Microbiology Lab Exam #1

Front 1

How does a light microscope work?

Back 1

It uses visible light or ultraviolet rays to illuminate specimens. They include brightfield, darkfield, phase-contrast, and flourescent instruments.

Front 2

How do the electron microscope work?

Back 2

It uses electron beams instead of light rays, and magnets instead of lenses to observe submicroscopic particles.

Front 3

Iris Diaphragm

Back 3

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.

Front 4

Condenser Position

Back 4

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.

Front 5

Lamp Intensity

Back 5

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.

Front 6

Define Resolution.

Back 6

Resolution in a lens is how well 2 adjacent objects can be seen as discrete points; equal to the wavelength/numerical aperture.

Front 7

What is numerical aperture?

Back 7

It is a measure of the ability to gather light.

Front 8

How does numerical aperture and wavelength influence resolution?

Back 8

The more light that can be gathered, the more detail that can be seen; the smaller the wavelength, the better the resolution.

Front 9

Explain why using oil increases the resolution of the oil immersion lens.

Back 9

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.

Front 10

Describe the procedure that allows you to focus your left eye independently using the binocular compound microscope.

Back 10

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.

Front 11

Describe the characteristics of an ocular and a stage micrometer.

Back 11

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.

Front 12

Can the same calibration factor be used to determine the size of a microorganism under all objectives?

Back 12

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.

Front 13

Would you expect the measurements for given organism to be the same if a size determination is made from a stained and unstained organism?

Back 13

No, because staining often causes the organism to appear larger because the stain collects around its outer surface.

Front 14

What are the components of nutrient agar and nutrient broth? What roles do they play in satisfying the nutritional needs of microorganisms?

Back 14

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.

Front 15

Differentiate between chemically-defined and chemically-complex medium.

Back 15

Chemically-defined is media of exact concentrations and chemically-complex is media of natural components and concentrations are not known.

Front 16

Define sterilization. Explain how autoclave sterilizes materials.

Back 16

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.

Front 17

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?

Back 17

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.

Front 18

Under what conditions would it be more appropriate to attempt to get isolated colonies using a pour plate rather than a strak plate?

Back 18

This would be preferred with motile bacteria.

Front 19

What are some precautions you need to take when doing a pour plate to ensure that you get isolated colonies?

Back 19

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

Front 20

What is the theory behind separation of organisms by streak plate method?

Back 20

The hope is that you can dilute out an isolated colony.

Front 21

Discuss some of the common mistakes that students make that result in a failure to get isolated colonies.

Back 21

They use too much inoculum and they don't utilize all the area of the plate.

Front 22

Why shouldn't you leave an inoculated tube or plate of agar in an incubator more than 48hrs?

Back 22

There's a chance it will dry out and kill the bacteria.

Front 23

What should you do with a tube once you have taken it out of the incubator? Why?

Back 23

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.

Front 24

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?

Back 24

This helps prevent aging and contamination.

Front 25

Compare and contrast basic and acidic dyes. Give some examples of commonly used basic and acidic dyes.

Back 25

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)

Front 26

What is a chromophore?

Back 26

It's the colored portion of a dye.

Front 27

Compare and contrast positive and negative stain techniques. What are the advantages of neg stain?

Back 27

Positive stains stain the object; negative stains stain background. Neg is better representation of actual size and shape of organisms.

Front 28

What is a structural stain and a differential stain.

Back 28

Structural stains stain structural components (flagella). Differential stains can distinguish between different cell types (neg/pos).

Front 29

Why are acidic dyes not commonly used to color bacteria?

Back 29

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.

Front 30

What is heat fixing and why is it done?

Back 30

It's when you run air-dried smear over flame; it attaches specimen to the slide.

Front 31

Why must you use a young culture when doing the gram stain?

Back 31

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.

Front 32

What would be the appearance of a gram (+) or (-) bacteria if you forgot to counterstain w/safranin?

Back 32

The positive cell would remain the same at the end, but the negative cell would not be colored at all.

Front 33

What would be the appearance of a gram (+) or (-) bacteria if the mordant were not used?

Back 33

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.

Front 34

What would be the appearance of a gram (+) or (-) bacteria if decolorizing step was omitted, too brief, or too long?

Back 34

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

Front 35

What would be the appearance of a gram (+) or (-) bacteria if crystal violet was replaced with methylene blue.

Back 35

Neg cells would be stained blue instead of red and pos cells would still be purple.

Front 36

What would be the appearance of acid-fast and non-acid-fast becteria if plain ethanol were used instead of acid-alcohol?

Back 36

Acid fast: wouldn't matter
Non Acid fast: carbolfuchin wouldn't be removed from cells that lack waxes in the cell wall

Front 37

What would be the appearance of acid-fast and non-acid-fast becteria if carbolfuchsin were not heated sufficiently?

Back 37

Acid fast: dye wouldn't penetrate wall b/c wax didn't melt
Non Acid fast: nothing would be affected

Front 38

What would be the appearance of acid-fast and non-acid-fast bacteria if methylene blue were used in place of crystal violet

Back 38

Both types would test positive for acid fast

Front 39

What would be the appearance of acid-fast and non-acid-fast bacteria if the counter stain were omitted?

Back 39

Acid fast: nothing
Non Acid fast: wouldn't become stained at all

Front 40

What would be the appearance of acid-fast and non-acid-fast bacteria if a thorough, long rinse occurred after counterstain?

Back 40

Acid fast: nothing
Non Acid fast: would wash out methylene blue

Front 41

Why are organisms acid fast?

Back 41

they have a lot of wax in their cell wall.

Front 42

Why is it important to know if an organism is acid fast?

Back 42

Mycobacterium and Nocardia contain important human pathogens. It also helps distinguish between these two groups from other bacteria.

Front 43

How does the relative size of an endospore compare to that of the vegetative cell that produced the endospore?

Back 43

The vegetative cell is twice the size of the endospore.

Front 44

Of what benefit is endospore formation to soil bacteria?

Back 44

When conditions change, get bad/cold/etc, the bacteria can form spores and survive.

Front 45

Do endospores from different organisms have the same shape?

Back 45

No, they don't

Front 46

Where do endospore formers usually live?

Back 46

In the soil

Front 47

What would be the results of the endospore stain if you failed to use heat w/the primary stain?

Back 47

The spore coat wouldn't expand, so the dye wouldn't penetrate

Front 48

What would be the results of the endospore stain if you rinsed inadequately after malachite green?

Back 48

Malachite green wouldn't be removed from the vegetative cell

Front 49

What would be the results of the endospore stain if you rinsed excessively after safranin?

Back 49

It's a weak dye and would be washed from the vegetative cell.

Front 50

Of what importance is the capsule to the bacteria?

Back 50

Helps in attachment to surfaces and other cells; to protect bacteria populations from predators

Front 51

What role might a capsule play in the virulence of a bacterium?

Back 51

It helps it stick to cells; this must happen in order to penetrate host cell. Almost all pathogens known have capsules.

Front 52

What is a glycocalyx?

Back 52

It is the stringy polysacchride material that binds bacteria to surfaces and other bacteria.

Front 53

What color did PHB granules and cytoplasm stain? Were the endospores stained?

Back 53

PHB: dark blue
Cytoplasm: red
Endospores remain unaffected

Front 54

How large are the PHB granules compared to endospores?

Back 54

they are much smaller than endospores

Front 55

Why do you need to use xylen?

Back 55

It decolorizes the smear

Front 56

Why are PHB granules stained by Sudan Black and not by safranin?

Back 56

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

Front 57

Why do cells store PHB? Of what value is PHB to the cell?

Back 57

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

Front 58

Explain chemistry behind how atmosphere in Gas-Pak anaerobic jar is created.

Back 58

H2 gas generated by the envelope reacts w/O2 to make water; rxn is calculated by pallidium.

Front 59

How can we tell if anaerobic conditions have been achieved in Gas Pak?

Back 59

There is an oxygen indicator that turns blue (oxidized) to white (reduced)

Front 60

Candle jar is used for growing what type of organism?

Back 60

Capnophile

Front 61

Explain why you can't use candle jar to grow obligate anaerobes.

Back 61

B/c O2 kills them and there's still 14% O2 in the jar.

Front 62

Obligate aerobe

Back 62

needs O2 and has detoxifying mechanisms

Front 63

Obligate anaerobe

Back 63

dies in O2 and doesn't have detoxifying mechanisms

Front 64

Aerotolerant anaerobe

Back 64

doesn't need O2, but can tolerate some oxygen; and can detoxify

Front 65

Facultative anaerobe

Back 65

grows better in O2 but doesn't need it; can detoxify

Front 66

Microaerophile

Back 66

needs reduced concentrations of O2; can detoxify

Front 67

What are some reasons why an organism is an obligate anaerobe?

Back 67

It cannot detoxify O2 toxins and can only do anaerobic respiration or fermentation.

Front 68

Distinguish between true motility and Brownian motion.

Back 68

Brownian motion is random vibration, whereas true motility is more than just that, it's actual movements, with directions.

Front 69

Polar flagellar arrangements

Back 69

amphitrichous kophotrichous

Front 70

Difference between regular and motility agar

Back 70

motility = 0.5% agar; semisolid; restricts diffusion of nonmotile cells
regular = 1.5% agar; solid

Front 71

What microscope adjustments should you make to have the best chance to see bacteria in hanging drop?

Back 71

Focus on the edege of the drop under low power and as you move up in objectives, decrease the light

Front 72

Positive Stain

Back 72

Crystal Violet

Front 73

Negative Stain

Back 73

NIgrosin

Front 74

Acid-Fast Stain

Back 74

Carbolfuchsin, acid-alcohol, and methylene blue

Front 75

PHB Stain

Back 75

Sudan Black, Xylene, Safranin

Front 76

Endospore Stain

Back 76

Malachite green, Safranin

Front 77

Capsule Stain

Back 77

Congo Red and Maneval's

Front 78

Gram Stain

Back 78

Crystal Violet 60sec, H2O brief, Gram's Iodine 60sec, water thorough, 95% ethanol 10sec, water thorough, safranin 60sec, water brief, drain and blot dry

Front 79

Gram stain results

Back 79

purple = positive and red = negative

Front 80

Acid fast stain results

Back 80

bright blue is negative and bright red is positive

Front 81

Capsule Stain results

Back 81

halo around cell is a positive and no halo is negative

Front 82

PHB stain results

Back 82

granules stain dark blue and cytoplasm is red; endospores are not stained

Front 83

How to calculate dilution factor?

Back 83

volume dispensed / total volume in tube after dilution

Front 84

How to calculate volume plated from last tube dilution?

Back 84

Number of colonies following incubation divided by the product of all the dilution factors of tubes

Front 85

How to calculate CFU/mL?

Back 85

Take the number of colonies and divide them by volume plated from last dilution tube