Gram Stain Procedure in Research and Labs

Gram staining is a microbiological procedure that categorizes bacteria based on the physical and chemical structure of their outer surface. This is done just with a slide, a microscope, and stains. This procedure is commonly used for detection and identification of bacteria both in research and clinical laboratories.

Microphotograph of example of staining bacteria using Gram method, at x1250 magnification
De Agostini Picture Library / Getty Images 

Gram-Positive and Gram-Negative

Bacteria are loosely categorized into two types, gram-positive and gram-negative.

  • Gram-positive bacteria have a thick layer made up of polymers of protein-sugar molecules called peptidoglycan. Gram staining of the peptidoglycan layer (which is the cell wall) with a chemical called crystal violet results in purple coloration of the gram-positive bacteria. Addition of acetone or alcohol dehydrates the bacteria, causing it to retain the purple color.
  • Gram-negative bacteria have an additional outer membrane whose properties resist acetone/alcohol-dehydration and result in loss of the crystal violet stain. A counterstain, made up of fuchsin, stains these bacteria red or pink.

The test involves looking at the bacteria on the slide under the microscope. Bacteria can be seen under a microscope if stained. These bacteria can be collected in samples such as from urine, sputa, and blood. These samples are used to identify if bacteria are in a particular part of the body. Infections are often localized to one part of the body such as the bladder, the lungs, or even bone, the gallbladder, the skin. Some infections are more generalized and spread throughout the blood.

Those bacteria that stain purple are gram-positive; those that stain pinkish are gram-negative. It's a simple way of knowing important information. It tells us what type of outer protective layer the bacteria has. This helps doctors decide which antibiotics to use and which bacteria is to blame for the infection — as some bacteria are expected to behave differently than others.

Some bacteria are gram-positive, like Staph Aureus (including MRSA) and Strep bacteria. Other bacteria is gram-negative like Salmonella, Shigella, and many other bacteria. The (genus of) bacteria that cause gonorrhea and a scary form of bacterial meningitis, Meninogococcus, is gram-negative. Some bacteria are not as well characterized by gram status, but it is useful for many types of bacteria.

Types of infections can often be caused by gram-negative or gram-positive infections but may be more likely to be caused by one type or the other. Pneumonia can be caused by either, but many suffer from gram-positive infections caused by Strep bacteria. Urinary Tract Infections are often caused by gram-negative infections. There are many other types of infections that can be predominantly gram-positive or gram-negative.

Why It Matters

Knowing if an unknown type of bacteria is gram-negative or positive can help identify the bacteria. Doctors often have to guess which infections a patient has. They often have to treat empirically, as we say, without knowing precisely what is causing the infection and which antibiotic is best. Knowing the gram stain status helps.

Different bacteria respond to different antibiotics. Gram-positive and gram-negative bacteria, in particular, respond differently to different antibiotics. Knowing whether bacteria are gram neg or pos can help decide which antibiotics will most likely work.

Gram staining can be done in laboratories without a lot of resources. It requires simply a microscope, slides, stains, and someone who knows how to do a gram stain.

1 Source
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  1. Boyanova L. Direct Gram staining and its various benefits in the diagnosis of bacterial infectionsPostgrad Med. 2018;130(1):105–110. doi:10.1080/00325481.2018.1398049

By Ingrid Koo, PhD
 Ingrid Koo, PhD, is a medical and science writer who specializes in clinical trial reporting