Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
85 Cards in this Set
- Front
- Back
|
How does a light microscope work?
|
It uses visible light or ultraviolet rays to illuminate specimens. They include brightfield, darkfield, phase-contrast, and flourescent instruments.
|
|
|
How do the electron microscope work?
|
It uses electron beams instead of light rays, and magnets instead of lenses to observe submicroscopic particles.
|
|
|
Iris Diaphragm
|
It regulates the amount of light entering the condenser; too much light will come around the specimen and too little will not penetrate the specimen. If you use a high magnification, then the iris will have to be opened more.
|
|
|
Condenser Position
|
It focuses the light coming from the lamp. Usually the condensor will need to be raised as you increase the magnification, but lowering it slightly may be helpful when viewing unstained specimens.
|
|
|
Lamp Intensity
|
It controls how bright the light appears. Too much light intensity would cause a glare which would obscure the object. Usually you will need to increase lamp intensity as you increase magnification.
|
|
|
Define Resolution.
|
Resolution in a lens is how well 2 adjacent objects can be seen as discrete points; equal to the wavelength/numerical aperture.
|
|
|
What is numerical aperture?
|
It is a measure of the ability to gather light.
|
|
|
How does numerical aperture and wavelength influence resolution?
|
The more light that can be gathered, the more detail that can be seen; the smaller the wavelength, the better the resolution.
|
|
|
Explain why using oil increases the resolution of the oil immersion lens.
|
The less light entering the objective lens that is scattered, the better the resolution. Oil and glass have the same refractive index (1.5), so the light doesn't scatter very much.
|
|
|
Describe the procedure that allows you to focus your left eye independently using the binocular compound microscope.
|
Look through the right eyepiece with right eye and adjust the course and fine adjustments until the specimen is in focus. Repeat with left eye and left eye piece.
|
|
|
Describe the characteristics of an ocular and a stage micrometer.
|
Ocular micrometer can be seen through eye piece and is dependent on the ocular being used. A stage micrometer is a glass slide that can be placed on the stage. It has sections of lines (small 10 um; large 100 um apart) that when lined up with are used to find the calibration factor.
|
|
|
Can the same calibration factor be used to determine the size of a microorganism under all objectives?
|
No, because each objective has a different magnification; thus, the higher the magnification, the smaller the distance between the lines gets and the smaller the calibration factor.
|
|
|
Would you expect the measurements for given organism to be the same if a size determination is made from a stained and unstained organism?
|
No, because staining often causes the organism to appear larger because the stain collects around its outer surface.
|
|
|
What are the components of nutrient agar and nutrient broth? What roles do they play in satisfying the nutritional needs of microorganisms?
|
Agar is a jelling agent. Broth is a liquid mixed 1.5-2%agar. Nutrients: water, energy source, nutritionally suitable sources of C, N, S, O, H, and trace elements; organic factors may be added. Both are chemically complex because exact concentrations are not defined. Agar is a polysaccharide from red algae. Both provide suitable environment and nutrients.
|
|
|
Differentiate between chemically-defined and chemically-complex medium.
|
Chemically-defined is media of exact concentrations and chemically-complex is media of natural components and concentrations are not known.
|
|
|
Define sterilization. Explain how autoclave sterilizes materials.
|
Sterilization is the killing or removal of spores and living organisms. The autoclave sterilizes by increasing the pressure and temperature by using steam. As the temperature increases, the water begins to boil and the steam form and the pressure increases. It is usually set to reach 15psi in 15mins. However, these vary with altitude; it must reach 121C to kill.
|
|
|
Why might something not be sterilized if you autoclave it at 15psi for 15min? What conditions should you be sure to achieve to ensure sterility?
|
If your autoclave isn't at sea level, then you may not be actually sterilizing. The celcius scale varies at different altitudes, so just make sure that you autoclave at 121C for 15min.
|
|
|
Under what conditions would it be more appropriate to attempt to get isolated colonies using a pour plate rather than a strak plate?
|
This would be preferred with motile bacteria.
|
|
|
What are some precautions you need to take when doing a pour plate to ensure that you get isolated colonies?
|
You need to make sure you use aseptic technique, that you mix the agar/inoculate mixture well and swirl/turn the petri dish to ensure it covers the entire area
|
|
|
What is the theory behind separation of organisms by streak plate method?
|
The hope is that you can dilute out an isolated colony.
|
|
|
Discuss some of the common mistakes that students make that result in a failure to get isolated colonies.
|
They use too much inoculum and they don't utilize all the area of the plate.
|
|
|
Why shouldn't you leave an inoculated tube or plate of agar in an incubator more than 48hrs?
|
There's a chance it will dry out and kill the bacteria.
|
|
|
What should you do with a tube once you have taken it out of the incubator? Why?
|
After using it, it should go to the refrigerator because if you leave it out at room temperature for extended periods of time they will die.
|
|
|
Why is it necessary to start a new stock and reserve culture of your unknown every 2-3 weeks even though those cultures have been in the refrigerator?
|
This helps prevent aging and contamination.
|
|
|
Compare and contrast basic and acidic dyes. Give some examples of commonly used basic and acidic dyes.
|
Acidic dyes: the colored portion is negatively charged and usually color background, not organism; don't stain cells b/c they're repelled by neg charge of cells.(congo red, sudan black)
Basic dyes: colored part positively charged; color organism because attracted to neg cell charge (methylene blue, crystal violet, safranin, fuchsin) |
|
|
What is a chromophore?
|
It's the colored portion of a dye.
|
|
|
Compare and contrast positive and negative stain techniques. What are the advantages of neg stain?
|
Positive stains stain the object; negative stains stain background. Neg is better representation of actual size and shape of organisms.
|
|
|
What is a structural stain and a differential stain.
|
Structural stains stain structural components (flagella). Differential stains can distinguish between different cell types (neg/pos).
|
|
|
Why are acidic dyes not commonly used to color bacteria?
|
they are not usualy used because they are repelled by the neg charge of the cells but are attracted to the positive charge of the slide.
|
|
|
What is heat fixing and why is it done?
|
It's when you run air-dried smear over flame; it attaches specimen to the slide.
|
|
|
Why must you use a young culture when doing the gram stain?
|
As cells get older, they may sustain damage to their cell walls, which could allow dyes to be easily leached from the cell by a decolorizing agent.
|
|
|
What would be the appearance of a gram (+) or (-) bacteria if you forgot to counterstain w/safranin?
|
The positive cell would remain the same at the end, but the negative cell would not be colored at all.
|
|
|
What would be the appearance of a gram (+) or (-) bacteria if the mordant were not used?
|
Nothing would help the dye attach to the charged groups in the cell wall, membrane and cytoplasm of both gram neg and gram positive cells.
|
|
|
What would be the appearance of a gram (+) or (-) bacteria if decolorizing step was omitted, too brief, or too long?
|
Omitted: crystal violet would not be washed out of neg cell
Too brief: crystal violet wouldn't be fully removed and may still remain in neg cell Too long: could wash crystal violet out of both cells |
|
|
What would be the appearance of a gram (+) or (-) bacteria if crystal violet was replaced with methylene blue.
|
Neg cells would be stained blue instead of red and pos cells would still be purple.
|
|
|
What would be the appearance of acid-fast and non-acid-fast becteria if plain ethanol were used instead of acid-alcohol?
|
Acid fast: wouldn't matter
Non Acid fast: carbolfuchin wouldn't be removed from cells that lack waxes in the cell wall |
|
|
What would be the appearance of acid-fast and non-acid-fast becteria if carbolfuchsin were not heated sufficiently?
|
Acid fast: dye wouldn't penetrate wall b/c wax didn't melt
Non Acid fast: nothing would be affected |
|
|
What would be the appearance of acid-fast and non-acid-fast bacteria if methylene blue were used in place of crystal violet
|
Both types would test positive for acid fast
|
|
|
What would be the appearance of acid-fast and non-acid-fast bacteria if the counter stain were omitted?
|
Acid fast: nothing
Non Acid fast: wouldn't become stained at all |
|
|
What would be the appearance of acid-fast and non-acid-fast bacteria if a thorough, long rinse occurred after counterstain?
|
Acid fast: nothing
Non Acid fast: would wash out methylene blue |
|
|
Why are organisms acid fast?
|
they have a lot of wax in their cell wall.
|
|
|
Why is it important to know if an organism is acid fast?
|
Mycobacterium and Nocardia contain important human pathogens. It also helps distinguish between these two groups from other bacteria.
|
|
|
How does the relative size of an endospore compare to that of the vegetative cell that produced the endospore?
|
The vegetative cell is twice the size of the endospore.
|
|
|
Of what benefit is endospore formation to soil bacteria?
|
When conditions change, get bad/cold/etc, the bacteria can form spores and survive.
|
|
|
Do endospores from different organisms have the same shape?
|
No, they don't
|
|
|
Where do endospore formers usually live?
|
In the soil
|
|
|
What would be the results of the endospore stain if you failed to use heat w/the primary stain?
|
The spore coat wouldn't expand, so the dye wouldn't penetrate
|
|
|
What would be the results of the endospore stain if you rinsed inadequately after malachite green?
|
Malachite green wouldn't be removed from the vegetative cell
|
|
|
What would be the results of the endospore stain if you rinsed excessively after safranin?
|
It's a weak dye and would be washed from the vegetative cell.
|
|
|
Of what importance is the capsule to the bacteria?
|
Helps in attachment to surfaces and other cells; to protect bacteria populations from predators
|
|
|
What role might a capsule play in the virulence of a bacterium?
|
It helps it stick to cells; this must happen in order to penetrate host cell. Almost all pathogens known have capsules.
|
|
|
What is a glycocalyx?
|
It is the stringy polysacchride material that binds bacteria to surfaces and other bacteria.
|
|
|
What color did PHB granules and cytoplasm stain? Were the endospores stained?
|
PHB: dark blue
Cytoplasm: red Endospores remain unaffected |
|
|
How large are the PHB granules compared to endospores?
|
they are much smaller than endospores
|
|
|
Why do you need to use xylen?
|
It decolorizes the smear
|
|
|
Why are PHB granules stained by Sudan Black and not by safranin?
|
Sudan Black stains the poly beta hydroxybutic acid dark blue because PHB is lipid-like and Sudan Black is a lipid-soluble dye; safranin will only bind to neg charges of cell
|
|
|
Why do cells store PHB? Of what value is PHB to the cell?
|
PHB is a polymerized form of bet hydroxybutric acid...the polymer doesn't alter pH or H2O pressure, which the normal form does; the reason this needs to be stored is b/c it's a readily available source of carbon and energy
|
|
|
Explain chemistry behind how atmosphere in Gas-Pak anaerobic jar is created.
|
H2 gas generated by the envelope reacts w/O2 to make water; rxn is calculated by pallidium.
|
|
|
How can we tell if anaerobic conditions have been achieved in Gas Pak?
|
There is an oxygen indicator that turns blue (oxidized) to white (reduced)
|
|
|
Candle jar is used for growing what type of organism?
|
Capnophile
|
|
|
Explain why you can't use candle jar to grow obligate anaerobes.
|
B/c O2 kills them and there's still 14% O2 in the jar.
|
|
|
Obligate aerobe
|
needs O2 and has detoxifying mechanisms
|
|
|
Obligate anaerobe
|
dies in O2 and doesn't have detoxifying mechanisms
|
|
|
Aerotolerant anaerobe
|
doesn't need O2, but can tolerate some oxygen; and can detoxify
|
|
|
Facultative anaerobe
|
grows better in O2 but doesn't need it; can detoxify
|
|
|
Microaerophile
|
needs reduced concentrations of O2; can detoxify
|
|
|
What are some reasons why an organism is an obligate anaerobe?
|
It cannot detoxify O2 toxins and can only do anaerobic respiration or fermentation.
|
|
|
Distinguish between true motility and Brownian motion.
|
Brownian motion is random vibration, whereas true motility is more than just that, it's actual movements, with directions.
|
|
|
Polar flagellar arrangements
|
amphitrichous kophotrichous
|
|
|
Difference between regular and motility agar
|
motility = 0.5% agar; semisolid; restricts diffusion of nonmotile cells
regular = 1.5% agar; solid |
|
|
What microscope adjustments should you make to have the best chance to see bacteria in hanging drop?
|
Focus on the edege of the drop under low power and as you move up in objectives, decrease the light
|
|
|
Positive Stain
|
Crystal Violet
|
|
|
Negative Stain
|
NIgrosin
|
|
|
Acid-Fast Stain
|
Carbolfuchsin, acid-alcohol, and methylene blue
|
|
|
PHB Stain
|
Sudan Black, Xylene, Safranin
|
|
|
Endospore Stain
|
Malachite green, Safranin
|
|
|
Capsule Stain
|
Congo Red and Maneval's
|
|
|
Gram Stain
|
Crystal Violet 60sec, H2O brief, Gram's Iodine 60sec, water thorough, 95% ethanol 10sec, water thorough, safranin 60sec, water brief, drain and blot dry
|
|
|
Gram stain results
|
purple = positive and red = negative
|
|
|
Acid fast stain results
|
bright blue is negative and bright red is positive
|
|
|
Capsule Stain results
|
halo around cell is a positive and no halo is negative
|
|
|
PHB stain results
|
granules stain dark blue and cytoplasm is red; endospores are not stained
|
|
|
How to calculate dilution factor?
|
volume dispensed / total volume in tube after dilution
|
|
|
How to calculate volume plated from last tube dilution?
|
Number of colonies following incubation divided by the product of all the dilution factors of tubes
|
|
|
How to calculate CFU/mL?
|
Take the number of colonies and divide them by volume plated from last dilution tube
|
