Sterilization of Spores and Toxins
Unfortunate for us but fortunate for certain bacteria, spores can be can be formed in adverse environmental conditions. This is often the case in dry environments. Bacterial spores are referred to as endospores because they actually form within the bacterial cell. An endospore bacterium can survive a number of harsh conditions such as heat, drying, radiation, and chemicals. Other organisms form spores, but the bacterial spore is generally more heat resistant and difficult to denature. The greater heat resistance is hidden in the very structure of an endospore. See drawing below.
Note the exosporium and its delicate thin membrane that is made up of protein. Inside of that is a spore coat that is made up of a number of layers of protein unique to that layer. The next layer in is called the cortex which contains loosely linked peptidoglycans (sugar proteins) that contain within them another molecule known as dipicolinic acid (DPA). DPA has the ability to cross-link with calcium that is embedded within the spore coat.The calcium cross-links contribute to the heat resistance of the bacterium making for a hard barrier to penetrate. Note that the bacterium is in the center of the endospore. The endospore makes it difficult to kill bacteria.
Endotoxins are molecules that are commonly found in gram negative cell walls. These molecules are meant to protect bacteria from various threats. Once a bacterium dies, it releases these endotoxins to the surround because their cell walls have been compromised. These molecules are toxic (poisonous) to humans and animals as well as other organisms. Endotoxins are only harmful once the bacteria dies. These toxic molecules can cause diarrhea, vomiting, fever, and inflammatory responses. Many endotoxins are in lipopolysaccharide (LPS) form, however there are other molecular types. It is possible to kill endotoxins with both dry and wet heat.
Exotoxins are different from endotoxins. An exotoxin is actually secreted by a bacterium. The presence of an exotoxic bacterium will create problems for a host when the bacterium is alive unlike endotoxic bacteria. Exotoxic bacteria also release endogenous exotoxins upon lysing as well. Most exotoxins can be deactivated by heating. An example of an exotoxin would be botulinum toxin which comes from Clostridum botulinum.
Pyrogens and Sterility
Pyrogens are molecules that cause fever. Common pyrogens are produced by bacteria and come in the form of endotoxins and exotoxins. Keep in mind that animals also produce pyrogens but that’s another story. One would think that sterilization would take care of these bacterial toxins since the bacteria have been destroyed. Not necessarily. In the process of sterilization, bacteria are destroyed but this releases endogenous exotoxins and endotoxins. Make sure you have used the appropriate temperature and times to deactivate not only bacteria but their toxins as well. When buying products for your lab, you need to pay attention to what it says on packaging. Some products may be listed as sterile, sterile and pyrogen-free, or pyrogen-free but not sterile. Sterile itself does not mean pyrogen-free.