
-faster
-more efficient
-reduces bacterial count for an extended period of time
-easier on hands
How should nails be kept?
short and clean, polish is ok but fake nailsare not
How do microbiologists define sterile
an environment completely free of all living organisms including vegetative bacteria, bacterial spores, fungi, and viruses
Which bacteria turn puple in a gram stain?
Gram positive
Which bacteria turn pink in a gram stain?
gram negative
Describe the proper microscope settings for viewing at low magnification
-voltage @ 4-6
- condenser in up position
- iris diaphragm closed down by 1/2
Describe the proper microscope settings for viewing at 1000x magnification
-voltage @ 8-10
-conderser in up position
-iris diaphragm wide open
Describe the gram staining process
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
How do Bacillus spp. gram stain?
gram positive rods (often appear gram -)
How do Staphylococcus spp. stain?
gram positive clusters of cocci
How does E. coli stain?
gram negative short rods
How do Listeria spp. stain
gram + rods
What should you do before storing your microscope
-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
What is the difference between Bacillus and bacillus
Bacillus (capital B) is a species, bacillus is a morphology
Discuss how crystal violet interacts with bacteria during a gram stain
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_
Discuss the role of iodine in the gram stain
The iodine acts as a mordant by complexing with the CV. This keeps the stain in the cell
Describe how the decolorizing step of the gram stain interacts differently with gram + and gram - organisms
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
Which has more peptidoglycan gram + or -
Gram positives has tons more peptidoglycan!
What is unique about the gram - bacteria membrane
There is an inner membrane and then a periplasm. Next there is an outer LPS membrane
What is the purpose of the safranin stain in the gram stain process
It stains the gram negative bacteria so they can be seen. (They become colorless during the decolorization step)
what is the magnifcation of the ocular lens of the microcope? How does this effect total magnification
The ocular lens has 10x magnification. To get total magnification you need to multiply the magnification of the objective by 10.
What is the purpose of the condensor
focuses and shapes the light entering the objective, should be in the up position for stained material
What is the purpose of the iris/ diaphragm
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
What can cause loss of cell wall integrity and therefore cause a gram + cell to appear gram -?
-antimicrobial exposure
-age of organism grown in culture (especially gram + bacilli)
-exposure to oxygen (obligate anaerobes)
What adjustments should you make when using another person's already focused microscope?
-fine focus
- interpupillary distance
- diopter if necessary
define pure culture
a population of cells all derived from a single parent cell
Define Asepsis and Aseptic technique
Asepsis- state of being free of microorganisms
Aseptic technique- procedures characterized by precautions for the exclusion of bacteria
Why is aseptic technique necessary?
-maintain pure cultures
-safety of personnel
Define aerosol
microorganisms suspended in air
What are the three goals of primary isolation from clinical specimens
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
What are the two ways to obtain pure cultures?
1. streak plate
2. spread plate
Describe a streak plate
-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
describe a spread plate (aka pour plate)
-used to quantitate the number of bacteria in an aqeous sample
-known quantity or dilution is spread over the entire surface of the agar
Why should you not mix or spread cultures vigorously?
it can create aerosols and destroy charateristic arrangemetns of cells such as chains or clusters
Describe how to prep a smear
-place small drop of water on slide away fro medge
-transfer tiny bit of MO with sterile wire (not loop)
-spread in even film
What are two common mistakes during streak plating
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
What is the fundamental difference between a streak plate and a spread plate?
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
what are two common sources of error during spread plate serial dilutions
1. pipetting errors.
2. failing to mix
How can sterilization procedures be monitored for performance
autoclave tape, spores
What are some important things to consider when using a disinfectant (in terms of efficacy)?
-dilution and concentration
-shelf life
-contact time
-water pH, temperature, physio-chemical concerns
-microbial load
-hazards associated with the chemical
how should a wire loop be introduced into a flame to sterilize
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
T/F disinfectants and antiseptics create a sterile environment
False, They do not necesarily create a sterile environment. The destroy or inactivate most of the organisms present
define disinfectant
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
define antiseptic
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.
Describe boiling as a disinfection method
requires 20 minutes at 100 degrees, consider pressure, temp, and humidity
Describe Non Ionizing radiation as a disinfection method
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
Describe pasteurization as a disinfection method
used to kill food pathogens especially in liquids, recommended 72C for 15 seconds
Describe filtration as a disinfection method
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
What MOs can filtration with a 0.22um pore not remove
viruses and small bacteria like mycoplasma
How can labs monitor the performance of autoclaves and drying ovens
expose bacterial endospores to the sterilization method, run through the sterilization cycle then incubate in nutrient medium to check for viability
What can an autoclave with steam and pressure be used to sterilize
heat stable media/supplies/ instruments
What are the usual conditions for autoclaving with steam and pressure
121C at 15 lbs/sq in, for 15-60 min depending on load size and makeup
What are some limitations to autoclaving
cannot use on heat labile materials (sugars, plastics, proteins etc), oil based materials will not be well penetrated
What can dry heat be used to sterilize
glassware, oils, instruments, powders
what conditions is dry heat sterilization carried out under
oven at 160-200 C for 1-4 hours depending on load size and make up
What are some drawbacks to dry heat sterilization
takes longer to kill spores than moist heat
What can gas vapor (ethylene oxide, plasma gas, formaldehye,peroxide, chlorine oxide) be used to sterilize
surgical supplies, plastics, instruments, bandages
How is gas sterilization carried out
Special sterilizer, 700 mg of Ethylene oxide/liter, chamber space at 60C for 2 hr
What are some drawbacks to gas sterilization
residual gas on materials can irritate skin, they are toxic, flammable, and expensive
What is incineration used to sterilize
infectious waste
How is incineration carried out
special facility, 870-980C
What are some drawback to incineration
emissions heavy metals, ash
what is ionizing/ gamma radiation used to sterilize
plastics, food
What are some drawbacks to ionizing radiation as a sterilization technique
facility expense and public acceptance
What is UV light/ non ionizing radiation used to sterilize
inanimate surfaces
What are some drawbacks to using UV light to sterilize
poor penetration
What 5 points should be considered when evaluating a potential pathogen
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)
Describe risk group 1 pathogens
not associated with disease in healthy adult humans
Describe risk group 2 pathogens
associated with human disease that is rarely serious and for which preventative or therapeutic interventions are often available
Describe risk group 3 pathogens
associated with serious or lethal human disease for which preventative or therapeutic interventions may be available (high individual but low community risk)
Describe risk group 4 pathogens
likely to cause serious or lethal human disease for which preventative or therapeutic interventions are not usually available (high individual and community risk)
What are the four most common routes of infection
1. inhalation
2. exposure of mucous membranes
3. ingestion
4. percutaneous self inoculation
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
what are the drawbacks to aldehydes even though they have broad germicidal activity
they are toxic to humans
how should unstained wet mounts or mycology slides be viewed?
minimize the amount of light by lowering the condenser and partially closing the diaphragm
describe dark field microscopy
-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
describe phase contrast microscopy
-used to observe living MOs
-light refracted by living cells differently, easily seen
-used to view tissue culture cell monolayers and mycology slides
describe fluorescence microscopy
-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
T/F streak plates are quantitative
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
T/F Serial dilution/ spread paltes are quantitative
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
How can growth be semiquantitativly described on a streak plate
Growth can be describe as +1 (few) to +4 (heavy) depending on the number of quarters that show growth
Describe E. coli (gram - short rod) colony morphology on BAP
Grey, irregular, umbonate
Describe Staphylococcus epidermidis (gram positive cocci clusters) colony morphology on BAP
white, small, slimy, round
Describe Micrococcus sp.(gram positive cocci in tetrads) colony morphology on BAP
small, yellow, flat, round
how are CFU/ml calculated
# colonies X reciprocal of dilutionX 1/volume plated
calculate the number of CFU/ml if 45 colonies were counted on the 10^4 plate and 0.5 ml was plated
45 X 10^4 X 1/0.5= 9.0 x 10^5 CFU/ml
How many colonies should be on the plate used for a spread plate count
30-300
How would a gram stain appear if you didn't heat fix it?
the bacteria would wash off and you wouldn't see anything
How would a gram stain appear if you omitted the decolorizing step
All of the cells would appear purple
Why is it important to streak plates away from dust and air drafts
fungal contamination and other bacteria
What are 3 reasons why we need pure cultures
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
Why do we need to do semi-quantitation of growth
-magnitude of infection
-pathogen vs. indigenous flora
-figure out which mo predominates
what is the difference between a differential and a seletive media
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
What information can a direct exam provide?
-information on the host response or inflammatory celluar components
-cellular morphology
-single or multiple MOs
-predominate organism
-gram stain rxn
-size, shape arrangement
Why are direct exmas fixed in methanol and not heat fixed
heat fixing destroys the morphology of the host cells
T/F a direct exam can reveal the genus and species of a specimen
false, the specimen must be cultured on appropriate media and then subjected to biochemical/ enzyme protocols before an identification can be made
What are clues that a sputum sample is really just saliva
-high number of epithelial cells
- a mix of many different types of bacteria (oral flora)
What constitutes a good sputum sample
-low number of epithelial cells
-1 or very few types of bacteria (e.g. gram + diplococci lancet shaped streptococcus pneumonia)
What would a normal (non infected) vaginal smear look like
-epithelial cells
-gram + lactobacilli, non adherent
What are clue cells
adherent MOs on epithelial cells, multiple species, be careful not confuse with stain particles
What might be a cause of overgrowth of bacteria without inflmamatory cells?
-improper prep that was heat fixed or an old sample
-fresh wound
-immune deficient
What might be the cause of WBCs without bacteria?
-cleared infection
-peripheral sample
what is the proper procedure for collecting a sputum sample
first cleanse the oral cavity, brush teeth and risne to avoid oral epithelial cells, saliva, and oral flora
What must be done before taking a sample from a wound
the skin must be cleansed so that skin flora does not contaminate the sample
What are three different ways to collect a specimin from a wound site
-swab (but cotton can trap MOs)
-aspirate
-biopsy
What macroscopic observations should be made from sputum
-color
-consistency
-mucous/ glycoprotein
-blood tinged?
What are the cut off numbers for cells in a sputum sample
very few epithelial cells
>25 WBCs for 100x mag
T/F epithelial cells are usally present in a wound sample
false
What speimen concentration do you need to see 1 mo per field @1000x
10^5 CFU/ml
Describe the indole test
-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
describe how kovac's reagent works
The amyl alcohol extracts the indole, the aldehyde complexes with it and forms the red ring
Describe the phenylalanine deaminase test
-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
describe the lactose fermentation test
-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
Describe the catalase test
-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
What can give a false positive in the catalse test?
-picking up RBC
-using a loop with nickel in it
Why does having catalse benefit bacteria
-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
T/F catalase test should be preformed on gram negative bacteria
false, it is only done on gram +
T/F the indole test should be done on gram + bacteria
false, it should only be done on gram negatives
T/F the phenylalanine deaminase test should be done on gram + bacteria
False, the phenylalanine test should only be done on gram - bacteria
T/F the lactose fermentation test should be done on gram + bacteria
false lactose fermentation should only be done on gram - bacteria
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 +
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
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
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
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
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
What are the disadvantages of a dichotomous key
-must interpret the tests as either positive or negative
-cannot account for strain differences
-single error can result in misdiagnosis
What are the advantages of using a profile matrix
-can compare many tests at once
-accomodates strain variants
What are the disadvantages of the profile matrixq
-as more tests are added it gets difficult to keep everything straight
-many do more tests than needed
What are the three main reasons for preforming suceptibility testing
-physicians need to know what the patient's infection is resistant to
-suceptibility patterns can help with identification
-set up appropriate treatment
What are three common techniques used to test antibiotic suceptibility
1.disk diffusion
2. broth/ agar dilution
3. E test (gradient diffusion method)
Describe the disk diffusion method for suceptibility testing
A paper disk is infused with an antimicrobial substance and zones of clearance are mesured
What are the advantages to the Kirby Bauer suceptibility testing method
cheap, simple, reproducible
What are the three categories that are used to describe zones of clearance in the kirby bauer test
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
Describe the broth dilution technique for suceptibility testing
-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
What is the diffrence between MIC and MBC
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)
What are the three ways broth dilutions to determine MIC can be carried out
1. macrodilution in test tubes
2. microdilution in plates
3. agar dilution
Describe the E test
strip with range of concentrations of antimicrobial is placed on lawn, bacteria growth in an elliptical shape, MIC is where elipse intersects strip
What are the 5 components of suceptibility testing that must be standardized
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)
T/F the disk diffusion test is quantitative
False, the test is not truly quantitative, it cannot be directly translated into ug/ml
Describe how to read a microdiution suceptibility test
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)
Describe the chromogenic beta-lactamase test
Tests for beta lactamase activity (penicillin resistance), substrate is hydrolyzed to produce pink color
Which antimicrobials were effective against both gram positive and gram negative
cephalothin, gentamicin
Which bacteria that we studied was resistant to many antimicrobials
Pseudomonas aeruginosa
What could cause hazy edges in a KB disk diffusion test
-multiple organisms
-could have resistant colonies
Why should a culture being used for suceptibility testing be less than a day old?
the culture needs to be in the log phase of growth
Which suceptibiltiy tests give quantitative results? qualitative?
quantitative- dilutions and E test
qualitative- disk diffusion
how can an infection with a beta lactamase producing bacteria be treated
give a combo of a beta lactam drug and a beta lactamase inhibitor