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GRAM STAIN

GRAM STAIN

The Gram staining method was developed and introduced by Hans Christian Gram and published in 1884. The method is based upon the chemical cell wall composition of bacteria. 

Because of the variability in the chemical structure of the cell wall, at least 4 types of gram stained organisms are identified by the method: Gram Positive, Gram Negative, Gram variable and Gram indeterminant groups. The technique is used as a tool to differentiate between two large groups of bacteria: Gram Positive and Gram Negative in microbiology labs and medical diagnosis of bacterial infections. 

With current technology, environmental microbiology uses other methods (e.g., PCR-DNA tests, genetic markers) to identify various bacteria. 

Medical Use of Gram Stain

The Gram stain is still used in medical microbiology as a quick method to obtain some idea of the bacterium involved in an infectious process. Its advantage is rapidity of results vs 24 or more hours awaiting bacterial cultures. Thus, a patient suspected of a bacterial infection will have body fluids or biopsy materials stained by the Gram method as well as microbiological cultures.

Gram-negative bacteria include the proetobacteria, cyanobacteria, spirochaetes, green sulfur and green-no sulfur bacteria. The group also contains medically relevant Gram-negative cocci, bacilli and many bacteria associated with nosocomial infections. These organisms stain pink and are the source of endotoxins.

Gram-positive bacteria include Bacillus, Staphylococcus, Streptococcus, Enterococcus, Diplococcus pneumoniae, Clostridium and Mycoplasma. These organisms stain purple.

Gram-variable bacteria result in a mixed pattern of staining--pink and purple--because their cell walls are particularly sensitive to breakage which results in both Gram negative and Gram Positive cells.

The Staining Mechanism

The stain method takes advantage of the structure of the cell wall of bacteria. The Gram-positive bacteria have a thick cell wall of peptiglycan which stains purple. The Gram-negative bacteria have a a thinner cell wall layer (stains pink) and they also have an outer layer containing lipids . 

The staining method involves four basic steps: 

(1) Microscope slide smear of the the culture is stained with Crystal Violet under heat (Bunsen burner) 

(2) A trapping agent (Gram’s Iodine) is applied next 

(3) Rapid de-colorization is done by placing treated slide(s )in alcohol or acetone 

 4) Counter staining with Safranin is done, or sometimes Basic Fuchsin is used.
 
The above information is provided because Dr. Thrasher has seen Environmental Laboratories use the Gram stain to identify large groups of gram positive and negative bacteria that have been cultured from bulk samples removed from water-damaged structures. However, PCR-DNA tests are probably more reliable for the identification of bacterial species (although PCR-DNA testing is expensive). The Gram stain method does give information as to which group most environmental bacteria belong.
Gram staining procedure

Gram staining procedure

Gram staining slides

Gram staining slides


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