Introduction

The Gram staining is one of the most crucial staining techniques in microbiology. It gets its name from the Danish bacteriologist Hans Christian Gram, who first introduced it in 1882, mainly to identify organisms causing pneumonia.[1] Often, the first test performed, gram staining, involves the use of crystal violet or methylene blue as the primary color.[2] The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms. The organisms that do not take up primary stain appear red under a microscope and are Gram-negative organisms.

The first step in gram staining is the use of crystal violet dye for the slide's initial staining. The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine complex to prevent easy removal of dye. Subsequently, a decolorizer, often a solvent of ethanol and acetone, is used to remove the dye. The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment.[3] Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content.[4] 

Initially, all bacteria take up crystal violet dye; however, with the use of solvent, the lipid layer from gram-negative organisms is dissolved. With the dissolution of the lipid layer, gram negatives lose the primary stain. In contrast, solvent dehydrates the gram-positive cell walls with the closure of pores, preventing diffusion of violet-iodine complex, and thus, bacteria remain stained.[5] The length of decolorization is a critical step in gram staining, as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria.[6]

The final step in gram staining is to use a basic fuchsin stain to give decolorized gram-negative bacteria a pink color for easier identification. It is also known as counterstain. Some laboratories use safranin as a counterstain; however, basic fuchsin stains gram-negative organisms more intensely than safranin. Similarly, Hemophilus spp., Legionella app, and some anaerobic bacteria stain poorly with safranin.

Specimen Collection

Various clinical specimens can be used to perform Gram staining. Some of the commonly used specimens are sputum, blood, cerebrospinal fluid, ascitic fluid, synovial fluid, pleural fluid, and urine. Swabs from nostrils, throat, rectum, wound, cervix, etc., can also be used. The collection of specimens should always be in sterile containers.

Procedures

Types of equipment needed for Gram staining include:

• Bunsen burner
• Alcohol-cleaned microscope slide
• Slide rack
• Microscope

 Reagents needed for Gram staining include:

• Crystal violet (primary stain) [1]
• Gram's iodine solution (the mordant) [1]
• Acetone/ethanol (50:50 v:v) (the decolorizer) [1]
• 0.1% basic fuchsin solution (the counterstain) [1]
• Water

Procedure

1. Preparation of a slide smear:

• An inoculation loop is used to transfer a drop of suspended culture to the microscope slide. 
• If a Petri dish or a slant culture tube has the colony, a drop or a few loopfuls of water is added to facilitate a minimal amount of colony transfer to the examination slide. 
• A minimal amount of culture is required. If culture can be detected visually on an inoculation loop, it indicates the collection of too much culture.
• Culture is spread with an inoculation loop to an even thin film over a circle of 15 mm in diameter. A typical slide can contain up to 4 small smears if examining more than one culture.
• The slide can be either air-dried or dried with the help of heat over a gentle flame. The slide should be moved circularly over the flame to prevent overheating or forming of ring patterns in the slide. The heat helps the cell adhesion to the glass slide and prevents the significant loss of culture during rinsing. 

2. Gram staining:

• Crystal violet stain is added over the fixed culture. 
• After 10 to 60 seconds, the stain is poured off, and the excess stain is rinsed with water. The goal is to wash off the stain without losing the fixed culture. 
• Iodine solution is used to cover the smear for 10 to 60 seconds. This step is known as "fixing the dye." The iodine solution is poured off, and the slide is rinsed with running water. Excess water from the surface is shaken off.[7]
• A few drops of decolorizer are added to the slide. Decolorizers are often the mixed solvents of ethanol and acetone. This step is known as "solvent treatment." The slide is rinsed with water for 5 seconds. To prevent excess decolorization in the gram-positive cells, stop adding decolorizer as soon as the solvent is not colored as it flows over the slide.
• The smear is counterstained with basic fuchsin solution for 40 to 60 seconds. The fuchsin solution is washed off with water, and excess water is blotted with the bibulous paper. The slide can also be air-dried after shaking off excess water. 

3. Microscopic examination of slide:

• The slide should undergo an examination under a microscope under oil immersion.
• The initial slide examination should use the X40 objective to evaluate the smear distribution, and then they should be examined using the X100 oil immersion objective.
• All areas of the slide require an initial examination. Areas that are only one cell thick should be examined. Thick areas in slides often give variable and incorrect results. 
• White blood cells and macrophages stain Gram-negative.
• Squamous epithelial cells stain Gram-positive.

Various modifications of gram staining are used, such as Atkin gram stain, Burke gram stain, etc. 

Indications

Gram staining is indicated whenever a bacterial infection is suspected for easy and early diagnosis.[8]

Potential Diagnosis

Gram staining aids in the diagnosis of a disease or a pathologic condition. 

Examples of gram-positive organisms are: 

• Cocci: Staphylococcus species, and Streptococcus species
• Bacilli: Corynebacterium species, Clostridium species, Clostridioides species, and Listeria species

Examples of gram-negative organisms are:[9]

• Cocci: Neisseria gonorrhoeae, Neisseria meningitidis, and Moraxella species
• Bacilli: Escherichia coil, Pseudomonas species, Proteus species, and Klebsiella species 

Examples of gram-variable organisms include:

• Actinomyces species

Normal and Critical Findings

A normal finding in a sterile body fluid should be the absence of any pathologic organism in the smear. The organisms are identified based on color and shape. Gram-positive organisms are either purple or blue in color, while gram-negative organisms are either pink or red in color. Bacilli are rod-shaped, while cocci are spherical. 

Findings on gram stain that suggest underlying bacterial infections:

• Gram-positive cocci in clusters: Usually characteristic of Staphylococcus species such as S. aureus.
• Gram-positive cocci in chains: Usually characteristic of Streptococcus species such as S. pneumoniae, B group streptococci
• Gram-positive cocci in tetrads: Usually characteristic of Micrococcus spp.
• Gram-positive bacilli, thick: Usually characteristic of Clostridium spp., such as C. perfringes and C. septicum.
• Gram-positive bacilli, thin: Usually characteristic of Listeria spp.
• Gram-positive bacilli branched: Usually characteristic of Actinomyces and Nocardia.
• Gram-negative diplococci: Usually characteristic of Neisseria spp., such as N. meningitidis.

(Please note: Moraxella spp. and Acinetobacter spp. are often diplococcal in morphology. Acinetobacter can sometimes appear as Gram-positive cocci, and they can be pleomorphic.

• Coccobacilli: Usually characteristic of Acinetobacter spp., and they can be gram-positive, gram-negative, or gram-variable.
• Gram-negative bacilli, thin: Usually characteristic of Enterobacteriaceae, such as E. coli.
• Coccobacilli: Usually characteristic of Hemophilus spp., such as H. influenzae.
• Curved: Usually characteristic of Vibrio spp.,; Campylobacter spp., such as V. cholerae and C. jejuni.
• Thin needle shape: Usually characteristic of Fusobacterium spp.

Gram variable organisms: these organisms do not group into either gram-positive or gram-negative organisms.

Interfering Factors

If the specimen collection is not sterile, multiple organisms can contaminate the specimen. Similarly, improper specimen collection and prior use of antibiotics can interfere with the growth of organisms. During the interpretation of the Gram stain, as described by the World Health Organization in 2003, the following steps should be followed:

1. The general nature of the smear requires analysis under low power magnification (10X)

• The background of the slide should generally be gram-negative or clear
• White blood cells, when present, should stain gram-negative
• Thin crystal violet or gentian violent precipitates should not be confused with gram-positive bacillus bacteria
• The smear should be one cell thick with no overlapping of cells

2. Low power magnification should be utilized to note the following:

• Relative numbers of polymorphonuclear neutrophils (PMNs), mononuclear cells, and red blood cells (RBCs)
• Relative numbers of squamous epithelial cells and normal microbiota bacteria
• Location, arrangement, and shape of the organisms

3. Oil immersion examination of multiple fields is necessary to note the following:

• Micro-organisms: If identified, please note numbers and morphology
• Shapes: coccus, bacillus, coccobacillus, filaments, and yeast-like
• The appearance of ends: rounded, tapered, concave, clubbed, and flattened
• The appearance of sides: parallel, ovoid, irregular, or concave
• The axis of the organism: straight, curved, or spiral
• Pleomorphism (variation in shape)
• Branching or cellular extensions

Complications

The interpretation of slides can be difficult if the microscopic smear is thick and clumped. Decolorization time should have very close monitoring to avoid under-decolorization or over-decolorization. Thicker smears require longer decolorizing time. Similarly, cultures should undergo evaluation while they are still fresh. Old cultures tend to lose the peptidoglycan cell walls, which predisposes gram-positive cells to be gram-negative or gram variable. Gram stain is not useful for organisms without a cell wall like Mycoplasma species, and for smaller bacteria like Chlamydia and Rickettsia species.

Gram stain may not falsely reveal organisms in the following scenario:

• Use of antibiotics before collecting a specimen
• Inappropriate age of culture: too young or too old
• Fixing the smear before it is dry
• The smear is too thick
• Low concentration of crystal violet
• Excessive heat fixation
• Excessive washing between steps
• Insufficient exposure to iodine
• Prolonged decolorization
• Excessive counterstaining
• Lack of experience in preparing the slide, and reviewing the slide

Sometimes results of Gram-stain may not match the final results of cultures and could potentially lead to inappropriate use of antibiotics.[10]

Clinical Significance

Gram stain is often the initial diagnostic test for the evaluation of infections. The use of Gram stain facilitates the rapid use of appropriate antibiotics. However, genetic sequences and molecular techniques are more specific than classic gram stain.