mastering lab review

A student is observing a stained smear that was prepared from a slant culture. This micrograph shows what was observed. Assuming he placed culture on the slide during preparation of the smear, which of these errors most likely explains the observed result?

He forgot to fix the slide.

If you were staining Gram-positive bacilli and Gram-negative cocci, which of these micrographs would correctly represent the appearance of these cells after staining with crystal violet, rinsing, and blotting

If you view the slide after the crystal violet step, you will have performed a simple stain on this smear. All cells would stain purple, as you will see in this video. The decolorization step is the step that rinses the crystal violet from the Gram-negative cell wall, but not the Gram-positive. This allows the differential staining of the Gram-negative cell wall with safranin.

You INCORRECTLY perform an acid-fast stain on a smear from a mixed culture of Mycobacterium smegmatis (acid-fast bacilli) and Staphylococcus aureus (non-acid-fast cocci). Your mistake is that you forget to use carbolfuchsin during the procedure. If you viewed this slide with a microscope, what would each cell type look like?

M. smegmatis (acid-fast) cells would be colorless. S. aureus (non-acid-fast) cells would be blue. -Normally, carbolfuchsin stains acid-fast cells red, but the question indicates that this step was omitted. Subsequent use of methylene blue will color only the non-acid-fast cells because methylene blue cannot penetrate the waxy cell wall of the acid-fast cells. This leaves the acid-fast cells colorless, and the non-acid-fast cells blue.

You INCORRECTLY perform an acid-fast stain on a smear from a mixed culture of Mycobacterium smegmatis (acid-fast) cells and Staphylococcus aureus (non-acid-fast) cells. Your mistake is that you forget to use methylene blue during the procedure. If you viewed this slide with a microscope, what would each cell type look like?

M. smegmatis (acid-fast) cells would be red. S. aureus (non-acid-fast) cells would be colorless. -Methylene blue is used as the counterstain in the acid-fast staining procedure. Forgetting to use methylene blue would leave the non-acid-fast cells colorless after the acid-alcohol decolorization step. In contrast, acid-fast cells are largely stained by the primary stain (carbolfuchsin), so the lack of methylene blue does not affect their appearance.

You INCORRECTLY perform an acid-fast stain on a smear from a mixed culture of Mycobacterium smegmatis (acid-fast cells) and Staphylococcus aureus (non-acid-fast cells). Your mistake is that you forget to use acid-alcohol during the procedure. If you viewed this slide with a microscope, what would each cell type look like?

M. smegmatis cells would appear red. S. aureus cells would appear purple. -Acid-alcohol is the decolorizing agent. If you forgot to decolorize, both cell types would retain the red primary stain, carbolfuchsin. The water-soluble methylene blue counterstain would not penetrate the cell walls of the acid-fast M. smegmatis, so these cells would remain red. In contrast, methylene blue would penetrate the non-acid-fast S. aureus. With S. aureus retaining both carbolfuchsin and methylene blue, these bacteria would appear purple.

With respect to the image of a microscope specimen, refraction of light by the objective lens enhances __________.

Magnification. -Magnification refers to the apparent size of the specimen when viewed through the microscope. Light bends at the convex surfaces of the objective lens, causing light rays to diverge and radiate outward. This divergence of light creates an image of the specimen that is larger than what would normally be seen by the naked eye.

Why do acid-fast cells NOT appear blue following the addition of methylene blue?

Methylene blue does not penetrate acid-fast cell walls. -The waxy mycolic acid layer of acid-fast cell walls is impervious to water-soluble dyes such as methylene blue and even to the intense acid-alcohol decolorizing agent. Acid-fast cells retain the carbolfuchsin primary stain and remain red at the end of the acid-fast staining procedure.

These stained bacteria are from a heat-fixed smear created from a slant culture. Which of the following would produce an improved smear?

Obtain a smaller amount of culture for the smear.

As bacterial cells age, their peptidoglycan begins to break apart. What would be the effect on decolorization?

Old Gram-positive cells will be decolorized. -Normally, Gram-positive cells aren't decolorized because they have many layers of peptidoglycan that hold the crystal violet/iodine complex during decolorization. However, old Gram-positive cells have less intact peptidoglycan, and the crystal violet/iodine complex is washed out more easily. These cells are indicated by the arrow in this micrograph.

Which of the following describes a proper fixation technique?

Pass the slide two or three times through a flame. -This method gently heats the slide, which denatures the proteins within the bacteria. Much like frying an egg, this causes the bacteria to become more rigid, preserving their natural shape. It also causes the bacteria to adhere to the glass slide.

What is the purpose of carbolfuchsin in the acid-fast staining technique?

Primary stain -Carbolfuchsin is a lipid-soluble dye that can penetrate the waxy cell walls of acid-fast bacteria. This red dye is used as the primary stain in the acid-fast procedure.

After you add crystal violet to the slide, what should be your next step?

Rinse with distilled water.

While performing the acid-fast stain procedure, you add acid-alcohol drop by drop until no more stain runs from the smear. What is your next step?

Rinsing with distilled water -necessary after each reagent is used in the acid-fast procedure. The acid-alcohol decolorizing agent, if not washed off, will interfere with the subsequent methylene blue counterstain.

How does safranin affect Gram-positive cells?

Safranin penetrates the cell wall, but is masked by the darker crystal violet stain. -In the Gram-positive cell walls, most of the spaces between the molecules that make up peptidoglycan are already occupied by crystal violet/iodine complexes

Under which of the following conditions would you be most likely to prepare (and use) a smear?

Staining bacterial cells to observe under the microscope. -Smears are prepared by spreading bacteria on a microscope slide. Once the sample is spread out and stained, the characteristic shape and arrangement of the bacteria can be seen under the microscope.

Immersion oil should be cleaned off an objective with which product?

lens paper -Using lens paper helps ensure the lens won't be scratched during cleaning. In addition, lens paper is essentially lint-free; no fibers will be left on the lens that could hurt the objective's performance.

The following is a micrograph of Trichinella spiralis larvae encysted in muscle tissue. Which of the image choices demonstrates the greatest increase in resolution?

This image has the greatest resolution, because it's been properly focused. This was accomplished by adjusting the fine focus knob (the image is seen under the 10X objective). The higher resolution allows the details of the tissue and encysted larvae to be seen with greater precision.

In a compound light microscope, what is the function of the condenser?

To concentrate light on the specimen. -Light diverges through the air as it exits the light source. The condenser gathers this divergent light and concentrates it on the specimen.

Which of the following best describes aseptic technique?

To manipulate bacteria without introducing contaminants.

You performed the Gram stain. You are expecting to find purple Gram-positive cocci and pink Gram-negative bacilli. Instead, you observe purple cocci but don't seem to see any bacilli. How could you explain this?

You forgot the safranin step.

You performed the Gram stain on a smear. You are expecting to find purple Gram-positive bacilli and pink Gram-negative cocci. Instead, you observe pink bacilli and pink cocci. Which of the following is NOT a possible explanation for this unusual result?

You skipped the safranin step.

While performing the acid-fast stain procedure, you add methylene blue to your smear for the appropriate amount of time and then rinse with water. What is your next step?

blot slide dry

Arrange these steps for preparing a smear from a broth culture in their correct order. Do NOT overlap any steps.

1. Flame loop & cool. Suspend broth culture 2. Remove cap & pass tube through flame 3. Obtain loopful of culture from tube 4. Pass mouth of tube through flame & re-cap 5. Spread culture on slide. Let air-dry 6. Flame loop.

Arrange the steps of the Gram staining procedure in their correct order. Do not overlap any steps.

1. Flood slide with violet crystals and rinse 2. Flood slide with iodine and rinse 3. Decolorize and rinse 4. Flood slide with safranin and rinse 5. Blot slide 6. View slide under microscope

Arrange the steps of loop sterilization in their correct order.

1. attach burner to gas source, adjust air intake 2. turn on gas 3. light burner with striker 4. adjust air supply 5. heat loop until red 6. cool loop

Arrange the steps of the acid-fast procedure in the correct order.

1. place slide over heated beaker of water 2. apply carbolfuchsin 3. remove slide from heat and rinse 4. apply acid alcohol and rinse 5. apply methylene blue and rinse 6. blot slide and view

If you view a specimen at 40X total magnification with a 10X ocular lens, then you are viewing the specimen with what objective?

4x

You should begin viewing a specimen with what objective lens?

4x

While performing the acid-fast stain procedure, you heat your fixed bacteria with carbolfuchsin for 5 minutes, let the slide cool, and then rinse with water. What is your next step?

Acid-alcohol -decolorizing agent in the acid-fast staining procedure. Acid-alcohol removes carbolfuchsin from non-acid-fast cells.

After you add crystal violet and rinse the slide, what should be your next step?

Add Gram's iodine. -Gram's iodine serves as a mordant. Notice in the image below how it forms an insoluble complex with crystal violet. This helps the crystal violet remain in the gram-positive cells during decolorization.

What is the best procedure for decolorization?

Add decolorizing agent until run-off is clear. -This method allows the decolorizing agent to dissolve the outer membrane of Gram-negative cells and rinse out the crystal violet from the thin layers of peptidoglycan. This causes the run-off to be purple. When that purple color ceases to appear in the run-off, decolorization should be stopped so as not to also cause the Gram-positive cells to lose their purple color. Watch the video to review the process of decolorization.

After Gram's iodine is added and then rinsed, what is the next step in the Gram stain procedure?

Add decolorizing agent. -The decolorizing step removes the crystal violet from the gram-negative cell walls, but not the gram-positive walls. Watch the video to review the addition of decolorizing agent.

While looking through a microscope at 400X total magnification, you see this image. To improve the quality of this image, what should you do?

Adjust the condenser aperture diaphragm. -The specimen is in focus, but contrast is poor. You can enhance contrast by moving the aperture diaphragm toward the closed position. This restricts the angles at which light strikes the specimen and thereby tends to eliminate the bright, washed-out appearance of the image. This is a better alternative than reducing lamp intensity. Reducing the lamp intensity would make the image less bright, but contrast would remain poor.

You have just rotated the 40X objective into position after viewing the specimen with the 10X objective. What would your next step most likely be?

Adjust the fine focus knob. -After changing objectives, you should always check to make sure your image is as well focused as possible. Your objectives are most likely parfocal, so not much adjustment should be necessary. The working distance between the 40X objective and the specimen is very small, so adjusting the fine focus (rather than the coarse focus) will help avoid smashing the objective into the slide.

If you FORGOT to do the decolorizing step, what colors would the Gram-positive and Gram-negative cells be when viewed at the end of the procedure?

All would be purple.

Transfer to an agar deep involves a slightly different procedure from transfer to an agar slant. Which of the following steps is unique to inoculating an agar deep?

An inoculating needle is used to stab the inoculum into the agar. -An inoculating needle is used for inoculating an agar deep. The agar deep medium is stabbed, not streaked. This technique is used in some biochemical tests to place the bacteria within the medium, rather than on the surface.

Smear preparation from a slant culture differs from smear preparation from a broth culture in what way?

Bacteria from a slant culture must be mixed with a loopful of water. -Because the slant culture is solid, not liquid, water is required to make the smear a uniform thickness. This video demonstrates how to add water to your slide.

What error in aseptic technique most likely caused this contaminant colony to grow on this agar slant?

Failure to flame the mouth of tube. -This yellow colony most likely originated from an airborne bacterium that landed at the top of the agar. Flaming the mouth of the tube prevents airborne bacteria from entering the tube while it is uncapped. Airborne bacteria may fall anywhere on this slant and grow to form a colony.

After you've removed a loopful of broth culture from the culture tube, what's your next step in preparing a smear?

Flame the opening of the tube -You should flame the mouth of the test tube before and immediately after removing a loopful of culture, as shown in this video. This helps prevent airborne contaminants from entering the culture.

You're preparing a smear from a broth culture. You've labeled a clean slide. You've flamed the inoculating loop and let it cool. What's your next step?

Flick the culture tube. -In preparing a smear from broth, you want to be sure enough cells make it onto your smear. Cells in broth cultures tend to settle to the bottom of the tube. This leaves very few cells in the top layer, where you'll be inserting the loop. Flicking the tube resuspends the cells evenly in the broth.

Sort each of these characteristics as either belonging to Gram-positive or Gram-negative bacteria.

Gram-positive bacteria -Crystal violet retained during decolorization -Many layers of peptidoglycan -Stain purple by Gram stain Gram-negative bacteria -One or two layers of peptidoglycan -Crystal violet NOT retained during decolorization -Stain pink/red by Gram stain

Assuming the Gram stain smear was blotted and observed following the decolorization step, which micrograph correctly illustrates how Gram-positive cocci and Gram-negative bacilli would appear?

The Gram-positive cells have thick walls, and they will retain the purple crystal violet stain if decolorization was carried out correctly. The Gram-negative cells will be decolorized and are colorless. Watch the video to review the effect of decolorization on both types of cells.

A smear from a mixed culture of bacilli and cocci is stained with the acid-fast procedure. This is an image of the stained smear at 1000X total magnification. Which of the bacteria, if any, are acid-fast?

The bacilli are acid-fast. -The acid-fast staining procedure stains the acid-fast bacteria red, because those bacteria retain the carbolfuchsin primary stain. The bacilli in the image are red, so they're acid-fast. In contrast, blue cocci are non-acid-fast.

A student prepares a smear from a broth culture; air-dries it, and fixes it. He then remembers that he forgot to flick the broth culture tube before removing the bacteria. What is the most likely consequence of this error?

The bacteria may be difficult to find on the slide

What is an inoculum?

The bacteria transferred to a new medium. -The bacteria that are transferred during inoculation are called the inoculum. The new sterile medium receives, or is inoculated with, the inoculum. The inoculum, it is hoped, consists only of the desired species of bacteria and does not include any contaminants.

This video shows a student incorrectly obtaining an inoculum from a broth culture. Prior to the start of the video, the inoculating loop was held in the flame until the wire part glowed orange. What is his error in aseptic technique?

The cap of the tube was not handled correctly. -The student placed the cap from the tube on the bench top. This may have contaminated the cap. Once the cap is placed back on the tube, the bacteria on the cap may contaminate the culture within the tube. Always hold caps in the hand that holds the loop. This minimizes the chance that environmental microbes might contaminate the cap.

What happens to the Gram-positive cell wall during decolorization?

The decolorizing agent dehydrates the peptidoglycan -Removing water from (or dehydrating) the peptidoglycan allows the decolorizing agent to shrink the spaces through which the crystal violet-iodine complexes might be able to pass. This makes it more difficult for the purple stain to be removed. Watch the video to view the effect of decolorization on the Gram-positive wall below.

What happens to the Gram-negative cell wall during decolorization?

The decolorizing agent dissolves the outer membrane. -The decolorizing agent dissolves the outer membrane of Gram-negative cells. This allows the decolorizing agent to penetrate the thin layers of peptidoglycan and rinse away the crystal violet/iodine complexes. Watch the video to review the effect of decolorization on the Gram-negative cell wall.

A student properly flames her loop but then forgets to let it cool. Instead, she immediately inserts the hot loop into her broth culture. What is the most important issue associated with this mistake?

The hot loop may create aerosols when it touches the culture. -When the hot loop touches the liquid broth culture, it will cause some of the broth (and bacteria) to boil briefly, creating a bacteria-containing aerosol. These airborne bacteria could enter the student's respiratory tract or land on her skin. If you hear a hissing sound when you place the loop into the broth culture, you'll know you haven't cooled the loop sufficiently.

A student properly flames her loop but then forgets to let it cool. Instead, she immediately inserts the hot loop into her broth culture. What is the most important issue associated with this mistake?

The hot loop may create aerosols when it touches the culture. -When the hot loop touches the liquid broth culture, it will cause some of the broth (and bacteria) to briefly boil, creating a bacteria-containing aerosol. These airborne bacteria could enter the student's respiratory tract or land on her skin.

This video shows a student incorrectly obtaining bacteria from a colony on a Petri plate. What has he done incorrectly?

The lid is too far away from the plate. -The lid should partially cover the plate while the student is removing inoculum, as shown in this photograph. This technique prevents airborne microbes from contaminating the surface of the agar.

A student inoculated an agar slant with inoculum from a colony on a Petri plate. The slant was incubated at an appropriate growth temperature for this species for 2 days. The results are shown

The loop dug into the agar during inoculation. -Notice the small gouge in the agar at the bottom of the slant, as indicated by the arrow in this photograph. The student most likely gouged the agar with the loop, depositing a good portion of the inoculating cells under the surface of the agar. After the gouge, very few cells were left on the loop to inoculate the rest of the slant surface.

If a student forgets to flame the mouth of a broth culture tube before transferring culture to it, what might result?

The medium in the tube might become contaminated. -Heating the mouth of the test tube creates an air current that helps prevents airborne bacteria from falling into the tube while the tube's cap is off. If airborne bacteria do fall into the tube, they will contaminate the medium.

A student prepared a smear from a broth culture, air-dried it, and heat-fixed it. This was followed by a staining procedure that colored the cells purple. Which of the following errors most likely caused the poor appearance the student's micrograph, shown here?

The slide was not cleaned completely.

Which of the following is the most appropriate way to carry a microscope?

The student correctly grasps the microscope arm with one hand and supports the base with the other hand. This minimizes the risk of dropping the heavy microscope during transport.

This video shows a student improperly sterilizing an inoculating loop. What is the student's mistake?

The wire portion of the instrument was not adequately heated. -To be properly sterilized, both the wire and the loop portions of the inoculating loop must be heated until red-hot. The nonglowing wire could still contain live bacteria that might contaminate the student's cultures.

When you look through the ocular lenses, you see this image of the endospore-forming bacteria Bacillus anthracis under oil. Which of the following qualities is most lacking in this image?

contrast -Contrast, which is lacking in this image, refers to the difference in light intensity between the specimen's features and between the specimen and the background. You can enhance contrast by adjusting the lamp intensity dial and the aperture diaphragm on the condenser.

What is the role of acid-alcohol in the acid-fast staining technique?

decolorizing agent