Lab 4 Work

The capsule is a coating around a bacterial cell that will repel any stains. True/False

False

The more bacteria you put on the glass slide, the better your results will be. True/False

False

After staining your fixed slide with methylene blue, you are unable to see any bacteria at 40X due to high background staining. What could you have done wrong? Not enough rinsing with water to remove excess stain. There are too many bacteria on the slide for the stain to be rinsed off. Nothing - at 100X, the individual bacteria will be visible. You wiped too hard with the bibulous paper and all of the bacteria are gone.

Not enough rinsing with water to remove excess stain.

A capsule imparts what protections to an invading bacterial cell? It can squeeze between our cells and invade deeper into our tissues. It increases the absorbance of nutrients from within our tissues. When exposed to air, it provides desiccation protection. Our immune cells are unable to phagocytize the bacteria.

Our immune cells are unable to phagocytize the bacteria.

What is the difference between doing a simple stain versus a differential stain on a bacterial slide? -The simple stain will only tell you morphological shape and size, while the differential stain will only tell you the type of the cell wall. -The simple stain will only tell you morphological shape and size, while the differential stain will allow for determination of size, shape, and type of the cell wall. -The simple stain allows for determination of size, shape, and composition of the cell wall, while the differential stain will only tell you morphological shape and size. -The simple stain allows for determination of the composition of the cell wall, while the differential stain will only tell you morphological shape and size.

The simple stain will only tell you morphological shape and size, while the differential stain will allow for determination of size, shape, and type of the cell wall.

The purpose of the mordant is to __________ fix the bacteria to the slide and allow it to be stained. intensify the color of the primary stain. disinfect the slide. kill the bacteria and turn it blue.

intensify the color of the primary stain.

The age of a Bacillus cereus culture to be spore stained in order to visualize endospores should be __________. less than 24 hours old to ensure that the culture is in the exponential phase of growth 48-72 hours old to ensure that the culture is in the death or decline phase to maximize the formation of spores 2-3 weeks old of no importance

48-72 hours old to ensure that the culture is in the death or decline phase to maximize the formation of spores

Because they have an extremely thick layer of peptidoglycan in their cell walls, Mycobacterium bacterial cells require the addition of heat in order for stains to penetrate the cells. True/False

False

Copper sulfate is used as a decolorizing agent in spore staining. True/False

False

During the heating step of the acid-fast stain, the carbol fuchsin is allowed to come to a boil and dry out. The bacteria are red after completion of the staining process when viewed under oil immersion, which means the bacteria are acid-fast positive. True/False

False

In a Gram stain, the wash steps are not required if you need to perform the stain quickly due to time constraints. True/False

False

Species of Bacillus form endospores as a means of asexual reproduction. True/False

False

Staphylococcus aureus is acid-fast negative and as such stains pink upon completion of the Ziehl-Neelsen method. True/False

False

The Gram stain allows for determination of bacterial species based on their metabolic activities. True/False

False

The rule of thumb for heat fixing a bacteria slide is, "The hotter the better when fixing a slide." True/False

False

The spore stain can be performed without heat, just like the acid-fast stain, as long as a wetting agent is used. True/False

False

Using a simple stain, you should be able to see multiple colors on a slide of mixed bacterial species. True/False

False

Vibrio bacteria are only "comma-shaped" when treated with detergents. True/False

False

You can use either a needle or a loop to transfer bacteria from broth to the glass slide and get the same results for staining. True/False

False

For simple staining, a well-fixed slide is important. How necessary is a well-fixed slide for negative staining? -Extremely important, just as it would be with any other staining procedure. -Somewhat important. Heat fixing is required for fixing some of the bacteria on the slide, but not for killing them. -Light heat fixing is required to ensure that the bacteria are nonmotile while viewing under oil immersion. -Heat fixing is not necessary with negative staining.

Heat fixing is not necessary with negative staining.

Why is heat fixing not required when staining capsules? The slide will air dry with the stain on it, just as is done with the negative staining. This is a simple stain. High temperatures may damage the capsule or lead to shrinkage of the cell that can be misread as a capsule. The stains do not require heat to penetrate the cell or capsule.

High temperatures may damage the capsule or lead to shrinkage of the cell that can be misread as a capsule.

What is the important difference between how stains used for simple staining and for negative staining penetrate bacterial cells? -There is no difference in how these stains penetrate the cells. -The inherent charges of the simple stains will ensure that those stains can penetrate into and remain within bacterial cells, while negative stains are repelled and will remain outside the cells. -Negative stains are darker in color and give more contrast than simple stains, which are lighter in color and have less contrast to them. -Negative stains are continuously pumped out of the cell while simple stains are pumped into the cells.

The inherent charges of the simple stains will ensure that those stains can penetrate into and remain within bacterial cells, while negative stains are repelled and will remain outside the cells.

A slide that is still cool to the touch after heat fixing may need to be heated again to correctly fix the bacteria to the slide. True/False

True

After performing a Gram stain, Gram-positive bacteria will not take the color of the counterstain. True/False

True

Debris on your slide from dead bacteria, cell remnants, or clumps of media may stand out on the negative-stain slide and confuse a student on first glance under oil immersion. True/False

True

In negative staining, the background will be stained, but the cell itself will be unstained. True/False

True

Mycobacterium tuberculosis may be stained using the acid-fast method. True/False

True

Negative staining with nigrosin will not allow for the determination of a bacterial genus or species from an unknown culture. True/False

True

Organisms cultured in a solid medium must be diluted by placing one or two loopfuls of water on the center of the slide in which the cells will be emulsified. True/False

True

Tap water is the only decolorizer required when spore staining. True/False

True

After the acid-fast stain, negative cells that have been decolorized with acid-alcohol are counterstained by the addition of __________. carbol fuchsin crystal violet safranin methylene blue

methylene blue

Spore formation by bacteria is primarily a mechanism for __________. dispersion of the bacteria infecting wounds survival of the bacteria when environmental conditions change and become unfavorable storage and weaponization of bacteria

survival of the bacteria when environmental conditions change and become unfavorable

To spread the bacterial cells and India ink across the slide, you should use _______________ enough cells and ink to flood the slide. the edge of another slide to pull the liquid across the slide. gravity to pull the liquid down the slide. the loop to smear the cells and dye around the slide.

the edge of another slide to pull the liquid across the slide.

Slides you make during lab should always be labeled with _____________ Your initials. the organism that is on the slide. your initials and the date. Nothing is required since the slide will be disposed of at the end of lab.

the organism that is on the slide.

A simple stain will allow you to determine the genus and species of an unknown bacterium. True/False

False

Acidic stains are positively charged and are attracted to the positively charged bacterial cell wall proteins. True/False

False

All bacteria have the capability of forming an endospore. True/False

False

All bacteria, whether collected from a colony on an agar plate or from a broth culture, must be diluted in a drop of water on the slide before being allowed to air-dry. True/False

False

Allowing the primary stain to sit on your slide for more than 3 minutes will have no effect on the results of your Gram stain. True/False

False

After heat fixing a slide, you notice that the bacterial smear has a slight brownish tint to it. Will the smear still be usable for staining? No; the brownish tint is the remnant of burned bacterial cells. Your best bet would be to start over with a new slide. Yes; the coloring is a normal effect of heat fixing. Yes; the browning is probably soot from the Bunsen burner. Yes; the coloring only means that some of the bacteria at the edges may have burned but the rest are probably intact.

No; the brownish tint is the remnant of burned bacterial cells. Your best bet would be to start over with a new slide.

One of the drawbacks for negative staining is that the shape of one species can look like the shape of another when the background is stained. Neither India ink nor nigrosin allow for the differentiation between bacterial species; thus, the cocci of Micrococcus luteus looks exactly like _________________________ Proteus vulgaris. Streptococcus epidermidis. Bacillus cereus. Escherichia coli.

Streptococcus epidermidis.

You are using an acid-fast staining kit that includes a wetting agent in the carbol fuchsin so you do not need to add heat. You leave the carbol fuchsin stain on for 1 minute before rinsing. What color is your Mycobacterium tuberculosis after this staining step? The bacteria would be purple. The bacteria would have no color. The bacteria would be red. The bacteria would be blue.

The bacteria would be red.

After placing bacteria on the slide, either with a drop of water or just from a drop of a broth culture, you are directed to let it air dry before heat fixing. What would happen to the bacteria on the slide if you heat fixed it before the slide had dried? The bacteria will appear bigger in size due to increased internal pressure from the boiling cellular water. The boiling action of the water would damage the bacteria and result in no bacteria being present for staining. Nothing; the bacteria will be more spread out on the slide and easier to identify. Nothing; it would just aid in drying the bacteria faster.

The boiling action of the water would damage the bacteria and result in no bacteria being present for staining.

Suppose you were performing the acid-fast procedure on E. coli using the Kinyoun method. In your haste, you forget to add Tergitol®. Assuming the rest of the experiment was performed correctly, what would you expect to see? The cells would not be stained. The cells would stain pink. The cells would appear yellow. The cells would appear blue.

The cells would appear blue.

Suppose a student performed the endospore stain on a culture of Bacillus cereus growing during the exponential phase. What color would you expect most of the cells to be after performing the spore stain? The majority of the cells would be red. The cells would stain blue. You would find free endospores that stain green. The cells would show a green spore surrounded by red.

The majority of the cells would be red.

If you fixed a sample from a week-old culture of staphylococcus aureus on a glass slide and Gram stained it, what would your results look like? -The organisms may appear to be Gram-variable; that is, some cells will appear purple, while others will appear pink. -The bacteria will retain the stains and give a false positive Gram stain. -Due to their age, the bacteria will not be stainable. -The bacteria would have their normal appearance; the age of the culture has no effect on staining.

The organisms may appear to be Gram-variable; that is, some cells will appear purple, while others will appear pink.

Under oil immersion, you notice what look like long strings of small rods. What are you looking at? The slide is made from a bacilli culture. The slide is made from a possible streptobacilli culture. The slide is a diplobacilli culture where the bacteria have dried together. More information is necessary to answer this question.

The slide is made from a possible streptobacilli culture.

After staining a bacterial slide from an unknown culture, you see under oil immersion what appear to be long, twisted cells. What are you probably looking at? Long chains of Streptococci have twisted due to the heat fixing. This is likely a slide of spirilla. Long chains of a streptobacilli species have twisted during heat fixing. This is likely a slide of spirochetes.

This is likely a slide of spirochetes.

A negative stain will not kill any bacteria on the slide, so all slides should be handled with care. True/False

True

A simple stain uses a single dye and is used to visualize the shape of bacteria and their arrangement. True/False

True

The Gram stain differentiates between bacteria based on the thickness of their peptidoglycan layer. True/False

True

The clumping of Mycobacterium cells when placed on the slide may affect the ability of carbol fuchsin to penetrate the cells and could result in a false negative for this test. True/False

True

The medical importance of the acid-fast stain is for the diagnosis of tuberculosis. True/False

True

The negatively charged dye India ink is unable to penetrate cells due to the overall negative charge inherent in cell membranes. True/False

True

The principle application of negative staining is to determine whether an organism possesses a capsule. True/False

True

A fellow student is unsure why his Gram-negative Escherichia coli is purple. Upon examining the Gram stain kit, you notice that it didn't have alcohol. What caused the bacteria to be purple instead of pink? -Without the alcohol during the decolorizing step, the crystal violet is not decolorized, so the safranin color cannot show through. -Without alcohol during the decolorizing step, the bacterial cell wall was not altered, which prevented the Gram stain's iodine and crystal violet from being rinsed away. -Alcohol shrinks the iodine crystals. Without alcohol during the decolorizing step, the iodine crystals were not shrunk and therefore couldn't be washed away. -Without alcohol during the decolorizing step, safranin will not be able to penetrate the cells.

Without alcohol during the decolorizing step, the bacterial cell wall was not altered, which prevented the Gram stain's iodine and crystal violet from being rinsed away.

If there are bacterial clumps visible in a drop of water on a glass slide, will this affect the staining of the bacteria? Yes; the clumps will hide the individual bacteria. No; the clumps will wash away later. Yes; the clumps of bacterial cells may retain the stains and lead to incorrect data being collected. The clumps will not stain.

Yes; the clumps of bacterial cells may retain the stains and lead to incorrect data being collected.

As long as you handle the slide gently during staining and use minimal water when rinsing, do you still have to heat fix? No; careful rinsing will keep the bacteria on the slide. No; you will lose a few bacteria, but there will still be enough for visualization after the stain is completed. Yes; unless fixed on the glass slide, the bacterial smear will wash away during the staining procedure. Yes; the heat fixing allows the bacteria to accept the stain.

Yes; unless fixed on the glass slide, the bacterial smear will wash away during the staining procedure.

After you have heat fixed your slide and used crystal violet to stain the bacteria, under oil immersion all you see are clumps of cocci that appeared layered on top of each other. What does this mean? -The bacteria are growing in clusters, and they are a Staphylococcus species. -You put too many bacteria on the slide. You should remake the slide and add fewer bacteria. -The rinsing step of the stain did not remove enough bacteria from the slide. -The bacteria have overgrown the broth, and that culture must be regrown.

You put too many bacteria on the slide. You should remake the slide and add fewer bacteria.

Differential staining using the acid-fast method allows for the determination of __________. bacterial shape and cell size only the cell wall composition of the bacteria bacterial shape, size, and cell wall composition whether there is mycolic acid present in the bacterial cell wall

bacterial shape, size, and cell wall composition

Malachite green with the addition of heat will penetrate and stain __________ without being washed away. only vegetative cells both free spores and endospores only free spores only endospores

both free spores and endospores

"Strepto-" is to chain as "staphylo-" is to ________ groups. coccus. cluster. sphere.

cluster.

When performing the acid-fast stain, the use of acid-alcohol as a decolorizing agent is required due to the presence of the alcohol-resistant __________. double membranes surrounding the peptidoglycan layer thickened membranes peptidoglycan layer mycolic acid waxy layer

mycolic acid waxy layer

For the Gram stain, purple means positive and pink means negative. For acid-fast staining _____________. blue means positive and red means negative the results are not based on coloration but the presence or absence of colored rings around the bacteria red means positive and blue means negative red means positive and yellow means negative

red means positive and blue means negative

The use of a negative stain will allow you to visualize and determine the following things about bacteria: _________________________ shape only. chromosomal arrangement only. shape, cell composition, and natural size. shape and natural size.

shape and natural size.