Direct Gram stain of a tissue biopsy shows Gram-positive bacilli with central spores; after 24 hours incubation, no growth on sheep blood agar aerobically or on chocolate agar incubated in increased CO2. Which organism is most likely responsible?

• A. Bacillus
• B. Lactobacillus
• C. Clostridium
• D. Prevotella

Answer: (C)

Obligate anaerobe that forms spores explains the best fit here. The direct Gram stain showing Gram-positive bacilli with spores points to spore-forming rods such as Bacillus or Clostridium. However, the culture result—no growth after 24 hours on blood agar in air or on chocolate agar with increased CO2—shows the organism does not tolerate oxygen and requires a strictly anaerobic environment. Bacillus species are typically aerobic or facultatively anaerobic and would grow under those conditions. Lactobacillus and Prevotella don’t match the Gram-stain description (one is non-spore-forming Gram-positive rods; the other is Gram-negative). Among the spore-formers, the inability to grow in the presence of oxygen points to Clostridium as the most likely cause, consistent with anaerobic tissue infections where Clostridium spp. thrive and form spores.