Bacterial Stains and Cultures

Author: Adnan Mousa, M.D.

Keywords: Bacterial staining, Gram stain, Ziehl-Neelsen stain, special culture media, microbiology

Overview

Bacterial staining techniques are essential for identifying and differentiating microorganisms based on their structural and chemical properties. The Gram stain, the most widely used method, classifies bacteria as Gram-positive or Gram-negative based on their cell wall composition. Additional staining techniques address bacteria that do not stain effectively with Gram stain, such as acid-fast stains for Mycobacteria and Giemsa stain for intracellular organisms like Chlamydia and Plasmodium. Bacteria are further classified through biochemical tests, including catalase, coagulase, and hemolysis assays, aiding in distinguishing pathogenic species. Specialized stains like Periodic Acid–Schiff (PAS) highlight carbohydrate-rich structures, useful for diagnosing conditions such as Whipple disease.

Bacterial Staining Techniques

Bacterial staining is essential for differentiating microorganisms based on structural and chemical properties. The most commonly used stains in microbiology include:

Gram Stain

Gram staining is the first-line laboratory test for bacterial classification. It differentiates bacteria based on their cell wall structure.

Steps:

●       Primary stain: First, a purple dye called crystal violet is applied to all the cells.

●       Mordant: Next, iodine is added to help keep the purple dye in the cells.

●       Decolorization: Then, alcohol or acetone is used to wash out the purple dye from Gram-negative bacteria.

●       Counterstain: Finally, a pink dye called safranin is added, which stains the Gram-negative bacteria pink.

Interpretation:

●     Gram-positive bacteria: Retain crystal violet stain due to a thick peptidoglycan layer and appear purple.

●     Gram-negative bacteria: Lose the crystal violet stain and take up the counterstain (safranin), appearing red/pink.

Figure 1. Staph Aureus: A gram positive bacteria.

Figure 2. E. Coli: A gram negative bacteria.

Some bacteria do not stain effectively with Gram stain due to structural differences:

●     Too thin to visualize: Treponema, Leptospira

●     Lack a cell wall: Mycoplasma, Ureaplasma

●     High lipid content in the cell wall: Mycobacteria

●     Primarily intracellular or lack peptidoglycan: Legionella, Rickettsia, Chlamydia, Bartonella, Anaplasma, Ehrlichia

Gram-Positive Lab Algorithm

Gram-positive bacteria are classified based on their shape (bacilli or cocci), staining properties, and biochemical tests.

Gram-positive bacilli (rod-shaped, purple/blue staining):

Aerobic: These bacteria require oxygen to grow.

• Listeria: Motile at room temperature, causes meningitis in newborns and pregnant women.

• Bacillus: Forms spores; B. anthracis causes anthrax, B. cereus causes food poisoning.

• Corynebacterium: Club-shaped; C. diphtheriae causes diphtheria (forms pseudomembrane in throat).

Anaerobic: These bacteria grow without oxygen.

• Clostridium: Forms spores; C. tetani (tetanus), C. botulinum (botulism), C. perfringens (gas gangrene), C. difficile (pseudomembranous colitis).

• Cutibacterium (formerly Propionibacterium): Part of normal skin flora; can cause acne and prosthetic joint infections.

Gram-positive cocci (circular, purple/blue staining):

●      Branching filaments:

• Nocardia: Weakly acid-fast (stains with acid-fast stain), causes pulmonary and cutaneous infections in immunocompromised patients.

• Actinomyces: Not acid-fast, forms sulfur granules, causes abscesses (e.g., cervicofacial actinomycosis).

●     Hemolysis on blood agar:

• Alpha hemolysis (partial, green) (Figure 9):

  - Streptococcus pneumoniae: Optochin-sensitive, bile-soluble, causes pneumonia and meningitis.

  - Viridans streptococci: No capsule, part of normal oral flora, causes endocarditis.
  

• Beta hemolysis (complete, clear):

- Group A (Streptococcus pyogenes): Bacitracin-sensitive, PYR-positive, causes pharyngitis, rheumatic fever, and glomerulonephritis.

- Group B (Streptococcus agalactiae): Bacitracin-resistant, causes neonatal meningitis and sepsis.

• Gamma hemolysis (no hemolysis):

- Enterococcus: Grows in 6.5% NaCl, PYR-positive, causes urinary tract infections (UTIs) and endocarditis.

- Non-enterococcus (Streptococcus gallolyticus): Grows in bile, PYR-negative, associated with colon cancer and endocarditis.

Catalase test:

Catalase-positive (bubbles when hydrogen peroxide is added):

• Staphylococcus: Forms clusters.

- S. aureus: Coagulase-positive, Novobiocin-resistant, causes skin infections, toxic shock syndrome, and food poisoning.

- S. epidermidis: Coagulase-negative, Novobiocin-sensitive, causes infections of prosthetic devices.

- S. saprophyticus: Novobiocin-resistant, causes UTIs in young women.

• Catalase-begative (no bubbles):

- Streptococcus: Forms pairs or chains, further classified using PYR, Optochin, bile solubility, and Bacitracin tests.

Key tests explained:

●       PYR test: Detects the enzyme pyrrolidonyl arylamidase.

○       Positive: Streptococcus pyogenes and Enterococcus.

○       Negative: Other streptococci.

●       Optochin sensitivity: Differentiates S. pneumoniae (sensitive) from other alpha-hemolytic streptococci.

●       Bile solubility: S. pneumoniae dissolves in bile; other streptococci do not.

●       Bacitracin sensitivity: Differentiates Group A (S. pyogenes, sensitive) from Group B (S. agalactiae, resistant).

●       Coagulase test: Differentiates S. aureus (positive) from other staphylococci (negative).

●       Novobiocin sensitivity: Differentiates S. epidermidis (sensitive) from S. saprophyticus (resistant).

Gram-Negative Lab Algorithm

Key shapes:

●       Diplococci (paired round cells):

○       Neisseria gonorrhoeae: Causes gonorrhea.

○       Neisseria meningitidis: Causes meningitis.

●       Coccobacilli (short, oval-shaped rods):

○       Haemophilus influenzae: Causes respiratory infections and meningitis.

○       Bordetella pertussis: Causes whooping cough.

○       Pasteurella: Associated with animal bites.

○       Brucella: Causes brucellosis.

○       Francisella tularensis: Causes tularemia.

●       Curved rods:

○       Campylobacter jejuni: Causes gastroenteritis.

○       Vibrio cholerae: Causes cholera.

○       Helicobacter pylori: Causes stomach ulcers and gastritis.

●       Bacilli (rods):

○       Escherichia coli: Common cause of UTIs and diarrhea.

○       Klebsiella, Enterobacter, Citrobacter, Serratia: Opportunistic pathogens causing various infections.

○       Pseudomonas: Causes infections in immunocompromised patients.

○       Burkholderia: Includes pathogens like B. cepacia and B. pseudomallei.

○       Shigella: Causes dysentery.

○       Yersinia: Includes Y. pestis (plague) and Y. enterocolitica (gastroenteritis).

○       Salmonella: Causes typhoid fever and food poisoning.

○       Proteus: Known for urinary tract infections and swarming motility.

Growth characteristics:

●       Aerobic: Grows in the presence of oxygen.

●       Maltose fermentation: Seen in Neisseria meningitidis.

●       Oxidase positive: Indicates the presence of cytochrome c oxidase enzyme.

○       Seen in Campylobacter jejuni, Vibrio cholerae, and Helicobacter pylori.

●       Grows at 42°C: Campylobacter jejuni can tolerate higher temperatures.

●       Grows in alkaline media: Vibrio cholerae thrives in alkaline conditions.

Fermentation characteristics:

●       Lactose fermenters (fast-growing):

○       Escherichia coli, Klebsiella, Enterobacter: These bacteria ferment lactose quickly,

producing acid and gas.

●       Lactose fermenters (slow-growing):

○       Citrobacter, Serratia: Ferment lactose more slowly.

●       H2S production on TSI agar:

○       Salmonella, Proteus: Produce hydrogen sulfide (H2S), which turns the medium black.

Important tests:

●       Oxidase test:

○       Positive in Neisseria gonorrhoeae, Moraxella, and Campylobacter jejuni.

○       Helps differentiate between oxidase-positive and oxidase-negative organisms.

●       Urease production:

○       Seen in Helicobacter pylori, which breaks down urea to produce ammonia.

Important pathogens:

●       Haemophilus influenzae: Causes respiratory and meningeal infections.

●       Bordetella pertussis: Causes whooping cough.

●       Neisseria gonorrhoeae: Causes gonorrhea.

●       Pseudomonas: Opportunistic pathogen causing severe infections.

●       Salmonella: Causes typhoid fever and gastroenteritis.

Giemsa Stain

Giemsa stain binds to nucleic acids, highlighting the nucleus and cytoplasm, making it ideal for visualizing intracellular organisms, certain extracellular bacteria, and protozoa. It produces purple-blue organisms against a lighter background, enhancing contrast for microscopic examination.

Common uses include detecting:

●       Chlamydia spp. – Obligate intracellular bacteria lacking peptidoglycan, making Gram staining ineffective.

●       Rickettsia spp. – Intracellular bacteria transmitted by arthropods (ticks, fleas, lice).

●       Borrelia spp. – A large spirochete visible with Giemsa stain, responsible for Lyme disease and relapsing fever.

●       Trypanosomes – Extracellular protozoa causing Chagas disease and African sleeping sickness, recognized by their flagellated morphology in blood smears.

●       Helicobacter pylori: Spiral-shaped Gram-negative bacteria detected in gastric biopsies.

●       Plasmodium spp. – Malaria parasites in RBCs, identified at various life stages (trophozoites, schizonts, gametocytes) in blood smears.

Figure 3. Gametocyte of plasmodium vivax on peripheral blood smear stained with giemsa.

Periodic Acid–Schiff (PAS) Stain

PAS stain detects glycogen, mucopolysaccharides, and other carbohydrate-rich structures, producing a magenta coloration. It is commonly used to highlight glycoproteins in basement membranes and fungal cell walls, and mucins in epithelial cells.

Clinical applications:

●       Whipple disease (Tropheryma whipplei) – PAS-positive foamy macrophages in the lamina propria of the small intestine aid in diagnosis.

●       Fungal infections – Stains the polysaccharide-rich cell walls of fungi, aiding in their identification.

●       Glycogen storage diseases – Highlights intracellular glycogen deposits in affected tissues.

Figure 4. Whipple’s disease: intensely PAS-positive macrophages occupying the lamina propria mucosae.

Review Article Whipple's Disease: Our Own Experience and Review of the Literature - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Whipples-disease-intensely-PAS-positive-macrophages-occupying-the-lamina-propria_fig14_241060805 [accessed 11 Mar 2025]

Ziehl-Neelsen Stain (Carbol Fuchsin Stain)

The Ziehl-Neelsen (Carbol Fuchsin) stain detects acid-fast organisms with mycolic acid in their cell walls, which resist decolorization. It is used to identify:

●       Mycobacteria (e.g., M. tuberculosis)

●       Nocardia

●       Cryptosporidium

Auramine-rhodamine stain is a more sensitive, fluorescent alternative but less specific, often requiring confirmation with Ziehl-Neelsen for accuracy.

Figure 5. Mycobacterium tuberculosis in Ziehl Neelsen stain.

India Ink Stain

The India Ink stain is used to detect the capsule of Cryptococcus neoformans, a fungus with a thick polysaccharide capsule. The ink stains the background, leaving a clear halo around the cells.

Alternatively, the Mucicarmine stain can highlight the capsule in red, providing a more specific visualization.

Figure 6. Crytpococcus neoformans under light India Ink stain.

Centers for Disease Control and Prevention. (1969). Cryptococcus neoformans using a light India ink staining preparation [Photomicrograph]. Public Health Image Library. https://commons.wikimedia.org/wiki/File:Cryptococcus_neoformans_using_a_light_India_ink_staining_preparation_PHIL_3771_lores.jpg

Silver Stain

The Silver Stain is a specialized staining technique used to highlight various microorganisms, particularly those that may be difficult to visualize with standard stains. It is especially useful for detecting:

●       Helicobacter pylori, a bacterium associated with peptic ulcers and gastritis.

●       Legionella, the causative agent of Legionnaires' disease, which can cause severe pneumonia.

●       Bartonella henselae, the bacterium responsible for cat scratch disease.

●       Coccidioides, a fungus that causes coccidioidomycosis (Valley fever), primarily in the southwestern United States.

●       Pneumocystis jirovecii, a fungus that causes pneumonia in immunocompromised individuals, especially those with HIV/AIDS.

●       Aspergillus fumigatus, a fungus that can lead to infections in immunocompromised patients, such as those with neutropenia.

●     Histoplasma capsulatum, a dimorphic fungus that causes histoplasmosis, commonly found in soil contaminated with bird or bat droppings, particularly in the Ohio and Mississippi River valleys.

Figure 7. A small intestine sample stained using the Grocott's methenamine silver stain demonstrating histoplasma (black round yeast with narrow budding) in a granuloma.

Häggström, M. (n.d.). Own work, CC BY 4.0. Retrieved from https://commons.wikimedia.org/w/index.php?curid=132594351

Fluorescent Antibody Stain

The fluorescent antibody stain uses antibodies labeled with fluorescent dyes to detect bacteria and protozoa. It's used to identify:

●       Treponema pallidum (syphilis) via the FTA-ABS test

●       Giardia and Cryptosporidium (gastrointestinal infections)

●       Pneumocystis jirovecii (pneumonia in immunocompromised patients)

The fluorescent signal allows for sensitive and specific pathogen detection under a microscope.

Figure 8. Treponema pallidum under fluorescent antibody stain.

Centers for Disease Control and Prevention (CDC). (1967). Photomicrograph of Treponema pallidum using fluorescent antibody staining [Image]. Public Health Image Library (PHIL). Retrieved from https://phil.cdc.gov/Details.aspx?pid=14967

Special Culture Requirements

Culturing bacteria requires specific growth conditions. Selective and differential media help isolate pathogens efficiently.

Table 1: Culture media and associated bacteria

Figure 9. Alpha-hemolysis characteristic of Streptococcus pneumoniae on the blood agar plate.

Figure 10. Chocolate agar in Petri dishes.

Figure 11. Escherichia coli on Macconkey agar.