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Questions and Answers
What is the purpose of heating the sample in a water bath at 80°C for 15 minutes?
What is the purpose of heating the sample in a water bath at 80°C for 15 minutes?
Which of the following species of bacteria is known to form endospores?
Which of the following species of bacteria is known to form endospores?
What is the purpose of using 0.1% PW in the sample preparation?
What is the purpose of using 0.1% PW in the sample preparation?
What is the temperature used for incubating the sample for thermophilic spores?
What is the temperature used for incubating the sample for thermophilic spores?
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What is the purpose of using malachite green in the endospore stain?
What is the purpose of using malachite green in the endospore stain?
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What is the purpose of incubating the sample in an upside-down position?
What is the purpose of incubating the sample in an upside-down position?
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What is the purpose of using 0.5% safranin in the endospore stain?
What is the purpose of using 0.5% safranin in the endospore stain?
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Why is it important to not overheat the slide during the endospore stain?
Why is it important to not overheat the slide during the endospore stain?
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What is the purpose of preparing smears of organisms for the endospore stain?
What is the purpose of preparing smears of organisms for the endospore stain?
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What is the purpose of incubating one set of plates at 30°C and the other set at 55°C?
What is the purpose of incubating one set of plates at 30°C and the other set at 55°C?
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Study Notes
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.
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Description
Procedure for enumerating mesophilic and thermophilic spores in food samples, involving heat treatment and serial dilution.
