Gram Staining

Question 1

He discovered that certain stains were retained by some types of bacterial cells but removed from others during the staining process. His published work served as the foundation of what
would become the most important stain in bacteriology, the Gram stain

Answer 1

Hans Christian Gram

Question 2

valuable diagnostic tool used in the clinical and research setting

Answer 2

Gram staining

Question 3

it must be decolorized and it must be counterstained with a red dye in order to be visualized by microscopy

Answer 3

gram-negative bacteria

Question 4

It retain a
purple dye complex

Answer 4

gram-positive bacteria

Question 5

1st stain in the gram staining process. It is crystal violet

Answer 5

primary stain

Question 6

it combines with the crystal violet and forms an insoluble complex in gram-positive cells. For example iodine

Answer 6

mordant

Question 7

comprises the cell wall of bacteria

Answer 7

peptidoglycan

Question 8

How old should cultures be for it to be still viable for gram staining

Answer 8

16–18 hours

Question 9

Why is the Gram stain considered a differential stain?

Answer 9

it distinguishes bacteria into Gram-positive and Gram-negative groups based on their cell wall properties, providing valuable information for medical treatment and microbiological research.

Question 10

How do gram-positive and gram-negative bacteria differ in cellular structure, and how does this contribute
to their differential staining properties?

Answer 10

particularly the thickness of the peptidoglycan layer and the presence or absence of an outer membrane—are critical to their differential response to the Gram stain. These structural features determine how the cells interact with the stains and reagents during the Gram staining process, resulting in the characteristic purple or pink coloration that distinguishes Gram-positive from Gram-negative bacteria.

Question 11

How does the age of a culture affect the Gram stain reaction? What is an optimum culture age for a valid Gram reaction?

Answer 11

Crucial to use bacterial cultures that are 18-24 hours old. This ensures that the cell walls of both Gram-positive and Gram-negative bacteria are in good condition, allowing for the correct retention or loss of the crystal violet-iodine complex during the staining process.Using cultures outside this optimal age range can lead to misleading results, with Gram-positive bacteria potentially appearing as Gram-negative due to compromised cell wall integrity.

Question 12

Which step in the Gram stain procedure is most prone to error?

Answer 12

The decolorization step in the Gram stain procedure is the most prone to error. This step involves the application of a decolorizing agent, typically alcohol or acetone, and is crucial for differentiating between Gram-positive and Gram-negative bacteria.

Question 13

What is the function of a mordant?

Answer 13

• Serves to stabilize and fix the primary stain within bacterial cells, particularly aiding in the differentiation of Gram-positive and Gram-negative bacteria by enhancing the retention of the crystal violet dye in Gram-positive cells.
• crucial for the accuracy and reliability of the Gram stain, allowing for effective bacterial classification and diagnosis.

Question 14

which reagent serves this purpose in the Gram stain procedure_mordant

Answer 14

Gram’s Iodine

Question 15

Gram Stain Process in order

Answer 15

1. Crystal Violet: Stains all bacteria purple.
2. Iodine: Forms a complex with crystal violet, enhancing retention in the cell.
3. Alcohol/Acetone: Decolorizes Gram-negative bacteria while Gram-positive bacteria retain the purple stain.
4. Safranin: Counterstains Gram-negative bacteria pink/red, while Gram-positive bacteria remain purple.

Question 16

In what type of cell, gram-positive or gram-negative, would you find lipopolysaccharide in its cell wall?

Answer 16

Lipopolysaccharide is a key component of the outer membrane of Gram-negative bacteria, distinguishing them from Gram-positive bacteria, which lack an outer membrane and LPS.

Question 17

Staphylococcus aureus before the primary stain

Answer 17

colorless or faintly translucent under the microscope.

Question 18

Pseudomonas aeruginosa after the primary stain

Answer 18

appear purple under the microscope

Question 19

Bacillus megaterium after the addition of the mordant

Answer 19

would appear dark purple or violet under the microscope.

Question 20

Staphylococcus aureus after decolorization

Answer 20

would still appear purple or dark purple under the microscope

Question 21

Moraxella catarrhalis after decolorization

Answer 21

would appear colorless or faintly translucent under the microscope.

Question 22

Pseudomonas aeruginosa after decolorization

Answer 22

appear colorless or faintly translucent under the microscope.

Question 23

Bacillus megaterium after adding the counterstain

Answer 23

would still appear purple or dark purple under the microscope.

Question 24

Escherichia coli under the microscope if you forgot to apply safranin

Answer 24

would appear colorless or faintly translucent under the microscope.

Question 25

Escherichia coli under the microscope if you forgot to apply decolorizer

Answer 25

would still retain the primary stain (crystal violet) and appear purple or dark purple under the microscope.

Question 26

Bacillus megaterium under the microscope if you forgot to apply iodine

Answer 26

would still retain the primary stain (crystal violet) and appear purple or dark purple under the microscope.