Question

For this discussion question, in your own words, describe why the Gram stain is so important to all of microbiology, how it is performed, how it works, and compare the cell compositions it contrasts between Gram positive and Gram negative cells. Include why it is of such particular importance for medical microbiology. Also, giving two specific medically-significant microbe examples and descriptions for each group.

Answer 1

Gram staining method, the most important procedure in Microbiology, was developed by Danish physician Hans Christian Gram in 1884. Gram staining is still the cornerstone of bacterial identification and taxonomic division.

This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition:

1. Gram positive bacteria (thick layer of peptidoglycan-90% of cell wall) - stains purple

2. Gram negative bacteria (thin layer of peptidoglycan-10% of cell wall and high lipid content) - stains red/pink

Classic Gram staining techniques involves following steps:

i. Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol. Methanol fixation preserves the morphology of host cells, as well as bacteria, and is especially useful for examining bloody specimen material.

ii. Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple

iii. Application of mordant: The iodine solution (mordant) is added to form a crystal violet iodine (CV-I) complex; all cells continue to appear blue.

iv. Decolorization step:

The decolorization step distinguishes gram-positive from gram-negative cells.

The organic solvent such as acetone or ethanol, extracts the blue dye complex from the lipid-rich, thin walled gram negative bacteria to a greater degree than from the lipid poor, thick walled, gram-positive bacteria. The gram negative bacteria appear colorless and gram positive bacteria remain blue.

v. Application of counter stain (safranin):

The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue.

Principle of Gram stain:

The differences in cell wall composition of Gram positive and Gram negative bacteria accounts for the Gram staining differences. Gram positive cell wall contain thick layer of peptidoglycan with numerous teichoic acid cross linking which resists the decolorization.

In aqueous solutions crystal violet dissociates into CV⁺ and Cl^- ions that penetrate through the wall and membrane of both Gram-positive and Gram-negative cells. The CV⁺ interacts with negatively charged components of bacterial cells, staining the cells purple.

When added, iodine (I^- or I₃^-) interacts with CV⁺ to form large crystal violet iodine (CV-I) complexes within the cytoplasm and outer layers of the cell.

The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram negative bacteria.

The outer membrane of the Gram-negative cell (lipopolysaccharide layer) is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram-positive cells. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell.

The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. The multi-layered nature of the peptidoglycan along with the dehydration from the ethanol treatment traps the large CV-I complexes within the cell.

After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.

Reporting Gram smears:

The report should include the following information:

i. Numbers of bacteria present, whether many, moderate, few, or scanty

ii. Gram reaction of the bacteria, whether Gram positive or Gram negative

iii. Morphology of the bacteria, whether cocci, diplococci, streptococci, rods, or coccobacilli. Also, whether the organisms are intracellular.

iv. Presence and number of pus cells

v. Presence of yeast cells and epithelial cells.

Importance in Medical Microbiology:

If a doctor suspects a possible infection, he will need to collect a sample of body fluid or tissue for analysis. Their collection methods will vary depending on the type of sample they need.

If your gram stain results are negative, it means no bacteria were found in your sample. If they’re positive, it means bacteria were present. The shape, size, and quantity of bacteria present will also provide information about the infection.

Examples of medically-significant microbe:

i. E. coli, which causes the majority of urinary tract infections.

Description (microscopic appearance):

Gram Stain:	Gram-negative.
Morphology:	Straight rods.
Size:	1.1-1.5 micrometers by 2.0-6.0 micrometers.
Motility:	Escherichia spp. are motile by peritrichous flagella or are non-motile. In addition to flagella, most strains have fimbriae (pili) that extend from the bacterial surface into the surrounding medium. Some fimbriae have specific functions as adhesive organs.
Capsules:	Capsules or microcapsules occur in many strains.
Spores:	None.
Other:	They occur singly or in pairs.

ii. Klebsiella pneumoniae, which causes many types of healthcare-associated infections, including pneumonia, urinary tract infections, and bloodstream infections.

Description (microscopic appearance):

Gram Stain:	Gram-negative.
Morphology:	Rod shaped.
Size:	0.5-2 micrometers
Motility:	Non-motile
Capsules:	Encapsulated
Spores:	None.
Other:	They occur singly or in pairs.