Enumeration of Mesophilic and Thermophilic Spores

Endospores Formation and Enumeration

• Only a few species of bacteria, such as Bacillus, Clostridium, Desulfatmaculum, and Alicyclobacillus, form endospores to resist harsh environmental conditions like UV-light, drying, freezing, and harmful chemicals.
• Endospores are highly resistant to extreme conditions, allowing the bacteria to survive and reproduce when favorable conditions return.

Enumeration of Mesophilic and Thermophilic Spores

• Weigh 25g of food sample and mix with 225ml of 0.1% peptone water (PW) in a sterile beaker or thermostable plastic bag.
• Heat the sample in a water bath at 80°C for 15 minutes to activate thermophilic spores.
• Perform 10-fold serial dilution of the sample (10⁻¹-10⁻⁵).
• Prepare two sets of plates for each dilution and add molten general media (TSA).
• Incubate one set at 30°C and the other set at 55°C in an upside-down position.
• Enumerate the colonies after incubating for about 24 hours.

Anaerobic Incubation

• Use anaerobic chambers or anaerobic jars to create anaerobic conditions for incubation.

Endospore Stain

• Prepare smears of organisms to be tested for endospores.
• Heat fix the smears.
• Saturate the paper with malachite green and heat the slide until steam can be seen rising from the surface.
• Reheat the slide as needed to keep the slide steaming for about three minutes.
• Remove the paper with tweezers and rinse the slide thoroughly with tap water.
• Counterstain with 0.5% safranin for 45 seconds.
• The vegetative cells will appear red, and the spores will appear green.