Types of stains

Which of the following is an example of a differential stain?

  1. Capsule Stain
  2. Spore Stain
  3. Gram Stain
  4. Flagella Stain

What is the primary stain in the gram stain?

  1. Alcohol
  2. Iodine
  3. Crystal violet
  4. Saffranin

What is the purpose of “fixing” a slide?

  1. To keep the bacteria alive to see it under the microscope
  2. To enlarge the bacteria to see it easier
  3. To attach a specimen (like cheek cells or bacteria) to the slide and kill the microbes
  4. To increase the pH of the specimen

With respect to timing the most critical part of the Gram stain is the application of:

a. crystal violet.

b. methylene blue.

c. iodine.

d. safranin.

e. alcohol

Which of the following is NOT TRUE in regards to the purpose of a mordant in the gram stain?

  1. It allows the primary stain to react chemically with the cell
  2. It keeps the crystal violet from being washed out of by the alcohol
  3. It makes the bacteria (or cell) stick to the slide better
  4. It forms a complex with crystal violet and peptidoglycan in the cell wall of bacteria

What color is a Gram positive organism after decolorizer is used?

a)Purple

b)Pink

c)No color

What color is a Gram positive organism after the counterstain is added?

a)Purple

b)Pink

c)No color

What color is a Gram negative organism before the primary stain is added?

a)Purple

b)Pink

c)No color

What color is a Gram negative organism after the primary stain is added?

a)Purple

b)Pink

c)No color

What color is a Gram negative organism after decolorizer is used?

a)Purple

b)Pink

c)No color

What color is a Gram negative organism after the counterstain is added?

a)Purple

b)Pink

c)No color

If you did a Gram stain on a culture that came out looking Gram negative even though you knew it should be Gram positive, which of the following could have caused the misleading results?

a. the culture was too old

b. you forgot to add ethanol

c. you used too much alcohol during decolorization

d. the organism is Acid fast +

e. two of these are possible reasons for the wrong Gram reaction.

What is the mordant for the Gram stain technique?

a)Crystal violet

b)Malachite green

c)Iodine

d)Heat

e)Safranin

What is the decolorizer for the Gram stain technique?

a)Alcohol

b)Acid alcohol

c)Water

What is the counter-stain for the Gram stain technique?

a)Crystal violet

b)Carbolfushin

c)Malachite green

d)Iodine

e)Safranin

What color are endospores after a Gram stain?

a)Green

b)Red

c)Colorless

You are given a culture of E. coli. You do a successful streak for isolation. You select a single colony and Gram stain it. You see equal numbers of cocci and bacilli, all Gram negative. The most reasonable conclusion is that ______.

a. you have contamination

b. you have a pure culture with some looking like cocci because of age

c. you did the gram stain incorrectly

d. you have a coccus that can also look like a rod

e. two of these are reasonable conclusions

List the reagents used in the Gram stain.

PRIMARY STAIN / Crystal violet
Color of Gram positive after primary stain / Purple
Color of Gram negative after primary stain / Purple
MORDANT / Gram’s iodine
DECOLORIZER / Alcohol
COUNTER STAIN / Safranin
Color of Gram positive after counter stain / Purple
Color of Gram negative after counterstain / pink

What is the function of a mordant?

Forms an insoluble complex with the primary stain in cells that are positive for the primary stain.

What layer of the cell wall does a primary stain color?

Peptidoglycan layer

Explain what is happening to both Gram positive and Gram negative cells during the Gram stain procedure.

Gram positive bacteria have more peptidoglycan in their cell wall, so they retain the crystal violet/iodine complex after decolonization with alcohol, and they appear purple.

Gram negative organisms have less peptidoglycan in their cell wall, plus they have an outer membrane, so the crystal violet/iodine complex washes away when alcohol is applied. Therefore, the Gram negative organisms must be counterstained with a red dye to be visualized. This red dye is not seen through the darker purple in Gram positive organisms.

How do gram positive and gram negative bacteria differ in their cell wall structure?

Gram positive have 90% peptidoglycan, Gram negative have 10%

How does culture age affect the results of a Gram stain?

Older cultures can’t hold the purple color as well

Which of the following is the most common type of stain?

  1. Iodine stain
  2. Gram stain
  3. Acid fast stain
  4. Capsule stain

True or false: The cell wall of a gram negative organism has many lipids

  1. True
  2. False

Which of the following is NOT a characteristic of gram positive bacteria:

  1. Purple stain coloration
  2. Thick peptidoglycan
  3. Very little lipids
  4. Lots of lipids

Which of the following is NOT a step in the gram stain procedure:

  1. Mordant
  2. Primary stain
  3. Counter stain
  4. Sealant
  5. Decolorizer

True or false: Gram positive bacteria have thick peptidoglycan

  1. True
  2. False

When you do a Gram stain, what would happen if you use an old culture?

  1. Cells will not hold a stain
  2. Gram positive cells would be purple and pink because some peptidoglycan has broken down
  3. Gram positive cells would be pink because they still contain peptidoglycan
  4. All cells will turn dark brown because no peptidoglycan remains

Match the following Gram stains. Each answer is only used ONCE.

  1. Alcohol or acetoneDecolorizer
  2. SafraninCounterstain
  3. Crystal violetPrimary stain
  4. IodineMordant

What color does a bacteria stain if it is Gram positive?

  1. Green
  2. Violet
  3. Yellow
  4. Black

What color will bacteria stain if it us Gram negative?

  1. Orange
  2. Brown
  3. Pink
  4. Black

If a bacterium is positive for the acid-fast stain, what does this indicate?

  1. It does not have mycolic acid in the cell wall
  2. It has mycolic acid in the cell wall
  3. It has peptidoglycan in the cell membrane
  4. It does not have peptidoglycan in the cell membrane

What is the primary stain in acid-fast stain?

  1. Acetone
  2. Carbolfuschin
  3. Mycolic acid
  4. Safranin

If a bacterium is gram stain positive, it will appear as a ______color; gram stain negative will indicate a ______color.

  1. blue, red
  2. pink, blue
  3. red, pink
  4. violet, pink

What is the first step in preparing a slide for staining?

  1. Wash the slide
  2. “Fix” the slide by passing it over a flame a few times
  3. Place the microbes on the slide
  4. Place 2-3 drops of acetone alcohol over the specimen on the slide

The simplest way to make a slide of bacteria is to prepare a wet mount. The main drawback to this method is that______

a. bacteria are generally colorless and transparent

b. all bacteria are pathogenic

c. live bacteria move around too quickly to be observed

d. reverse osmosis occurs, killing and distorting bacteria

e. cover slips are too expensive to use regularly

Before heat fixation, a wet smear must first be

a)Air-dried

b)Blotted dry

c)Rinsed with water

d)Stained with a basic dye

e)None of the answers are correct

What is the purpose of a smear prep?

To prepare an organism for staining

What are two problems that arise when a slide is not heat fixed properly?

a) burn the cells off

b)cells don’t stick

What causes a stain to adhere to bacterial cells?

Charged ions in the dye are attracted to the charges in the bacteria

What is the purpose of using a pure culture technique?

To separate a mixture of organisms into pure cultures

What does TSA stand for?

Triptic soy agar

The purpose of heat-fixing a bacterial smear slide is to:

a. get the bacteria to stick to the surface of the slide so they don’t wash off during

the later staining.

b. kill and preserve the bacteria.

c. break up clumps of bacteria by evenly melting them across the slide.

d. a & b

e. a, b, & c

ETOH is

  1. A type of gram stain
  2. An amino acid
  3. Ethanol alcohol
  4. A type of bacteria resistant to stain

What procedure is done when you “Fix” a slide?

  1. Pass it through a flame a few times.
  2. Clean it with Lysol
  3. Glue pieces together
  4. Put it away

What is the purpose of “Fixing” a slide?

  1. So that you can see it.
  2. To attach the cells (or the bacteria) to the slide and kill the microbes.
  3. To fix the slide so it is not broken.
  4. To get it ready for the next class.

If an ion has a positive charge it is called a:

  1. Cation
  2. Anion

If an ion has a negative charge it is called a:

  1. Cation
  2. Anion

A simple stain uses:

  1. One stain
  2. Two stains
  3. Three stains
  4. Four stains

What is the pH of a cationdye?

  1. an acidic dye (pH lower than 7)
  2. a basic dye (pH higher than 7)
  3. doesn’t create a dye

An acidic dye is also called a negative stain?

  1. True
  2. False

Smears from a slant are:

a. placed directly onto a clean slide and spread into a dime-sized area.

b. placed onto a drop of water on a clean slide and spread into a dime sized area.

c. made by using as much culture as can be lifted with a loop to insure a good

smear.

d. made by using a sample from a broth culture.

e. Both B and C are correct

Which of the following morphological shapes classify a round bacterium?

a)Coccus

b)Bacillis

c)Helical

A rod-shaped bacterium that occurs in strands is which classification?

a)Streptococcus

b)Streptobacillus

c)Spirochete

A bacterial colony shaped like a cluster of grapes is which classification?

a)Streptococcus

b)Streptobacillus

c)Spirochete

What are the two different types of basic stains?

  1. negative and positive
  2. simple and differential
  3. colored or non colored
  1. cation and anion

What does a negative stain do?

  1. Stains the bacteria
  2. Creates a more negative charge
  3. Stains the background instead of the bacteria
  4. Stains the cell membrane of the bacteria

What is the purpose of using a negative stain?

a)Determine the size of an organism

b)Determine the shape of an organism

c)Counting the number of cells present

d)A and B are correct

e)A, B, and C are correct

Which of the following are true about negative stains?

a)It is acidic and has a positive charge

b)It is acidic and has a negative charge

c)It has a basic pH and a positive charge

d)It has a basic pH and a negative charge

A stain used to determine the true cell morphology and size is the:

a. negative stain.b. simple stain.c. acid fast stain.d. Gram stain.

Dyes for bacteria contain which of the following?

a)Positively charged chromophores that are attracted to the negative charge of bacteria

b)Positively charged chromophores that are attracted to the positive charge of bacteria

c)Negatively charged chromophores that are attracted to the negative charge of bacteria

d)Negatively charged chromophores that are attracted to the positive charge of bacteria

Which of the following are negative stains?

a)Nigrosin

b)India ink

c)Congo red

d)Carbolfuschsin red

e)(a), (b), and (c) are correct

f)All of those are negative stains

What is the function of a spore stain? To visualize bacteria that make spores

What is the function of a capsule stain? To visualize bacteria that have a capsule

What is the function of a flagellar stain? To visualize bacterial flagella

Which one is not part of the immunocompromised patients?

A. Teenagers

B. Elderly people

C. Infants

What happens when an environment becomes too harsh too survive for bacteria?

A. Make a defense mechanism

B. Make an endospore

C. They die

What happens to an endospore when the environment improves?

A. It germinates

B. Try to take care of what was damaged

What can kill a spore?

A. Alcohols

B. Detergent

C. Sterilization

What organisms make spores?

  1. Gram positive rods
  2. Gram positive cocci
  3. Gram negative rods
  4. Gram negative cocci
  5. Acid fast bacteria
  6. All of the above

What does a capsule resist?

A. Diffusion

B. Phagocytosis

C. Osmosis

What does the capsule stain color?

A. The inner cell

B. The outer membrane

C. The background

What is the purpose of the capsule stain?

A. Assist with identification of the organism

B. Cure the disease

C. Find the disease

What is the definition of a flagellum?

A. Small object that allows movement of material inside the cell

B. Whip-like tail that helps the cell move

Can the tail of a flagella be easily seen?

A. Yes

B. Sometimes

C. No

Basic dyes are used to stain bacteria because they have:

a. positively charged chromophores that are attracted to the negative charge of bacteria.

b. negatively charged chromophores that are attracted to the positive charge of bacteria.

c. positively charged chromophores that are attracted to the positive charge of bacteria.

d. negatively charged chromophores that are attracted to the negative charge of bacteria.

e. none of the above

Which of the following are basic dyes?

a)Methylene blue

b)Crystal violet

c)Eosin

d)(a) and (b)

e)All of the above

Which of the following are acidic dyes?

a)Methylene blue

b)Crystal violet

c)Eosin

d)All of the above

Which species of bacteria produce endospores?

a. Bacillus anthracis.

b. Streptococcusmutans

c. Mycobacteriumsmegmatis

d. Staphylococcus aureus

e. two of these are spore formers

What is the mordant for the acid-fast technique?

a)Crystal violet

b)Malachite green

c)Iodine

d)Heat

e)Safranin

At what temperature is a motility stab incubated?

a)Body temperature

b)Room temperature

c)50 °C

What external structure makes Mycobacterium different from other organisms?

a)It has a capsule

b)It has mycolic acid in the cell wall

c)It has a larger amount of peptidoglycan than most

Endospores are produced by bacteria in the genus

a)Bacillus

b)Clostridium

c)Mycobacterium

d)Both (a) and (b)

e)Both (a) and (c)

Acid-fast staining is useful for identifying the causative agent of

a)Leprosy

b)Tetanus

c)Tuberculosis

d)Both (a) and (c)

e)All of the above

What are the three purposes of a spore stain?

To show endospore presence, location, and size

Name an organism which can be identified with an acid-fast stain

Mycobacterium

What is the purpose of a motility test?

To determine the presence of a flagella to assist with identifying the organism

Why is Bacillus more resistant than Staph to environmental conditions?

Can produce endospores when environmental conditions are not favorable.

What is the difference between true motility and Brownian motion?

In true motility, the organism moves from one location to another. Brownian motion is just water molecules hitting the organism, causing it to bounce around. It is not true motility.

Multiple Choice: Select the single best answer to the following.

Tuberculosis and leprosy are both disease caused by:

a. viruses.

b. Streptococcus.

c. Klebsiella.

d. Mycobacterium.

e. Bacillus.

Endospores are:

a. the result of sexual conjugation in bacteria.

b. found only in gram negative bacteria.

c. resistant to chemicals and heat.

d. all of the above are correct statements about endospores.

e. none of the above

Matching: Match the organisms listed in Column 1 with the appropriate description in Column 2.
Descriptions in column 2 may be used once, more than once, or not at all.

COLUMN 1 COLUMN 2

36. MycobacteriumEA. Gram negative bacillus

37. S. aureusDB. Gram positive bacillus

38. B. cereusBC. Gram negative coccus

39. E. coliAD. Gram positive coccus

E. Tuberculosis

What color do Acid fast bacteria stain if they are positive for being Acid fast?

  1. Blue
  2. Red
  3. Pink
  4. Yellow

Match the stain to the result of color for each

A. PurpleB. PinkC. Colorless

1. Gram + with Crystal Violet A-PURPLE

2. Gram + with IodineA-PURPLE

3. Gram - with Alcohol-acetone C-COLORLESS

4. Gram - with SafraninB-PINK

5. Gram + with Alcohol-acetoneA-PURPLE

6. Gram + with SafraninA-PURPLE

MATCH THE IMAGE WITH THE FLAGELLA ARRANGEMENT BELOW:

7. AmphitrichousC

8. LophotrichousB

9. MonotrichousA

10. PeritrichousD

What is an acidic stain used for?

  1. When you want to stain the cell
  2. When you can’t see the cell
  3. When you want the cell to change color
  4. when you want to stain the background instead of the cells

A differential stain uses methylene blue.

  1. True

B. False

Immunocompromised patients include the following

  1. Elderly people or infants
  2. AIDS or HIV infected patients
  3. organ transplant recipientson immunosuppressing agents
  4. cancer patientson chemotherapy
  5. Patients with malnutrition
  6. Patients on certain medicines (some antibiotics)
  7. Post-op patients
  8. All of the above

Special stains are

  1. Spore or endospore, capsule, and flagella
  2. Gram stain
  3. Acid fast stain
  4. B and C

Only sterilization can kill a spore. T

Spores are only produced by

  1. Cocci bacteria
  2. Bacillus bacteria
  3. Cocci bacteria and Bacillus bacteria
  4. None of the above

When the environment becomes too harsh to survive, some bacteria have the ability to eliminate all their cytoplasm and condense all their essential DNA and organelles in to a highly resistant structure called a spore. T

Spores are metabolically inactive. T

Streptococcus pneumoniae are bacteria which have a capsule which resists phagocytosis. T

Flagella stain reveals bacterial flagella, the tail which not easily seen in ordinary stains. T

Capsule stain colors the background but the capsule remains clean. This reveals the presence of a capsule, assisting in the diagnosis. T

What is the name of the primary stain used for a gram stain?

  1. Crystal lightC. Crystal white
  2. Crystal redD. Crystal violet

To determine a gram positive stain, the color must be:

  1. PinkC. Purple
  2. ColorlessD. Blue

A Gram negative stain will show what color?

  1. PurpleC. colorless
  2. PinkD. Red

Name the order of staining for the Gram Stain.

  1. Alcohol-acetone, safranin, iodine, Crystal violet
  2. Safranin, alcohol-acetone, crystal violet, iodine
  3. Crystal violet, alcohol-acetone, safranin, iodine
  4. Crystal violet, iodine, alcohol-acetone, safranin

A flagella that has a tail at one end is called: