Micro lab midterm

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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 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.

Staphylococcus aureus cells after the decolorizer is used.

purple

Pseudomona aeruginosa after primary stain is added.

purple

Pseudomonas aeruginosa after the counterstain is added.

pink

Bacillus megarerium after the mordant is added.

purple

Bacillus megaterium 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.

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.

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.

List four diseases caused by spore forming bacteria.

Anthrax, Tetnus, Botulism, Gas gangrene.

Endospores are extremely resistant to heat but can be destroyed 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.

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.

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.

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.

Differentiate 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 chemicals 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

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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