ch 10-14 microlab

Your capsule stain is complete and correct. Which of the following statements would apply to the image you see?Select all that apply.

- A.The bacterial cells are stained with crystal violet. - The background is stained dark with nigrosin. - You can observe encapsulated organisms in this field.

Why is the aseptic technique important during laboratory activities like smear preparation?

- Aseptic technique helps prevent accidental infection of the handler - proper aseptic technique aids in keeping microbes from spreading to other surfaces, where they might be contacted by others in the lab - the proper aseptic technique helps prevent contamination from being introduced into your sample during preparation

Please order the following choices to reflect the appropriate sequence of materials used in the Gram staining procedure.

- C.V - Safranin - Gram's Iodine - Alcohol

Select the statements that represent goals in correct smear preparation.

- The cells must be heat fixed to the slide to prevent removal when stain is washed off the slide.The cells must be heat fixed to the slide to prevent removal when stain is washed off the slide. Correct - It is important to minimize any artifacts or distortion of the cells for accurate viewing after staining. - Smears must be thin to allow for observation of single cells.

sample of pond water, looking for living bacteria activity such as observing for mobility

- This Image shows a cell arrangement of streptococci - This image likely deserves a Gram stain bacterial smear - This image shows a cell morphology of Cocci

You observe this slide under the microscope. It was prepared with a negative stain used to visualize capsules.

- This bacterium is not encapsulated. - The capsules are dark purple. - The background of the slide is white, indicating that the slide is densely covered with cells.

Your Gram stain is complete and correct. Which of the following statements would apply to the image you see?Select all that apply

- You can observe gram-negative bacilli on this field. - You can observe gram-positive cocci on this field.

Put the following steps in the correct order for performing a smear preparation

- appropriately label slide - apply bacteria to the slide using aseptic technique - air drying - heat fixation - apply staining method

tools used in Gram staining

- appropriately label slide - apply bacteria to the slide using aseptic technique - air drying - heat fixation - apply staining method

capsule staining is different from other classi. microbiology staining protocols because

- capsules are specialized microorganism structures and require diff techniques to visualize - does not use heat fixation, as other staining protocols do, to preserve capsule structure - a neagtive-staining protocol, compared to many other staining protocols that are positive staining

which of the following is/are correct regarding your stain

- capsules are visible - counterstaining with crystal violet was likely omitted

Move the reagents into the figure to illustrate the correct sequence when conducting a Gram stain.

- crystal violet - gram's iodine - decolorizer - safranin

examples of dyes used in technique: positive staining

- crystal violet - methylene blue

you have just preformed an acid-fast staining and gotten the follwoing result

- decolorization step was omitted - the sample contains only acid-fast bacteria

Move the descriptions into the correct boxes to assess your understanding of the differential staining techniques: gram stain

- differentiates cells based on thickness of peptidoglycan layer - does not require heat to be used in staining procedure - result is purple and red/pink cells - a chemical is used as a mordant

Move the descriptions into the correct boxes to assess your understanding of the differential staining techniques: acid-fast stain

- differentiates cells with high lipid content in cell wall - important diagnostic tool in Mycobacterium infections - uses carbolfuchsin, acid-alcohol, and methylene blue

Now that you have seen the difference in the smear preparation after simple methylene blue staining, which of the following is correct regarding your smear now?

- helps show the intact appearance and localization of the cells, compared with no staining -helps visualize the shape and arrangements of bacterial cells that were difficult without stain

endospores

- highly resistant to heat and chemicals - stain with mala hite green - require autoclave for destruction - formed when conditions become unfavorable - heat is mordant to facilitate staining of those

As you create your smear from a sample on solid media, it is important to use an appropriate amount of material and evenly disperse the organisms onto the surface of the slide. Which of the following can occur due to using excessive material as you create a smear preparation?

- it will be difficult to apply the stain correctly to the slide for visualization - it will be difficult to distinguish bacterial cell morphology and arrangements of cells on the smear sample - it will be difficult to remove excess dye stain in later steps of the smear procedure

examples of dyes used in technique: negative staining

- nigrosin - india ink

what is the purpose of adding a strip of paper to the spore stain procedure

- paper keeps the dye from evaporating too quickly during the heating step - the paper allows for more contact time between the dye and spores

Based on what you learned, for which of the following samples would you skip the heat fixation step?

- sample of pond water, looking for living bacteria activity such as observing for mobility

Vegetative cells

- stain with safranin - higher water content - actively metabolizing

you have just preformed an acid-fast staining and gotten the following result

- the acid alcohol step was performed for too long - the bacteria are non acid-fast - the carbolfuchsin step was omitted

which of the following is/are correct regarding acid-fast staining?

- the acid-staining procedure would be categorized as a differential stain - the correct of an acid-fast stain is primary staining, iodine fixation, decolorizing, counterstaining

this image shows the results of a spore stain preformed on a known endospore producing stain of bacteria. What statement below would correctly explain this image

- the heating step was not preformed to allow dye to penetrate spore coats

What is the purpose of adding a strip of paper to the spore stain procedure?

- the paper keeps the dye from evaporating too quickly during the heating step - the paper allows for more contact time between the dye and spores

this image depicts the results of a spore stain performed on a smea producing bacterial species. Which of the following could have led to the results shown here

- the smear was preformed correctly, but the culture conditions did not encourage spore formation - too many organisms were used - the malachite green step was skipped

Based on what you understood from the video, which of the following statements are correct? Select all that apply.

- this image could be representative of Bacillus species - shows primarily endospores - shows endospores are green in color

Move the descriptions into the correct boxes to assess your understanding of the differential staining techniques: spore stain

- uses malachite green - distinguishes between active metabolic cells and dormant structures

Your acid-fast stain is complete and correct. Which of the following statements would apply to the image you see?Select all that apply.

- you can observe acid-fast bacterial cells o this field - you can observe o-acid fast bacterial cells o this field

Your spore stain is complete and correct. Which of the following statements would apply to the image you see?

- you can observe spores on this field - you can observe vegetative bacteria on this field

Gram-positive organisms might incorrectly stain pink.

- you forgot the crystal violet step - you forgot the Gram's iodine - you applied the decolorizer for too long

Move the images of bacterial cells into the chart to illustrate the color of gram-positive and gram-negative cells after each step of the Gram stain process.: Gram-Negative

None (heat-fixed cells): clear crystal violet (30 sec): purple gram's iodine (1 min): purple Ethyl Alcohol: (5-15 sec) clear safranin (1 min): pink

Move the images of bacterial cells into the chart to illustrate the color of gram-positive and gram-negative cells after each step of the Gram stain process.: Gram-Positive

None (heat-fixed cells): clear crystal violet (30 sec): purple gram's iodine (1 min): purple Ethyl Alcohol: (5-15 sec) purple safranin (1 min): purple

You make a smear of E. coli and then simple stain the slide with methylene blue. If you failed to use correct aseptic technique by not sterilizing the loop before picking up the bacterial sample for the smear, what results may be seen under the microscope?

There may be cells of different shapes, sizes, and arrangements.

What is heat fixation?

Passing the slide through the flame of a burner several times after the smear has completely dried.

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

Water is applied to the slide before emulsifying cells from a solid medium.

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

air-dried.

When staining a known acid fast organism, in the presence of acid alcohol decolorizer, carbolfuchsin dye should in the sampled bacteria.

be retained

What is the purpose of skipping the heat fixation step in the capsule stain procedure?

capsules are fragile and ca be destroyed with heating

The primary stain, carbolfuchsin, binds to in the sampled bacteria.

cell wall lipids. such as mycolic acid

If methylene blue was omitted from the acid-fast stain, non-acid-fast cells would be _____ at the end of the staining process.

clear

If safranin was omitted from the endospore stain, what color would the endospores appear?

clear (colorless)

appearance of cell: negative staining

clear/colorless

appearance of cell: positive staining

colored by dye

The Gram stain is an example of a _______ staining procedure, which takes advantage of the fact that cells or parts of cells react differently and can be distinguished by the use of two different dyes.

differential

It is important that the smear is thick in order to ensure that the stain will be retained on the slide. (T or F)

false

It is important that the smear is thick in order to ensure that the stain will be retained on the slide. (T or F)

false

This is how your smear preparation looks in the microscope immediately after completion. True or False: This image, as it appears, is able to be interpreted for information about the organism(s) on the slide.

false

Which of the following is a layer found outside of the bacterial cell wall and membrane consisting of repeating sugar units and/or proteins that is resistant to staining.

glycocalyx, capsule, and slime

A _______ stain is one that colors the background surrounding the cell, leaving the cell itself unstained.

negative

background appearance: positive staining

net stained

Please choose the answer that best fills in the blanks of this sentence in the correct order. Gram-________ cells stain purple whereas gram-_______ cells stain pink or red when using the Gram stain technique.

positive; negative

The rationale for using methylene blue as a counterstain in the acid-fast protocol is to

provide contrasting color to non-acid-fast bacterial cells

Based on what you understand about the acid-fast staining process, acid-fast cells will appear ____, whereas non-acid fast cells will appear _____

red; blue

After correct Gram staining, the gram-negative cells will appear ___ , whereas the gram-positive cells appear ____.

red; purple

background appearance: negative staining

stained black/dark

you are observing streptococcus pneumoniae on a slide that has been stained using the capsular staining method. However, you do not see capsules surrounding the cells as you expected. what is likely for this reason

the slide was heat fixed, which can shrink or destroy capsules

The capsule can be considered a virulence factor and microorganisms containing one are often pathogenic. (T or F)

true

This is how your smear preparation looks in the microscope after simple staining with methylene blue. True or False: This image, as it appears, is able to be interpreted for information about the organism(s) on the slide

true

Creating a bacterial smear from solid media, such as an agar plate or slant, often feels more tangible than creating a smear from liquid media. Which of the following is appropriate when creating a smear in this fashion?

using the loop, you should disperse the bacteria sample material into water drop on the slide

you may not be able to view any organisms on the slide

you did not heat fix the smear correctly

Gram-negative organisms might incorrectly stain purple.

you forgot the decolorization step

Gram-negative organisms might not be visible.

you forgot to apply safranin

Match the following reagents with their role in the Gram stain procedure.

Counterstain- Safranin Primary Stain- Crystal Violet Decolorizer- alcohol/acetone Mordant- Gram's iodine

An acid-fast staining method was performed on a bacterial smear using the dyes carbolfuchsin and methylene blue. The resulting stained slide contained only cells that appeared blue when visualized using a microscope. Which of the following could explain why only blue cells were present? 1. No acid-fast bacteria are present on the slide 2. Methylene blue was not used 3. Carbolfuchsin was not used 4. Acid-alcohol was not used

1 and 3

Place the steps of the spore stain in the correct sequence

1. Apply malachite green and a small strip of paper covering to a prepared smear Steam over heat source Rinse Apply safranin to smear Rinse and blot dry

Order the reagents used in the acid-fast staining procedure.

1. Carbolfuchsin 2. Acid-alcohol 3. Methylene blue

Order these statements to indicate correct aseptic procedure for removing organisms from a culture for application to a slide.

1. Heat inoculating loop and wire to red-hot. 2. Remove the cap of the culture tube and flame the mouth of the tube. 3. Remove a loopful of organisms from the culture. 4. Flame the mouth of the culture tube. 5. Return the cap to the culture tube. 6. Place organisms onto the slide in the center of the target circle. 7. Flame the loop red-hot before setting aside.

Correct Order of Steps to Perform an Acid-Fast Stain

1. Stain with carbolfuchsin 2. Rinse off excess primary stain 3. Add acid alcohol reagent 4. Rinse with water to stop the decolorization process 5. Stain with methylene blue 6. Rinse off excess counterstain 7. Blot dry 9. Perform microscopy

Place basic steps of capsule staining in correct sequence

1. apply primary stain (india ink or nigrosin) to a clean slide 2. use innoculating loop to aseptially mix bacterial sample with primary stain 3. create a thin smear by dragging a clean slide through the sample 4. allow sample to throughly air dry 5. apply secondary stain (crystal violet) to smear 6. rinse with water and prepare for microscopy

Order steps of a id-fast staining

1. stain with carbolfuchsin 2. rinse off excess primary stain 3. add acid alcohol reagent 4. rinse with water to stop the decolorization process 5. stain with methylene blue 6. rinse off excess counterstain 7. blt dry 8. preform microscopy

Acid-fast cells definition

Cells which contain a thick layer of mycolic acid or cord factor

Gram-positive cells definition

Cells which contain a thick layer of peptidoglycan and teichoic acids

Gram-negative cells definition

Cells which contain both an inner and outer membrane as well as a thin layer of peptidoglyan

You are Gram staining a known sample that contains BOTH gram-positive and gram-negative bacteria. Are you complete the staining, you realize that you did not perform the safranin step. Which of the following statements would correctly describe what you would see?

Graham negative organisms might not be visible

You are Gram staining a known sample that contains BOTH gram-positive and gram-negative bacteria. After you complete the staining, you realize that you performed the decolorizer step for 10 minutes instead of 10 seconds! Which of the following statements would correctly describe what you would see?

Gram-positive organisms my incorrectly staying red

You collected a sample from a patient with a suspected STI. After Gram staining, you observed this under the microscope. What can you conclude about this microbial agent?

It is gram-negative.

Please choose the answer that best completes the blanks of this sentence in the correct order. _______ is used to stain the endospore, and _______ is the stain used to stain the vegetative cell in spore staining.

Malachite green; safranin

You are Gram staining a known sample that contains BOTH gram- positive and gram-negative bacteria. Are you complete the staining, you observe the following. Which of the following statements would you apply to the image you see?

This image could be explained by the omission of the grams iodine step, causing all cells to appear gram-negative

You are Gram staining a known sample that contains BOTH gram-positive and gram-negative bacteria. After you complete the staining, you realize that you did not use the alcohol bottle at all. Which cells would appear purple

gram-positive cells