GRAM POSITIVE AND GRAM NEGATIVE BACTERIA

Gram positive and Gram negative  Bacteria as seen under Light Microscope after Gram staining

Bacteria are classified either as Gram negative or Gram Positive based on Gram staining techniques; they possess cell wall outside their plasma membrane which functions to

1. Protects the cell from toxic substance.
2.  Determines the shape of the cell 
3. Protect cell from Osmotic lysis
4.  Contributes to the ability to cause infection (Pathogenicity)

 Cell wall of Gram Positive Bacteria

Gram Positive organisms have a simpler and thicker cell wall made up of Peptidoglycan and teichoic acid. Teichoic acid acid is occur in large amount and is negatively charge. and because they cover the surface of the peptidoglycan layers confers their negative charge on the Gram Positive cell wall.

The periplasmic space of these prokaryotic organisms lies between the plasma membrane and the cell wall and is smaller than that of the Gram negative bacteria; they secrete exoenzymes which break down polymeric units that are too large to be transported across the plasma membrane.

Cell Wall of Gram Negative cell Wall

Gram negative bacteria are more complex than the former, they posses thinner cell wall with bimolecular layer of peptidoglycan, there is a second outer membrane external to the peptidoglycan layer made up of lipid bilayer, Proteins and lipopolysaccharides (LPS).

The lipid bilayer is attached to peptidoglycan by lipoproteins that cross the periplasmic space, the protein includes porin which forms transmembrane channel that are important in the transport of ions, and hydrophilic compounds from the outside of the cell to the periplasm.  Three porin proteins cluster together and span the outer membrane to form a narrow channel for the passage of transport molecules, only molecules smaller than about 600 -700 Dalton can pass through the channels. Large molecules such as Vitamin B₁₂ can only pass through the membrane with the help of specific carrier molecules.

 Lipopolysaccharide (LPS) is made up of three parts, a lipid portion, (Lipid – A), a polysaccharide rich core and an O side chain. LPS, not only contributes to negative charge on the surface of the bacteria, but also stabilizes the membrane structure.

 The lipid portion consists of two glucosamine derivative each with three fatty acids and phosphate or pyrophosphate attached. It is buried in the outer membrane and the remaining of the LPS projects from the outer surface.

Lipid portion, is resistant to heat, toxic and responsible for the biological effect of the endotoxin including symptoms that arises in Gram negative bacterial infections. The outer membrane functions as a barrier that regulates the entry of bile salt, antibiotics and other toxic substances that might kill or harm the bacterium.

The core polysaccharide is joined to the lipid and is made of 10 sugars with unusual structures.

The O side chain or O-antigen is a short polysaccharide chain that extends outward from the core. It has several peculiar sugars which differ in composition from one bacteria strain to another. O side chain is easily recognized by host antibodies. Gram negative bacteria may evade host defence by rapidly changing the nature of their O side chain. Antibody interaction with LPS before reaching the outer membrane proper may also increase the bacteria resistance to attack.

The periplasmic space of Gram negative bacteria is bounded on either side of the peptidoglycan and may constitute 5-10% of the wall’s weight, some periplasmic proteins takes part in nutrient acquisition for example hydrolytic enzyme and transport proteins, others takes part in energy conservation. 

 Gram Staining Techniques

Principle

Gram stain was devised by Danish scholar, Christian Gram in 1884, the stain is the most widely used tool in bacteriology, it is important because it differentiate between two bacterial which are morphologically indistinguishable yet of different species. Bacteria differ from one another chemically and physically and thus react differently to a given procedure, this is the principle of differential staining, it divides bacteria into two, (a) those that retain the crystal violet dye – iodine complex known as Gram Positive and (b) those that loose the crystal violet Iodine complex upon washing with alcohol and stained the color of Safranine (counter stain) called Gram Negative.

 Gram Staining Procedure

The first step in this procedure involves staining with basic dye crystal violet, which is a primary stain. This is followed by treatment with Iodine solution, which acts as a mordant that increases the interaction between the bacteria cell wall and the dye so that the dye is more tightly bound or the cell is strongly stained. The smear is then decolorized by washing with 95% ethanol or acetone, The Gram positive bacteria retains the crystal violet iodine complex when washed with decolorizer, whereas  Gram Negative bacteria lose their crystal violet Iodine complex and become colorless when counter stained with Safranine.  Safranine will stain the colorless cell pink but does not alter the purple color of Gram positive cells.

The end reaction is that Gram positive organisms are deep purple in color while Gram negative organisms appears red or pink in color.

The age of culture is very important in Gram staining, the age of the culture and the acidity and alkalinity (pH) of the media in which the bacteria are grown affects their reaction to Gram Stain.  Older cultures of gm +ve organisms particularly if they show autolysis and those in acid media appear either as gm –ve or as both gm –ve and gm +ve (Gram variable), in  the same culture. However, gm +ve organisms do not become gm –ve regardless of age or medium of culture. 

Only fresh cultures of about 12 – 18 hours old should be used for Gram staining. Indistinct Gram staining result can be confirmed by a simple test using KOH, this is done by placing a drop of KOH on a clean class slide, after 30seconds, a loop is slowly  pulled through the suspension up and down from the slide,  gm-ve organism will produce mucoid string while gm +ve organism will produce fluid. 

Procedure

• Clean a glass slide with a cotton wool

•  Place a drop of water on he slide using a sterilized wire loop also known as inoculating loop, (the wire loop is usually sterilized by burning it in the blue zone of a flame).

•  Us the wire loop to Place a smear from an appropriate plate on one half of the microscope slide.

• Heat fixes the slide by passing it over a flame.

• Flood the slide with crystal violet and allow contact time of 1minute, pour off the crystal violet and wash of excess stain with water.

• Flood the slide with Iodine solution and allow contact time of 1minutes, wash the slide with water blot dry to remove excess water.

• Flood the slide with 95% alcohol (ethanol) for 10 seconds and quickly pour it off.  watch for continued evidence of dye removal, this is shown by the alcohol solution changing color to purple, continue pouring off the alcohol while adding fresh one until the CV, stop coming  out of the cell and then wash the slide in water to stop the decolorization process.

• Counter stain with Safranine for 1minute, wash the slide in water, blot dry or allow to air dry.

• Examine the slide under a microscope.

NOTE: It is not easy to tell when the decolorization process (coming out of die from the cell) has stop; it involves proper training of the eyes. A smear that is too thick will protect the cells covered by other cells from getting decolorized, and a Gram negative organism will appear Gram positive.

Removal of Bacteria Cell Wall

Bacteria cell wall protects the delicate underlying cytoplasmic membrane, which is the site of intracellular metabolism, its removal results in bacterial lysis.

Removal of cell wall in Gram positive bacteria result information of protoplast, this requires isotonic medium in other to become spherical.

Due to the complexity of Gram negative organisms, there is innate resistance to enzymatic destruction of cell wall.  Gram negative bacteria with damaged cell wall become spheroplast, they take on spherical shape even in non isotonic solution. 

Question and Answers

What is a differential stain?

Answer: a differential stain is one that divides bacteria into two groups, Gram +ve and Gram –ve, it involves more than one dye (staining agent).

What is gram variable?

Answer: This means that some cells in culture are gram negative and some gram positive (both gm +ve and gm –ve) depending on the age of the culture.

Which part of a cell is mostly involved in Gram staining?

Answer: The cell was is mostly involved due to its permeability and ability reacts with dye. It is negatively charged.

What is the purpose of mordant in Gram staining?

 

Answer:   To increase the interaction between the cell and the dye so that the dye is more tightly bounded or the cell is more strongly stained.

Assignment: Explain the Mechanism of Gram Staining?