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Gram Stain

Bacterial infections Biomarker

Sample Needed

Collection Type: Sputum

Body System

Related System: Bacterial infections

Overview

The Gram stain is a rapid, microscopic staining technique that classifies bacteria as Gram-positive (purple) or Gram-negative (pink) and shows basic bacterial shape (cocci, bacilli) and arrangement. It is applied to clinical specimens (sputum, urine, CSF, wound swabs, synovial fluid, blood culture smears) to detect and give early clues about bacterial infection and guide empiric therapy. Clinicians order it for symptoms such as fever, cough with purulent sputum, dysuria, meningitis signs, wound drainage, or sepsis. Results depend on specimen type, specimen quality, prior antibiotics, and patient factors (age, mucosal colonization, indwelling devices); neonates and immunocompromised patients may have atypical findings.

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Test Preparation

No special preparation is required

Why Do I Need This Test

Profile: Bacterial infections - Symptoms: fever, purulent cough, neck stiffness, dysuria, wound drainage, signs of sepsis - Diagnoses/monitoring: rapid presumptive ID of bacterial infection, assessment of specimen quality, guidance of initial antibiotic choice - Causes of abnormal result: true infection, contamination, poor collection technique, recent antibiotics - Biological meaning: organisms present and whether intracellular organisms/PMNs indicate active infection - Lifestyle/factors: catheter use, poor hygiene, sexual activity, recent antibiotic use - Family history: recurrent infections or immunodeficiency may prompt testing

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Understanding Test Results

No organisms seen and low WBCs (<5 WBCs/HPF or CSF WBC 0–5/µL) generally indicates no active bacterial infection or a non-bacterial cause.
Presence of numerous organisms plus many neutrophils (e.g., >10 WBCs/HPF in urine or >25 neutrophils/LPF and <10 squamous epithelial cells in sputum) suggests active bacterial infection and likely pathogenic organisms.
Gram-positive cocci in clusters presumptive Staphylococcus; cocci in chains Streptococcus; Gram-negative rods Enterobacteriaceae or Pseudomonas (latter often slender, sometimes oxidase-positive on culture).
Finding intracellular bacteria in neutrophils is strong evidence of invasive infection.
Mixed flora with many squamous epithelial cells typically indicates contamination.
A negative Gram stain does not rule out infection—low bacterial load, intracellular organisms, fastidious organisms, or prior antibiotics can produce false-negative results; culture and molecular tests may be required.

Normal Range

No organisms seen; <5 WBCs/HPF; <10 squamous epithelial cells/LPF; CSF: no organisms; WBC 0–5 cells/µL

FAQs

Q: What are the 4 steps of Gram staining?

A: Gram staining involves four steps: (1) apply crystal violet primary stain to a fixed bacterial smear, (2) add iodine mordant to form a crystal violet–iodine complex, (3) decolorize briefly with alcohol or acetone to remove stain from Gram-negative cells, and (4) counterstain with safranin so Gram-positive remain purple and Gram-negative appear pink/red for microscopic differentiation.

Q: What is a Gram stain used for?

A: Gram staining is a rapid, inexpensive laboratory technique to classify bacteria as Gram‑positive (purple) or Gram‑negative (pink/red) based on cell‑wall properties. It reveals bacterial shape and arrangement, guides initial diagnosis, helps narrow antibiotic choices, and directs further testing. Gram stains are applied to clinical specimens and cultures to provide prompt, actionable information for infection management and microbiology workflows.

Q: What is the difference between Gram stain positive and negative?

A: Gram-positive bacteria retain crystal violet and appear purple because they have a thick peptidoglycan cell wall and teichoic acids. Gram-negative bacteria do not retain crystal violet, appear pink after counterstain, and have a thin peptidoglycan layer plus an outer membrane containing lipopolysaccharide (LPS). This structural difference influences permeability, immune reactions and antibiotic susceptibility.

Q: What is the normal range for Gram stain?

A: A normal Gram stain shows no organisms and only background flora; microscopy should reveal few inflammatory or epithelial cells. General thresholds: ≤10 squamous epithelial cells per low‑power field (sputum acceptability), neutrophils >25/LPF support lower respiratory infection, while sterile fluids or urine typically have very few white cells (generally <10 per high‑power field). Many organisms or many WBCs are abnormal.

Q: What color is gram-positive?

A: Gram-positive bacteria appear purple or violet after Gram staining. Their thick peptidoglycan cell wall retains the crystal violet–iodine complex during the alcohol decolorization step, so they keep the primary stain. In contrast, Gram-negative bacteria lose the crystal violet and take up the counterstain (safranin), appearing pink or red. Gram staining thus differentiates bacteria by cell wall properties.

Q: What are the 4 components of a Gram stain kit?

A: The four components of a Gram stain kit are: crystal violet (primary stain), Gram’s iodine (mordant), a decolorizer—usually ethanol or acetone–alcohol, and a counterstain such as safranin (or fuchsin). Together these reagents allow differentiation of Gram-positive (retain crystal violet–iodine complex) from Gram-negative bacteria (decolorize and take up the counterstain).