BIO 150 Staining Quiz

Basic dyes uses what type of dyes?

methylene blue, crystal violet, and safranin

Acidic dyes use what kind of dyes?

nigrosin and india ink

Basic dyes are associated with what kind of charged ion

cation- positively charged ions

Acidic dyes are associated with what kind of charged ion?

anion - negatively charged ions

Name 3 reasons why smearing is good?

1. morphology of cells and other bacterial shapes

2. arrangement of cells

3. internal structures (endospores and cell inclusions)

What are the consequences of a thick smear?

• Can obscure details about arrangements and the presence of internal structures

• thick smear with large clumps — stains can get trapped in clumps of cells, preventing the removal of destaining and washing

For the preparation of a smear on a slide, what is the purpose of heat fixation?

• to kill bacteria

• adhere the bacterial cells onto the slides

• prepare cells to take stain

What problems can arise when the slide is heated in a flame?

• can destroy the bacterial cells

• risk of exposing yourself to bacteria

Define simple staining

the use of single stain to color a bacterial cell

List the 3 morphological types of bacterial cells

• bacilli

• cocci

• spirals

Know the shapes of bacterial morphology

• coccobacillus

• bacilli

• fusiform

• diplococci and tetrads

• streptococci

• staphylococci

• comma

• spirillum

• spirochetes

What is a chromophore?

color-bearing ionic groups

What are the basic steps in making a smear from solid media and from liquid media?

• Liquid:

  • Place loopful of culture directly on slide

  • air dry smear

  • heat fix

• Solid:

  • place dot of water on slide

  • mix dot of water with colony of bacteria

  • air dry

  • heat fix

Bacteria are usually stained with a basic dye – why?

because bacterial cells are negatively charged

What is the difference between a basic and an acidic dye?

• Basic dyes: have positively charged chromophores; they stain negatively charged bacterial cells

• Acidic dyes: have negatively charged chromophores; they stain the background, leaving cells clear

What is the difference between a simple and a differential stain?

• Simple stain uses 1 dye to increase contrast of cells. A simple stain determines size, shape, and arrangement of cells but cannot differentiate between types of bacteria.

• A differential stain uses 2 or more dyes to differentiate between organisms or between cell structures.

What is the purpose of a gram stain?

To differentiate bacteria into Gram-positive and Gram-negative

What is the ending result of gram positive stain?

purple

What is the ending result of gram negative stain?

pink

What are the procedures and stains used in the Gram stain procedure?

1. Crystal violet (primary stain) – stains all cells purple (30 secs)

   • wash

2. Iodine (mordant) – forms CV-I complex to fix dye in thick walls. (1 min)

   • wash

3. Alcohol/ethanol (decolorizer) – removes stain from Gram-negative cells. (5-15 secs)

   • wash

4. Safranin (counterstain) – stains Gram-negative cells pink/red. (1 min)

   • wash

What is the purpose of spore staining?

To detect the presence of endospores

What is the result of spore staining?

• Spores: Green (retain malachite green).

• Vegetative cells: Pink/red (stained with safranin).

What is the purpose of negative stain?

• To observe cell shape and size without distortion and to highlight capsules (if present).

• Ideal for delicate cells that might be damaged by heat fixation

What are the results of negative stain?

• Background: Dark.

• Cells: Appear clear or light-colored against the dark background.

• Capsules (if present): Appear as halos around cells.

What are the procedures/stains for negative staining?

• Mix bacteria with acidic dye (e.g., nigrosin or India ink).

• Smear gently across the slide.

• Do not heat-fix.

• Let air dry and observe under the microscope.

What is the purpose of acid fast stain?

To identify acid-fast bacteria, especially Mycobacterium species (e.g., M. tuberculosis), which have waxy cell walls rich in mycolic acid

What are the results of acid fast stain?

• Acid-fast bacteria: Red or pink (retain carbol fuchsin).

• Non–acid-fast bacteria: Blue (take up methylene blue).

What are the procedures/ stains for acid fast stain?

• Carbol fuchsin (primary stain) – applied with heat to penetrate waxy wall.

• Acid-alcohol (decolorizer) – removes stain from non–acid-fast cells.

• Methylene blue (counterstain) – stains non–acid-fast cells blue.

What is the function of a mordant?

The mordant is used to form insoluble complex with the primary stain, allowing it to become trapped or fixed into certain cell structures.

Which reagent serves this purpose in gram stain procedure?

Iodine is used to form insoluble complexes with crystal violet, allowing gram positive to remain a purple color.

What is the color of staphylococcus aureus before the primary stain?

colorless

What is the color of pseudomonas aeruginosa after the primary stain?

purple

What is the color of bacillus megaterium after the addition of the mordant?

purple

What is the color of Staphylococcus aureus after decolorization?

purple

What is the color of Moraxella catarrhalis after decolorization

colorless

What is the color of Pseudomonas aeruginosa after decolorization?

colorless