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204 Cards in this Set
- Front
- Back
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What is the most researched organism concening malaria?
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plasmodium malaria
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Spell out the "e" in E. coli.
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Escherichia coli
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which has cilia euglena or paramecium?
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paramecium
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What is the form of Bacillus that we we wiill be working with and where is it found?
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B. subtilis; found in soil
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How does B. sutilis look under the microscope after gram stain?
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gram positive
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What organism is found on the skin and mucous membranes of humans and organisms?
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Staphylococcus
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is "staph" gram + or -
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Gram positive
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is Bacillus gram positive or negative?
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Gram negative
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is E. coli gram positive or negative?
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Gram negative
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Where is E. coli found in the human body?
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lower intestine (poop)
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what is the pathogenic strain of E. coli?
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serotype O157:H7
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what is a serotype?
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The kind of microorganism as characterized by serologic typing (testing for recognizable antigens on the surface of the microorganism).
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Which organism looks like paintbrushes?
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Penicillium
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Which organism looks like poof balls?
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Aspergillus
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What is Peziza?
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cup fungus; ascomycetes
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What disease is caused by trypanosoma?
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sleeping sickness
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In Peziza, what are the pink ("bristles")?
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spores
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In Peziza, what is the blue space below the bristles?
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hyphae
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hyphae
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long, branching filamentous cell
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What are 3 examples of typical molds?
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Rhizopus, Aspergillus and Penicillium
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characterize trypansoma
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obligate parasites with 2 hosts (insect vector and mammalian host)
hemoflagellate |
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hemoflagellate
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A flagellate protozoan, such as a trypanosome, that is parasitic in the blood.
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What preparations do you never heat fix?!
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negative staining
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What are 2 reasons that we "heat-fix"?
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kill the bacteria; fix organisms to slide so no shape change like shrinkage of the membrane
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What is the charge of the bacterial cell membrane?
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negative
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basic dyes
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positively charged dyes that will stain the membrane
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examples of basic dyes
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methylene blue, basic fuchsin and crystal violet
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acidic dyes
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negatively charged dyes that will not stain the membrane used for negative staining
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examples of acidic dyes
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india ink and nigrosin
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why would you use negative staining?
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no heat fixation so no shrinkage of cell capsule
see spirochetes that don't show with simple staining |
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simple staining
differential staining |
one stain
multiple stains |
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What are 2 bacteria that we will definitely be using in class?
_______ coli and Staphylococcus____ |
Escherichia
aureus |
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Who came up with the Gram-Stain?
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Sir Christian Gram
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differential stain why?
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1. identify unknown
2. morphology **use 2 different dyes |
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gram + are what color?
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purple
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gram - are what color?
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pink to red
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what part of the cell is actually aquiring the Gram-Stain?
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the peptidoglycan layer
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list the reagents used (in the correct order) for the Gram-Stain procedure
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-crystal violet (primary stain)
-Gram's iodine (mordant that complexes with CV) -95% alcohol (decolorizer)-removes complex from Gram - -Safranin (counterstain) |
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What is the 40x objective lens called?
the 100x called? |
high-dry
oil-immersion |
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list the steps of the Gram-stain?
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1. smear and heat fix
2. CV (30 sec) 3. DD H2O 4. Gram's iodine (1 min+) 5. DD H2O 6. 95% alcohol (really quick rinse) 7. DD H2O 8. Safranin (30sec) 9. DD H2O 10. Blot with bibulous paper |
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is Escherichia coli gram pos or neg?
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negative (rod-shaped)
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is Staphylococcus Aureus gram pos or neg?
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positive (coccus shaped)
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mordant
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fixes/sets dyes to bacterium
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What are 2 examples of bacterium that form endospores?
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Gram +; Bacillus and Clostridium
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what are some properties of an endospore?
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heat, radiation, acid/disinfectant resistant
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what is the purpose of an endospore?
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ensure survival through environmental stress
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Define endospore.
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a resting stage (not metabolically active) because the organism has exhausted all of its essential nutrients
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what complex forms to control the water content of the endospore?
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dipicolonic acid and calcium
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Are endospores produced internally or externally?
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internally
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do gram negative bacteria form endospores?
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no
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What are the layers of an endospore?
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exosporium
spore coat (sieve to exclude toxic chems) cortex (peptidoglycan) core wall protoplast (core) --> DNA in acid soluble spore proteins to protect DA from UV rad and heat + normal cell structure such as ribosomes and enzymes |
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autoclave
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b/c endospores can't be killed with normal disinfectant,
Warmup 15min Sterilization 15min 15psi 121C Cool down 15min |
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spheroplast
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cell with C.W. partially removed (typically Gram - w/penicillin b/c little peptidoglycan)
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protoplast
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cell with C.W. completely removed (typically Gram + w/penicillin)
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What are the 2 methods to stain endospores?
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1. Schaeffer-Fulton (green endospore + pink vegetative cells)
2. Dorner (pink endospore, clear vegetative cell, nigrosin negative stained in background) |
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what method did we use in lab to stain the endospores?
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schaeffer-fulton using malachite green and safranin(counterstain)
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what microorganism did we stain for endospores?
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Bacillus subtilis
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what is acid-fast staining?
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Ziehl-Neelson method; stain bacteria w/C.W. w/ a high lipid content that can't be stained w/ normal staining methods
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what lipid must be in the C.W. for acid-fast staining to work?
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mycolic acid; complex lipid with fatty acids and fatty alcohols with hydrocarbon chains up to 80 carbons
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what is meant when called "acid-fast" bacteria?
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have mycolic acid in CW. and stain isn't removed by acid-alcohol
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what is meant by "non-acid-fast"?
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easily discolorized by acid-alcohol because don't have mycolic acid complex
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what color are....bacteria?
acid-fast.. non-acid-fast... |
pink/red
counterstained w/methylene blue (otherwise clear) |
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what is the procedure for "acid-fast" staining?
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1. carbolfuschin (w/phenol)
2. heat (want steam 5 min) 3. H2O rinse 4. acid alcohol rinse 5. H2O rinse 6. methylene blue (counterstain) 7. H2O rinse |
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what does the phenol do when combined w/ carbolfucshin?>
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enables the primary stain to enter the cell wall
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what is the mordant in acid fast staining?
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heat(just like in endospore staining)
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what is one difference when heating in endospore and acidfast staining?
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a papertowel with repeated saturations is used for malachite green in endospore staining
carbolfuchsin goes directly on the slide once |
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what is the makeup of the acid-alcohol in acid-fast staining?
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30% HCL and 70% alcohol
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What 2 bacterium were used in acid fast staining?
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Mycobacterium smegmatis and Staphylococcus aureus
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of the 2 bacteria used during acid fast staining, which is acid fast and which is non acid fast?
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Mycobacterium smegmatis is acid fast (look pink)
Staphylococcus aureus is non acid fast (look blue b/c of counterstain) |
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What is the Lowenstein-Jensen medium
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LJ medium; the only medium to grow Mycobacterium on
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What is the microorganism that causes TB?
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Mycobacterium tuberculosis
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What is the microorganism that causes leprosy?
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Mycobacterium leprae
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what are 2 reasons acid fast staining is differential?
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1. 2 different stains
2. using 2 different organisms (ex: M. smegmatis and S. aureus) |
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What is and where is M. smegmatis?
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nonpathogenic acid fast rod found in soil and external human genitalia; used to study members of Mycobacterium in the lab
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Concerning acid fast staining, what is staphylococcus?
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non acid fast coccus a part of the normal flora of human/ can be a potential pathogen
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How long are flagella?
How thin are flagella? |
10 microns
less than 2 microns (less than the typical microscope resolution) |
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what do flagella help the cell with?
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motility and chemotaxis towards or away from a stimulation
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what are the "rings" of the flagella?
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L ring (in the LPS layer--Gram - only)
P ring (in the Peptidoglycan layer--Gram - only) S Ring M Ring |
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What drives the flagella?
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the proton motive force with protons diffusing through the Mot proteins that are located between the S and M Rings
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How do flagella of prokaryotes differ from flagella of eukaryotes?
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prokaryotes have a rotating motion
eukaryotes have a whiplike motion |
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what are the ways to determine motility?
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Wet mount
Hanging Drop technique Inoculation of Semisolid Agar |
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What is a wet mount?
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place a drop directly on slide and cover with a coverslip
**dries out quickly |
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What is a hanging drop technique?
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place drop on coverslip, then place coverslip on a slide with a concave depression in it and seal with petroleum jelly
**prevents dessication |
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What is the Inoculation of Semisolid Agar?
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The agar is 0.4% so that bacteria are still motile and able to swim through the medium.
If the bacteria are motile, the medium will turn turbid; if not the bacteria will stay by the inoculation point |
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What are the bacteria that we used for motility?
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Proteus vulgaris and Micrococcus luteus
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What are the 2 bacteria that acid-fast staining are crucial?
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Mycobacterium leprae and Mycobacterium tuberculosis
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pure culture
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contains only 1 type of an organism
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contaminated culture
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contains desired organism but also has some unwanted organisms
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streak plate
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different pattern on an agarose gel designed to isolate single colonies
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pour plate
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organisms grow dispersed with in the medium
allows the growth of microaerophiles |
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what bacteria did we use in the mixed media for our pure culture technique?
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Serratia marcescens, Escherichia coli and Micrococcus luteus
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colony
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visible mass of microorganisms growing on a solid medium
thought to be clones of one cell |
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what are the 3 methods to isolate a pure culture
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pour plate
streak plate spread plate |
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culture media
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nutritional substrate w/ substances req'd to grow microbes in the lab
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what are the different types and forms of culture media
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complex
defined selective differential agar SIM broth |
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complex media
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nonsynthetic media
rich extract of meat/plants to supply all A.As nucleotides bases vitamins and other growth factors |
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defined media
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synthetic media
specific nutritional req's or chem composition needed for growth is known ex: what is used for E. coli |
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selective media
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supports the growth of some media and inhibits the growth of others
ex: MSA selects for S. aureus |
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differential and selective media
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both selects and differentiates between bacteria
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differential media
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causes some bacteria to take on a different appearance
ex: Maconkey agar - make gram- look pink |
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what 2 different companies are used in our lab?
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Difco Laboratories (Detroit MI)
Baltimore Biological Laboratories (Cockeysville MD) (BBL) |
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How is using pours plates a practice of dilution?
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as pour plates increased in number, the number of colonies formed decreased.
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What colors were each of the colonies that we isolated with the streak plates?
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Serratia marcescens (red)
Escherichia coli (clear) Micrococcus luteus (yellow) |
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At what temperature did Serratia marcescens produce it's color change?
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25C
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Are Serratia marcescens and Escherichia coli gram + or -?
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gram negative
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lyophilization
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freeze drying to remove H2O from cells so less metabolism and physiologyical mutations
**revitalize by adding sterile H2O and replating |
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what is a reserve culture?
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the culture that you go into as little as possible
**the whole purpose is to maintain the culture |
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what is a working culture?
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the culture that you make inoculations from, get cells to stain/test
***THIS IS THE ONE THAT YOU USE!! |
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When we were making cultures what bacteria did we use?
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Escherichia coli and Bacillus megaterium on tryptone agar slants
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what is the purpose of an agar slant?
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allows bacteria to grow on medium, but if you don't get enough growth on the medium, there will be some in the 'dip liquid'
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What is the level of oxygen in atmospheric gases?
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20%
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What are obligate aerobes?
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need oxygen for respiratory metabolism
ex: Pseudomonas Micrococcus and Bacillus |
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microaerophiles
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need oxygen but at less than in the atmosphere (5-10%)
ex: Helicobacter pylori |
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why may microaerophiles need oxygen at lower levels?
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may have oxygen sensitive proteins and enzymes or limited respiration ability
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Helicobacter pylori
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causes stomach ulcers
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What type of aerobic bacteria are enhanced growth in a candle jar?
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microaerophiles
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facultative aerobes
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grow in anaerobic or aerobic environments depending on the condition
ex: Escherichia coli |
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Aerotolerant anaerobes
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can grow in oxygen but still carry on fermentation
ex: Streptococcus pyogenes |
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Streptococcus pyogenes
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causes strep throat and kidney infections
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obligate anearobes
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can't tolerate oxygen (anoxic)
ex: Clostridium and Bacterioles |
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How do obligate anaerobes metabolize?
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fermentation or anaerobic respiration using inorganic compounds instead of oxygen as last e- acceptor
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Why may obligate anaerobes be sensitive to oxygen?
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sulfhydryl groups in proteins
may not have detox mechanisms to react to toxic forms of O2 |
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catalase
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takes 2 H2O2 --> O2 + 2H2O
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superoxide dismutase
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breaks superoxide into hydrogen peroxide and oxygen then catalase works on it
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peroxidase
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uses NAD+ to break down H2O2 to H2O
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aerotolerant anaerobes don't have ________
Obligate Anaerobes lack _____ detox mechanisms |
catalase
all of the |
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How do you cultivate anaerobic bacteria?
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1. anaerobic incubator or anaerobic jars using chemical catalysts to get rid of oxygen
2. media w/thioglycalate 3. in a candle jar |
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why is thioglycalate used to cultivate aanaerobic bacteria?
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reacts with oxygen to create anaerobic conditions
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How does cultivation in a candle jar work?
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light candle jar w/ culture then put the lid on which extinguishes the flame b/c O2 partially consumed by the combustion
O2 concentration decr and CO2 incr to ~3.5% in a jar |
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What are bacteria that we may culture in an anaerobic incubator?
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Clostridiudm and Bacteriodes
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Bacteriodes
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Gram-negative, bacillus non-endospore-forming, anaerobes, motile or non-motile
resistant to a wide variety of antibiotics may become a reservoir for resistance in other, more highly-pathogenic bacterial strains. |
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How does an anaerobic jar work?
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creates an O2 free environment using a gas generator which makes H and CO2. the hydrogen removes the O2 by making H2O. The CO2 is req'd for growth of fastidious bacteria. A palladium pellet catalyzes the reaction at romm temp. Methylene blue indicator strip becomes colorless when all done
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What medium did we use to inoculate anaerobic bacteria?
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fluid thioglycollate medium (FTM)
both aerobes and anaerobes can grow on it glucose, cystine and sodium thioglycollate resazurin (indicator for O2 presence) |
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What medium is used to grow Mycobacterium?
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Lowenstein Jensen
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What bacteria did we use for acid0fast staining?
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Mycobacterium smegmatis
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What 3 bacteria did we use for the TFM medium?
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Clostridium sporogenes
Escherichia coli Bacillus subtilis |
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what medium did we use to plate doing anaerobic bacteria?
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Brewer's Anaerobic agar
thioglycollate with resazurin as an oxidation/reduction indicator |
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What was the method used when cultivating anaerobic bacteria?
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boil FTM tubes until liquid is the color of nutrient broth to signal that the oxygen is gone and inoculate.
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monotrichous
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polar--1 flagella/cell
ex: Pseudomonas and Vibro Cholerae |
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amphitrichous
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2 flagella at opposite ends
-only works one at a time |
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peritrichous
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highly motile, b/c flagella all over the place
ex: Proteus vulgaris & Escherichia coli |
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lophotrichous
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tuft ("lopho") of hair at one end (maybe both)
ex: Spirillium |
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axial filaments
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aka...endoflagella
falgella w/in c.w. cause spirochete body to rotate like a cork screw as they twist instide the outer sheath ex: spirochetes (trepanoma) |
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chemotaxis
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towards/away from a certain stimulus
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true motility
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move spontaneously and individual using internal stores to affect the movement
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brownian motion
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random (false) because of currents (molecular bombardment)
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why did we gram stain when isolating the colonies for a pure culture?
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to check for uniformity of the colony
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What is the importance of determining the number of bacteria:
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test purity of different things such as milk, bladder infections and H2O
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What is an indirect method to enumerate bacteria?
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look at turbidity, absorbance and % transmittance
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colony forming unit
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aka CFUs because don't know how many cells formed a colony
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what is the range of CFUs that determine a valid plate count?
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30-300
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TFC
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too few count;
in plate counting the number of CFUs is less than 30 means there was a sampling error |
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TMC
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too much count;
in plate counting the number of CFUs is greater than 300 means there could've been overcrowding that could've inhibited growth |
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What is one problem with turbidity?
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doesn't tell if cells are viable or not because dead cells are included
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What is a way to measure turbidity?
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using a spectrophotometer to measure the optical density and/or the % transmittance
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spectrophotometer
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measures the OD/absorbance of a culture by passing light through a cell suspension and detecting the unscattered light that emerges
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what are units that you could measure in the spectrophotometer?
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% transmittance-how much passes through
absorbance-how much light doesn't pass through |
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If % transmittance is high, the test tube is _______
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clear
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If absorbance is high, the test tube is ______
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turbid
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What is the cycle for an autoclave?
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15min warmup
15min sterilization--121C 15psi 15min cool-down |
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What method was used to do spore staining?
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Schaeffer-Fulton
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What were the nonacid fast bacteria used?
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Staphylococcus aureus
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What was the acidfast bacteria used?
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Mycobacterium smegmatis
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culture media
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nutritional substrate containing substances required to grow microbes in the lab
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what are the 4 different types of culture media?
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complex, defined, selective, differential
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what are the 3 forms of culture media?
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broth, semisolid, solid
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What is the direct method to enumerate bacteria?
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Standard Plate Count
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What is the other name for SPC?
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viable count because only count viable cells
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Standard Plate Count
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most common method to determine bacterial number in a sample
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How do you determine the # of bacteria in a SPC?
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multiply the colony # by the dilution factor
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What could the bacterial population be diluted with?
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PBS or 0.1% Protease Peptone
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What did we dilute the bacterial population with?
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Proteases Peptone tubes
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What are different methods to enumerate bacteria?
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Microscopic Counts, Most Probable Number, Standard plate counts, indirect methods
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Most probable number:
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relate some growth parameter to statistical probability table
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What are some uses of MPN?
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determine drinking water safety by presence of coliforms
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coliforms
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found in the intestines of humans and warm-blooded animals whose presence suggests disease potential; they are lactose fermentors and make acid and gas (turn EMB media from red to yellow)
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EMB media
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media on which if lactose fermenters such as E. coli are grown on it will turn the media from red to a yellow/green sheen
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What is the methodology used to enumerate bacteria?
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1. 6 tubes with 9ml 0.1% protease peptone
2. took 1 ml of original culture of E. coli 3. placed in first tubes and mixed (10ml) 4. took 1 ml from this first tube and mixed in 2nd (repeat till last tube) ***last tube has 10ml all others are 9ml 5. put 1ml of tubes 4, 5, and 6 inoculated on plate count agar with hockey stick (95% alcohol burned off) |
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Calculate the total count of a sample if 30 total cells and dilution factor of 1:10,000?
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30 * 1/(10^-4) = 300000 = 3.0 * 10^5 CFUs
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What is the difference between PCA and NA?
|
same composition in general but PCA used for plate counting
both are complex media and you can grow many things on each of them |
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What media was used to determine motility?
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Motility media
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What is the type of microscope that we typically use?
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Brightfield microscope
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what it is a dark field microscope?
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specimen is bright and backround is dark
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what is a phase contrast microscope?
|
converts slight differences and refraction index easy to see--it's very detailed
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How do you calculate the total magnification?
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multiply the ocular by the objective for eyepiece is 10x therefore if using 40x objective, total mag is 400x.
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photoluminescence
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light emitted as molecule returns to ground state
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fluorescence
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returns immediately to the ground state, wavelength is always longer than the excited light wavelength
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fluorochromes
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dyes used to stain microbial material in fluorescent microscopy
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quenching
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occurs with fluorescent microscopy b/c continued exposure to UV light eventually diminishes fluorescence
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mechanical stage
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holds slide and moves slide
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light souce
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voltage control vary light intensity
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cular magnification?
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10x
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condenser
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under stage, directs light
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diaphragm
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w/in condenser that regulates the amt of light reaching slidew
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focusing knobs
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coarse and fine adjustment
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resolving power
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ability to completely separate 2 objects
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what does resolving power depend on?
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wavelength of light and numerical aperture
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why may you place a blue filter on a microscope?
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creates a shorter wavelength and therefore increases resolution
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star diaphragm
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insert into filter slot of the condenser. opaque disk blocks the central rays of light
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phase contrast microscopy
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Zernike; look at differences between refraction of light in different matter and amplifies this
**wavelength can be slowed 1/4 wavelength when passing through some objects which make is darker |
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coincidence
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2 rays brought into same phase and amplitude is the sum of the 2 waves
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interference
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cancels out so looks darker
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phase plate
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special optical disk located on the obj lens to advance or slow direct rays by 1/4 wavelength
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bright phase = ________ image
dark phase= _________ image |
lighter
darker |
