Direct Detection Methods for Bacillus and Similar Organisms
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p268-269
2025-05-29
599
The Gram stain is the only specific procedure for the direct detection of Bacillus spp. in clinical specimens. Microscopically the organisms appear as large gram-positive rods in singles, pairs, or serpentine changes (Figure 1).
Fig1. Gram stain of Bacillus cereus. The arrow is pointed at a spore, the clear area inside the gram-positive vegetative cell.
Bacillus spp. are the only clinically relevant aerobic organisms capable of producing endospores in the presence of oxygen. Sporulation is inhibited by high concentrations of CO2. The production of spores may be induced by growth in triple sugar iron (TSI), urea, or nutrient agar containing 5 mg/L manganese sulfate. Spores may appear as intra or extracellular clear oval structures upon Gram staining. Special staining is required in order to visualize endospores. The smear is covered with malachite green, and a piece of filter paper is placed over the stain. The microscope slide is then heated for several minutes to force the dye into the cell walls of the spore. During the heating process, it is important to keep the filter paper moist so that the stain is steamed rather than baked into the endospores. A safranin counterstain follows the primary stain. The endospores stain green and the vegetative cells will appear pink from the secondary stain, safranin (Figure 2).
Fig2. Spore stain of Bacillus cereus. The arrows are pointed at green spores in a pink vegetative cell.
The vegetative cell width of B. anthracis, B. cereus, B. mycoides, B. thuringiensis, and B. megaterium is usually greater than 1 µm, and the spores do not cause swelling of the cell. The vegetative cell width of B. subtilis, B. pumilus, and B. licheniformis is less than 1 µm, and the spores do not cause swelling of the cell. The cell width of B. circulans, B. coagulans, B. sphaericus, B. brevis, P. macerans, P. alvei, and P. polymyxa is less than 1 µm, and the spores cause the cell to swell. When determining cell width, only the cells that stain gram-positive should be measured. Organisms that fail to retain the crystal violet appear narrower.
Direct detection of B. anthracis in clinical and environ mental samples is also available using molecular and antigen-based methods. The immunohistochemical method available from the CDC uses antibodies specific to the organism’s cell wall antigen or capsule for the detection of B. anthracis. A positive molecular amplification assay, PCR, from a normally sterile site is considered a presumptive diagnosis for anthrax infection.
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