Lab test 2

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How does smear preparation of cells from a liquid media differ from preparation of cells from a solid medium?

you get less bacteria from liquid and you don't need to start with a drop of water.

Why is it important to limit the quanitity of cells used to prepare a smear?

if a smear of bacteria is too thick when it is stained it will retain too much of the stain when the rinse steps are done.

For preparation of a smear on a slide, what is the purpose of heat fixation?

makes cells stick to the slide when rinsing.

What problems can arise when the slide is heated in a flame?

the heat can change the shape of the bacteria.

What causes a stain to adhere to bacterial cells?

the bacteria cell walls make make the stain stick during the rinse.

Why are all colored dyes not necessarily useful for simple staining?

Simple stain does not determine if the bacteria has a thick or thin cell wall.

Which type of microscope produces an image of unstained cells that is most similar to the one achieved by negative staining?

Darkfield is similar to negative staining.

A basic dye is utilized to stain bacterial cells.

simple

A stain that does not penetrate cells is used to color the background.

negative

Useful for visualizing spirochaetes

capsule

Heat fixation of the slide is not recommended

negative

Water is used to remove excess stain from the slide.

simple

For the simple stain procedure one can use

methylene blue

For the negative stain procedure, one can use

both india ink and nigrosin

Before heat fixation, a wet smear of bacterial cells on a slide must first be

air dried

Which of the three differential stains would likely be the first used when identifying an unknown bacterium?

The gram stain.

What is the function of a mordant?

is what fixes the stain into the cell wall of the bacteria.

For differential staining, how does a counterstain differ from a primary stain?

It's a different color from a primary stain.

How do gram-positive and gram-negative bacteria differ in cellular structure?

Gram- are thin cell walls and are pink (fusia). Gram+ are thick cell walls and are purple and made of peptiglycan.

Which is the most critical step in the Gram-stain procedure? Why? If this procedure is done incorrectly, how might that affect the final results?

The most critical step in the Gram stain is the decolorizer step with acetone alcohol. If the decolorizer is left on too long Gram positive bacteria will come out pink and if it is not left on long enough the Gram negatives will come out purple.

How does culture age affect the results of a Gram-stain?

gram+ will get old and will look pink. The cell wall gets old and thin and stains pink.

How does culture age affect the results of a spore stain?

old culture have more spores, bacteria produce more endospores when they are old.

Why must smear thickness be considered before performing a Gram-stain ?

If they are too thick gram - will not decolorize properly.

What color are bacterial endospores after a Gram-stain is performed? What does this tell you about the physical properties of endospores?

clear inside and around them. They are so thick they will not take up the stain.

What makes Mycobacterium particularly resistant to staining? How are the bacteria in this genus grouped in terms of Gram classification?

They have an extra thick cell wall with mycolic acid.

How do you think the acid-fast nature of Mycobacterium contributes to its virulence?

to survive enzamatic digestion once it is phagocytized and it can then grow and take over the macrophage.

Staphylococcus aureus before primary stain is added.

no color.

Pseudomona aeruginosa after primary stain is added.

purple

Bacillus megarerium after the mordant is added.

purple

Staphylococcus aureus cells after the decolorizer is used.

purple

Moraxella (Branhamella) catarrhalis after the decolorizer is used.

no color

Bacillus megaterium after the counterstain is added

pink

Pseudomonas aeruginosa after the counterstain is added.

pink

Gram stain chart

Crystal Violet, Iodine, Acetone alcohol, Safranin, Gram+ purple, Gram - pink.

Spore stain chart

Malachite Green, Heat, NO decolorizer, Safranin, Endospores are green, Cells are pink.

Acid Fast Stain chart

Carbolfuchsin, Heat, Acid alcohol, Methylene Blue, Acid Fast is Fuchia, Non acid fast is blue.

Primary stain

crystal violet, Malachite, carbol fuchsin

Mordant are

Iodine, Heat, Heat

Decolorizer

Acetone alcohol, None, acid alcohol

Counterstain

Safranin, Safranin, Methylene blue

Cell type = color after completion of stain

Gram + is purple endospores, Gram - is Pink, Sporangium+ green and Vegetative cell=pink, Acid-fast bacteria=fusia and Non-acid-fast bacteria= blue.

Bacterial cell wall is composed of

peptidoglycan.

The exosporium, or endospore coat, is composed of

proteins.

Endospores are produced by bacteria in the genus

Bacillus and Mycobacterium.

Acid-fast staining is useful for identifying the causative agent of

leprosy and tuberculosis.

What is the role of oxygen for cellular respiration?

Oxygen is a final or terminal electron acceptor.

What type of metabolixm occurs in the absence of oxygen?

Anaerobic or anaerobic metabolism.

Name two enzymes that are present in obligate aerobes but lacking in obligate anaerobes.

Superoxide dismutase and Catalase.

dismutase

superoxide ion is toxic to strict anaerobes because they lack

Catalase

enzyme found in most plant and animal cells that functions as an oxidative catalyst

Differentiate between a microaerophile and an aerotolerant organism

Microaerophiles need small amount of oxegen and aerotolerant organisms to no need oxygen but can live in the presence of it.

obligate anaerobe

an organism that cannot grow in the presence of oxygen

obligate aerobe

organism that REQUIRES A CONSTANT SUPPLY OF OXYGEN in order to live

facultative aerobe

an organism that prefers to live in a non-oxygen environment but can also live in an oxygen environment

microaerophile

it needs oxygen, but can only tolerate low concentrations of it.

When performing a standard plate count, why are the counts reported as colony forming units (CFUs)?

because one colony could grow form a group of cells.

How would you inoculate a plate to get a 1:10 dilution? AND a 1:100 dilution?

Just plate 0.1ml for a 1:10. To do a 1:100 do a 1:10 dilution then plate 0.1 dilution then plate 0.1 ml.

Define the optimal growth temperature for each of the following classes of bacteria...

hyperthermophile 80 and above, Thermophile between 50 and 80. Mesophile between 20 and 50. Psychrophile -5 to 20.

Differentiate between psychrophile and psychrotroph.

Psychorophiles grow best at low temps and psychrotrophs are mesophiles that can grow at low temps.

Why are psychrotrophic bacteria of concern to those in the food-service industry?

They can spoil food.

What is the optimum growth temperature for most human pathogens?

37* is the optimum tempurature for most pathogens because that is human body thempurature.

Name three cellular components involved in metabolism that are influenced by temperature changes.

Ribosomes, enzymes and cell membranes are all affected by temperature.

What is the importance fo inoculating a control plate in theis experiment?

to show that organisms that are not exposed to heat can still grow.

Bacillus megaterium has a high thermal death point and long thermal death time, but it is not classified as a thermophile.

it can form endospores.

Give three reasons why endospores are much more resistant to heat than are vegattive cells.

Thery have a tougher spore coat. They have a low moisture content. They have a large amount amount of calciium and dipicolinic acid that protects them from heat.

List four diseases caused by spore forming bacteria.

Anthrax, Tetnus, Botulism, Gas gangrene.

Endospores are extrememlyt resistant to heat but can be destryed by heat if the proper conditions are applied. Describe the heating conditions necessary to kill endospores when the following are sterilized.

Glassware treated in an oven needs high heat for hours, Media in an autoclave needs at least 15 minutes at 121* and 15 psi, Aseptic transfer of bacteria in done with a loop that was heated to red hot.

How does pH negatively affect the metabolism of microorganisms?

causes proteins and enzymes to denature and causing a loss of enzymantic activity.

Define three groups of microorganisms in regard to their optimum pH for growth.

Acidophile, neutrophiles, alkalinphiles.

Acidophiles

`bacteria that survive in a pH below 6.0 and make foods such as sour cream and yogurt

Neutrophiles

grow optimally at pH 5.5-8 +63

0.00 .Alkalinphiles

. like more basic enviroment up to 10 pH

Why are bacteria generally resistant to hypotonic environments whereas animal cells are not?

Because they have a cell wall and animal cells do not.-

How do hypertonic environments negatively affect most bacteria cells?

the water leaves the cell causing the membrane to pull away from the cell wall (plasmolsis) thus dissrupting the cell membrane.

Why are staphylococci well-suited for the colonization of skin?

are halotolerant and can live on the skin just fine.

Differentiate between halophiles and osmophiles. Which type would most likely cause spoilage of jams and jellies?

Halophiles are bacteria that prefer to grow in high salt. Osmophiles can grow in high sugar and are the ones that are found in spoiled jams and jellies.

Halophiles

live in exreme salt water (Dead Sea, Great Salt lake)

Halotolerant

can survive at higher salt concentrations but grow best at low or zero concentations

Osmophiles

live in habitats with high solute concentration.

How many times more resistant were B. megaterium spores than S. aureus vegatative cells?

Bacillus megaterium should have been more resistant to UV because it forms endospores.

Why was half of each plate covered with an index card?

to show the bacteria would grow when not exposed to UV.

What was the purpose of leaving the cover on one set of petri dishes?

the plastic cover was left on one plate to show that plastic blocks UV.

Describe the damaging effects of UV radiation on living cells.

UV causes damage to DNA by causing thymine dimers (pyrimidine dimers).

Why does exposure to UV radiation cause death in vegetative cells but not endospores?

UV does no cause death to endospores because they have proteins around the DNA that provide protection from the damaging affects from UV.

At which wave length is UV radiation most germicidal?

260 nm, this is the wavelenth that DNA absorbs.

What limited protection do cells have against the damaging effects of UV rediation?

some cells use enzymes that are activated by normal white light. These enzymes repair the thymine dimers when they are activated.

What types of damage to human tissues can result from prolonged exposures to UV radiation?

skin cancer and cataracts.

What protective measures can be taken to limit these types of damage, both during the experiment as well as in everyday life?

eye protection.

Which B. megaterium culture, logarithmic or stationary phase, would show the best survival following exposure to UV radiation?

the stationary phase would have the best survival after exposure to UV because many of the bacterium would have formed endospores.

Why did lysozyme affect the test organisms differently?

Gram negative bacteria have an outer membrane that does not let lysozyme tin to destroy the peptidogylcan.

What was the function of peptidoglycan in bacterial cells?

is to protect the bacteria from osmotic pressure.

How does lysozyme specifically affect peptidoglycan?

Lysozyme destroys the bonds between NAG and NAM.

Where in nature can lysozyme be found?

Lysozyme is found in skin tears, mucus membranes (saliva)

Why is it produced in these environments?

It is useful because it protects therse areas from bacterual infection.

Based on the results, what might one conclude about the types of bacteria that are involved in eye infections?

Gram negative infections because they are not sensitive to lysozyme.

What effect does alcohol have on the level of skin contaminants?

it is effective to kill bacteria, but completely sterile.

Is there any difference between the effects of dipping versus swabbing?

we didn't dip.

Which method appears to be more effective, dipping or swabbing??

I would think swabbing would be.

There is definitely survival of some microorganisms even after alcohol treatment. Without staining or microscopic scrutiny, predict what ty[es of microbes are growing on the medium where you made the right thumb impression after treatment.

alcohol can't kill all bacteria or fungi.

For what pruposes is alcohol a useful antiseptic?

use on the skin.

What advantages does alcohol have over hand soap for antisepsis of the skin?

kills bacteria on skin and you don't need water.

Why does treatment of human skin with alcohol not create a completely sterile environment?

there are cracks and crevises in the skin that harbor bacteria. Alcohol will not kill all endospores and spores from fungi.

Differentiate between the following and provide one example of each.....antibiotics and antimicrobial

Antibiotics are natural, antimicrobial drugs are synthetic.

Differentiate between the following and provide one example of each....broad and narrow spectrum antibiotics

broad spectrum antimiotics can kill a widr range of bacteria. Narrow spectum antibiotics are for specific group

What factors influence the size of the zone of inhibition for an antibiotic?

the size of the drug and how well i differs out into the agar and the resistance of the microorganism.

Two antibiotics are tested for their efficacy against a single bacterial species. If antibiotic A and antibiotic B produce zones of inhibition with the same diameter, how can the bacterium be considered resistant to antibiotic A, but sensitive to antibiotic B?

even though on drug gives a large zone of inhibitions that doesn't mean that it will be effective in killing the pathogen when it is iside of the organism.

Why are certain gram-negative bacteria more resistant than gram positive bacteria to antibiotics that attack cytoplasmic targets?

because their outer membrane does not allow the drugs in.

Why are gram positive bacteria typically mre resistant than gram negative bacteria to antibiotics, that disrupt plasma membranes, such as polymyxin B?

polymixin B disrupts the cell membrane and causes cell lysis of the bacteria. Gram positives are more resistant to this drug because their thick cell wall protects then when they take on water.

If a bacterial isolate shows intermediate to moderate resistance to an antibiotic, how might this antibiotic still be successfully used in the treatment of this microbe?

a drug can still be effective by simply increasing the dosage.

Differenetiate between antiseptic and disinfectant. Include examples of each in your answer. Indicate whether any chemicals can be used as both.

antiseptics are used on tissue such as alcohol and Iodine. Disinfectants are used on Fomites such as bleach.

What factors influence the size of the zone of inhibition produced by a chemical?

by the size of the molecule of the chemical used to kill the organism. The smaller it is the faster it can diffuse out.

How might the physical differences between gram positive and gram negative bacteria contribute to differences in chemical resistances?

Gram -'s maybe more resistant due to the outer membrane that can keep shemicals from reaching the inside of the cell.

Name the three types of microbes most commonly associated with skin.

Dipthroids, Staphlococcus, Micrococcus and yeast.

Dipthroids

bacteria found in the mouth

Staphlococcus

purple grape like clusters

Micrococcus

type genus of the family Micrococcaceae, gram + cooci found in pairs of clusters,nonpathogenic, grows well at 25*C, cant tolerate high salt

How are normal skin flora beneficial to the host?

they take up space and nutrients from potential pathogens.

What important opportunistic pathogen is associated with skin?

Staphylococcus Aureus

Differentiate between normal flora and transient bacteria found on skin. Which type is more difficult to remove?

normal flora are microbes that are always there and transient bacteria are ones that are picked up from the environment.

Why is it so important that surgeons scrub their hands prior to surgery even through they wear rubber gloves?

there could be small holes in the gloves.

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