Microbiology lab, Bacterial staining

Which would be the proper amount of time required for methylene blue dye to remain on the bacterial smear during a simple stain?

30 seconds to 2 minutes

Excessive heat fixation can cause damage to which of the following structures, lowering the quality of the stained preparation?

Cell membrane Capsule Cell wall

In order to ensure that dye penetrates the spore coat and stains the endospore, which of the following is needed?

Heat

Stains are commonly used for ask of the following except

The treatment of disease

Differential stain

Uses two due to discriminate between cell types or structures

In order to withstand hostile conditions and facilitate survival, some bacteria produce resistant structures known as

endospores

Omitting the steaming in an endospore stain would result in which of the following?

endospores would not take up stain

A simple stain would be ineffective for observing which of the following organisms?

intracellular pathogens viruses

Place the steps of the endospore stain in the correct sequence.

1. Apply malachite green to smear 2. Cover smear with small piece of paper towel 3. Steam over water bath for 5 minutes remove the paper with a tweezer, then 4. Rinse 5. Apply safranin to smear, wait 30 seconds 6. Rinse and blot dry

Place the following steps of the acid-fast stain in the proper sequence

1. Stain with carbolfuchsin. Wait 5 minutes. 2. Rinse off excess primary stain 3. Decolorize with acid alcohol. Wait 1 minute. 4. Rinse with water to stop the decolorization process 5. Counterstain with methylene blue. Wait 30 seconds. 6. Rinse off excess counterstain 7. Blot dry

Which slide shows both vegetative cells and endospores?

A

Heat fixation should take place right after which step in a smear preparation?

Air drying of smeared bacterial cells.

Which of the following is not a common staining method used for visualization of bacteria?

All of the above are commonly used (simple, negative, capsule, endospore, acid-fast)

Why might it be important to perform a Gram stain on bacterial cells?

As part of the information used to choose an appropriate antibiotic treatment

Structural stain

Assists in the observation of structures external to the cell wall

Which of the images represents bacilli?

B

What is a bacterial smear?

Bacteria spread on a slide

To prepare a ....., a sample of bacteria is taken from a culture and spread onto a clean microscope slide.

Bacterial smear

Which of the following apply to bacterial smears?

Bacterial smears provide scientists with the means to look at microorganisms Preparation of bacterial smears is required for bacterial staining procedures. Preparing smears disperses bacterial cells on a slide so that individual cells can be easily visualized under the microscope.

Staining can be used to identify which of the following

Cellular arrangement Cell shape Cellular structures

Which of the images depicts a slide that has been properly strained using a simple stain procedure?

D

If organisms are washing off of the slide during rinsing of the stain, which step in the smear preparation may have been excluded?

Heat fixation Air drying

Which of the following describes the correct sequence of the Gram stain procedure?

Heat fixed slide; use of Crystal Violet, iodine, decolorizer, and safranin

Which of the following is true regarding proper heat fixation?

Heating too briefly may result in bacteria not adhering to the slide (Not heating long enough will not allow the bacteria to adhere to the slide and they may be rinsed off during staining)

............... is used to stain the endospore in the endospore staining procedure.

Malachite green

You have just performed an acid-fast stain, and all the cells appear to be blue. Which of the following could explain your results?

The bacteria are non acid-fast.

What might be the result of not allowing a slide to air dry sufficiently prior to heat fixation?

The bacteria could be serialized. The bacteria may rinse off of the slide. The shape and arrangement of the cells may be disrupted.

Which of the following describes the correct method for heat fixation?

The heating should be very brief. Passing the slide though the flame for 3-5 seconds is enough

What is the most likely reason for the poor appearance of the bacteria on this slide?

The slide was heat fixed for too long.

Staining allows for the visualization of bacterial cells because it increases contrast during microscopic imaging.

True

Which of the following would be appropriate for choosing a sample to perform a smear preparation?

Use only a small amount of bacteria in the loop Use bacteria from an isolated colony.

Which of the following structures makes it difficult to stain endospores?

endospore coat

The simple stain provides enough information about a microbe to clearly identify its species.

false

Which of the following are diseases caused by an acid-fast bacterium?

leprosy and TB

The counterstain used in the acid-fast stain is

methylene blue

Which of the following could contribute to a poorly stained slide?

-Stain was rinsed too soon -Bacteria were not "fixed" to the slide -Too many cells in smear preparation

Which of the following are characteristics of simple stains?

-Used to visualize morphology of bacteria (simple stains are frequently used to identify cellular morphology) -Used to identify cellular arrangements of bacteria (simple stains can be used to identify how bacteria are arranged. For example, clusters of cells versus chains of cells)

What are the steps in the proper preparation of a slide from a bacterial colony?

1. Label the microscope slide. 2. Add a drop of water to the center of the slide using the loop. 3. Sterilize the loop and obtain a small amount of bacteria from the colony on the agar plate. 4. Mix the bacteria into the water drop and spread out to make a thin smear. 5. Sterilize the loop again before putting it back in the rack. 6. Air dry for 5 minutes. 7. Use a clothes pin to heat fix the slide using the Bunsen burner. 8. Add methylene blue to the smear, wait 1 minute, and rinse with water. 9. Blot with paper. 10. Observe the bacterial smear under the microscope.

What are the steps to properly prepare a bacterial slide?

1. Label the slide 2. Obtain a loopful of bacteria from the tube using aseptic technique. 3. Smear the bacteria on the center of the microscopic slide. 4. Let the bacterial smear air dry for 5minutes. 5. Heat fix the slide using the Bunsen burner. 6. Add methylene blue to the smear, wait 1 minute, and rinse with water. 7. Blot the paper. 8. Observe with the microscope.

What are the proper steps in gram staining?

1. add Crystal Violet. Wait 30 seconds, then rinse with water. 2. add iodine. Wait one minute, then rinse with water. 3. add decolorizer. Wait 5 to 10 seconds, then rinse with water. 4. add safranin. Wait one minute, then rinse with water. 5. Blot with paper and move to the microscope.

If carbolfuchsin was omitted from the acid-fast stain, what color would non acid-fast cells appear?

Blue

Of these images, which smear represents the best quality for observation of bacterial cells?

C

Which of the following could be determined through simple staining of a microbial smear?

Cell morphology Cellular arrangement

The acid-fast cell wall contains large amounts of mycolic acid. Which of the following can be attributed to the presence of mycolic acid?

Chemicals cannot readily penetrate the cell wall of these cells. Mycolic acid makes the cells more difficult to stain.

If carbolfuchsin was omitted from the acid fast stain, what color would acid fast cells appear?

Clear

Place these reagents in the proper order of their use in gram staining technique

Crystal violet Grams iodine Decolorizer Safranin

The type of stain designed to show differences between various organisms or cellular structures is referred to as an........ stain, whereas a ....... stain uses only one dye to stain all cell types.

Differential Simple

Staining is commonly used microbiological technique that is used for which of the following?

Differentiation of microorganisms Identification of some bacterial structures Visualization of microorganisms

Endospores are difficult to stain due to which of the following?

Endospores have a thick spore coat. Endospores exclude dye.

What might happen if you omitted the safranin step while performing the Gram stain on a mixed culture of gram-positive and gram-negative cells?

Gram negative organisms might not be visible.

If you forget to use the decolorizer, which cell type will appear purple?

Gram positive and gram negative

What outcome could be expected if you forgot the Crystal violet while performing a Gram stain?

Gram positive and gram negative organisms would stain pink

What is used as the mordant in the gram staining technique?

Gram's iodine

What type of bacterial cell wall is composed of a thin layer of peptidoglycan and an outer membrane?

Gram-negative

Which of the following correctly describe how the Gram stain works to color different types of cells?

Gram-negative cells are decolorized and stained by the pink / red secondary stain. Gram-positive cells retain the purple dye mordant complex because of their thick layer of peptidoglycan.

What type of cell wall contains a thick layer of peptidoglycan and no outer membrane?

Gram-positive

Which of the following clinically important organisms are considered acid-fast?

Mycobacterium tuberculosis Mycobacterium leprae

Which of the following can occur due to using excessive material to create a smear preparation?

Poorly stained slide Inability to distinguish the morphology and arrangement of cells. Inability to remove excess stain on slide.

Which of the following are terms used to describe the color dyes applied in the gram staining technique?

Primary stain Counterstain

Staining techniques do NOT provide information on which of the following structures?

Ribosomes Cell membrane

..... is used to stain the vegetative cells in the endospore staining procedure.

Safranin

Which occurs first in the endospore stain?

Staining the endospore

The morphology and arrangement of the organism represented in the image would be characteristic of the genus

Streptococcus

How would you describe the morphology and arrangement of the bacteria on this image?

Stretobacilli

What will happen if the heat fixation step is omitted?

The cells may wash off of the slide.

What is stained by the counterstain in the acid-fast stain procedure?

The non-acid-fast organisms on the slide

This image depicts the results of an endospore stain performed on a smear of known endospore producing bacterial species. Which of the following could have led to the results shown here?

The smear was performed from a young culture that was not yet forming endospores. Too many organisms were used. The primary stain was omitted.

What is thought to cause gram-positive cells to retain the primary stain and the mordant complex whereas gram-negative cells are decolorized?

The thickness of the Peptidoglycan layer

Which of the following apply to endospores?

They are used for surviving adverse environmental conditions.

Why is it important to air dry the bacteria before they are immobilized by heat fixation?

This ensures the optimal preservation of the bacterial morphology.

What is the purpose of the Gram staining technique?

To characterize unknown bacteria

How do you smear a bacterial colony onto the microscope slide?

Using the loop, you disperse the bacteria into the water drop on the microscope slide

How do you smear a bacterial culture onto the microscopic glass slide?

Using the loop, you spread the bacteria onto the center of the microscope slide.

Bacterial cells that are curved rods or comma shaped are called...... whereas helical bacteria are referred to as spirilla or spirochetes.

Vibrio

There are many types of staining techniques used in microbiology. Which of the following is not a commonly used staining technique?

Wright's stain

You just performed an acid-fast stain on an acid-fast organism, yet some of the bacteria appear blue. Which of the following could explain your results?

You have a mixed population of bacteria in which some organisms are acid fast and some are not. The primary stain was not allowed to stain long enough.

What is the initial stain used in the acid-fast stain?

carbolfuchsin

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

clear

What is the primary dye used in the gram staining technique?

crystal violet

In a differential staining technique, a ..... is used to revive the primary stain from the bacteria cell.

decolorizer

Which of the following represents consequences of incorrect performance of the decolorization step?

decolorizing too briefly: false positive results decolorizing for too long: removal of Catholicism (primary stain) from all cells decolorizing for too long: false negative results

The purpose of heat fixation is to

inactivate microorganisms placed on the slide adhere the organisms to the slide

Which of the following is a significant lipid component of the cell walls of acid-fast bacteria?

mycolic acid

Gram- ....... cell walls contain a thin layer of peptidoglycan whereas Gram-...... cell walls contain a thick layer of peptidoglycan.

negative positive

This slide depicts a mixed culture of bacteria that were properly stained using the acid-fast stain. Which are more numerous, acid-fast or non acid-fast cells?

non acid fast

This slide depicts bacteria that were properly stained using the acid-fast staining technique. The bacteria appear to be what?

non acid fast

If you decolorize for more than 30 seconds, gram-positive organisms will most likely appear what in color?

pink

If you omit the grams iodine step while performing the Gram stain, gram-positive cells will most likely be what in color?

pink

gram-negative cells appear...... after gram staining whereas gram-positive cells appear.....

pink purple

If decolorization was omitted from the acid-fast stain, what color would non acid fast cells appear?

pink/red

If methylene blue was omitted from the acid-fast stain, what color would acid-fast cells appear?

pink/red

In the image, the figure on the left represents a Gram-....... Cell wall whereas the figure on the right represents a Gram-....... Cell wall.

positive; negative

The first dye applied in a differential staining technique is referred to as the _______ stain.

primary

If decolorization was omitted from the acid-fast stain, what color would acid-fast cells appear?

red

Before bacteria from a solid culture are applied to a slide, a drop of sterile water is typically added to the slide in order to

reduce the tendency of the bacteria to stick together help the bacteria spread evenly

The name of the counterstain out secondary stain, typically used in the Gram stain technique is

safranin

Spherical cells arranged in a chain are called

streptococci

This image likely depicts a Gram stained slide

true

Two clinically significant genera of bacteria that are capable of producing endospores are Clostridium and bacillus.

true

simple stain

uses only a single due to color the cells