Physical Methods to Kill Microbes or Taking Microbes by Force

In previous posts, the control of a microbial population such as in a pond was discussed by using chemical means or disinfectants (“How to Properly Use a Cleaner – Things One Should Know About Disinfectants”), and by using biocontrol or by adding viable microbes as with probiotics (“You Get More Flies with Honey and You Get Better Microbes with Probiotics”). This post will go over another means of controlling microbes, such as the physical means used to kill microbes, or utilizing forces and energy to control their populations. Many of the physical means to kill microbes are not used in the case of the pond enthusiast, but some are, and those that are not still have valuable applications in science, medicine, and in food processing. So, a brief description will be given for each one and its applications. Finally, how these apply to the pond enthusiast will be discussed.
The most common physical method used to treat microbes is by heat. Heat kills microbes by drying them out and by denaturing the proteins within the microbial cells. Protein denaturation is the process where proteins lose their usual three dimensional shapes. In order for a protein to function biologically, it must retain its specific shape (Figure 1). Therefore, heat inactivates proteins within a cell. Several methods utilizing heat are used:

Figure 1: Protein denaturation. The protein on the right maintains it’s biologically active, three dimensional shape with various chemical interactions. Certain conditions, like excessive heat, will cause the protein to lose its form, as seen on the left, and become denatured and biologically inactive.

1. Incineration (>500°C):
This is simply killing microbes by burning them. It vaporizes organic matter but destroys many other substances other than microbes in the process. This is utilized in the lab for sterilizing inoculating wires used to inoculate new microbial cultures in order to ensure there is no contamination in microbiological and other types of experiments.
2. Boiling (100°C):
Doing this for 30 minutes will normally kill all microbes except for some endospores. Endospores are a stripped down, dormant form that some bacteria can adopt when conditions are unfavourable. Bacteria as endospores can remain dormant for a long time until favourable conditions trigger a bacterium to exit the endospore state and enter the active or replicative vegetative state. Endospores pose a large challenge in sterilization because they are highly resistant to many adverse effects like heat and pressure. In order to completely sterilize and kill the endospores, boiling for very long periods of time (>6hrs) is recommended or for intermittent periods of time with cooling.
3. Autoclaving (121°C/15psi/15 minutes):
This is a very efficient method of sterilization. It is used very commonly to sterilize biohazardous waste, surgical dressings, microbiological media, and other liquids in hospitals and laboratories. This method employs heat with pressure in a synergistic fashion to kill microbes. The steam created in an autoclave can reach higher than normal temperatures due to the high pressure. Due to this, autoclaving is very effective at killing the heat and pressure resistant endospores. The standard autoclave cycle is 121°C at 15psi (pound per square inch) for 15 minutes. Therefore, it is a very fast method to completely sterilize objects. The principle of autoclaving comes from the concept of latent heat of vaporization. This means that steam contains much more energy than boiling water. For example, it takes about 80 calories of energy to boil 1 litre of water but much more energy to convert the same amount of boiling water to steam, 540 calories. Therefore, steam contains much more energy and heat to kill microbes.
4. Dry heat or a hot air oven (160°C/2hours or 170°C/1hour):
This is what is used when cooking food, essentially. It does not utilize the more effective wet heat of an autoclave, so longer treatment times are needed. This method is employed to prepare food, and to sterilize glassware, and metal objects. Essentially, objects that will not melt at these high temperatures can be sterilized in a hot air oven.
Cold can be considered a physical method of controlling microbes though it is not a specific force but rather a lack of one, heat. The cold, conversely, does not sterilize or kill microbes but simply slows down or ceases microbial growth. Microbes that have been cooled will generally resume replication upon returning to more ideal temperatures. This method is very popular for food preservation as most of us who have a refrigerator or freezer will know. Many bacteria that do not require oxygen for survival, or otherwise known as the strict anaerobes (ex: Clostridium perfringens and some strains of Clostridium botulinum, or the bacterium that causes botulism), cannot reproduce below 12°C. However, some types of bacteria (ex: Listeria monocytogenes and Yersinia enterocolitica) can replicate below 1°C. Some microbes which are commonly associated with food spoilage can still multiply, albeit slowly, at temperatures as cold as -7°C. At -18°C, however, food can be properly stored since all microbial growth ceases at and below this temperature. Slow loss of quality to food will still occur, but this is not due to microbes but rather from enzymatic degradation in the food.
Other forces used to kill microbes include pressure. High pressure is a well established, non-thermal method used to kill bacteria, yeasts and moulds. Aside from bursting the cells, high pressure alters macromolecules (ex: proteins, polysaccharides), which are present in microbes among other organisms, but it has little effect on smaller molecules. Since the chemical compounds which affect flavours and odours are often smaller molecules, pressure is an excellent force used to pasteurize foods like fruit juices, jams, and dressings. Pressure is also used in the laboratory to kill and burst microbes to study their insides. The French Press is the machine used to do this in the laboratory. It forces a pressurized cell suspension through a narrow orifice (Figure 2). The pressure drop, once the suspension reaches the orifice, combined with the shearing forces, causes the necessary damage to open up the microbes.

Figure 2: The French Press. Pressure is applied from the top and the cells are forced through a narrow orifice at the bottom. The pressure drop once the cells reach the orifice, and the shearing forces, causes the cells to burst open.

Sonication is another method used in the laboratory to burst open cells. This method employs sonic energy. The cells, which will be burst open, are suspended in water and sonic pressure waves are applied, which causes the rapid formation and collapse of micro bubbles. These bubbles, when they collapse, generate shock waves that will disrupt the cell walls of the microbes.
Aside from pressure, heat and sonic energy, yet another force can kill microbes. This force is osmotic pressure. Osmotic pressure often deals with biological processes because it involves a semi-permeable membrane like the cell membranes that living things possess. Small molecules like water can traverse this membrane so, when a concentration gradient like that caused by the presence of a salt or sugar exists across this membrane, the water will pass through this membrane in order to equalize the concentration gradient. Therefore, taking a cell from a solution higher in salts or sugars, which will allow the chemicals to slowly permeate into the cell since they are small enough molecules, then transferring the cells to distilled water will result in a rapid influx of water into the cells due to an osmotic gradient, and the cells will burst (Figure 3). This is often used to break open mammalian cells in the laboratory. Bacterial and fungal cell walls are stronger and will generally not burst due to osmotic pressure. Osmotic pressure is used to preserve food by adding sugars or salts to foods. Though microbes may not burst and die from this, adding sugars or salts will result in essential water being removed from the microbes, since there will be a higher concentration in the solution and thus a concentration gradient.

Figure 3: The effects of osmotic pressure on red blood cells. In the centre panel, the osmotic pressure is perfectly balanced and the water entering and leaving the cells is equal. On the left, water leaves the cells and they dry out, due to a higher concentration of solutes outside of the cell. On the right, water enters the cells and they burst due to a higher concentration of solutes inside the cell.

Finally, come some physical methods used to control microbial growth in the pond, one being filtration. Filtration is simply passing a liquid or gas through cavities where, depending on the size of the cavities, certain impurities or contaminants cannot pass through. Filters are used to remove unwanted substances in our air when we are in certain buildings (office buildings, laboratories, hospitals, etc.). Filters in a laboratory setting can be used to sterilize solutions that would otherwise be destroyed if heat were used to sterilize them (ex: enzymes and vaccines). However, these filters are extremely fine in order to catch bacteria that are usually one micron, or 0.001 millimetres in size! Filters are also used in ponds, not necessarily to eliminate microbes, but to remove larger particulate matter that may detriment the appearance of a pond. Filters, in this case, actually serve as a haven for the biocontrol microbes that may be added to a pond to regulate microbial growth (As reviewed in the last post “You Get More Flies with Honey and You Get Better Microbes with Probiotics”). The biocontrol microbes will get caught in the filter and will colonize it, which is necessary for the microbes to achieve high enough numbers.
Another physical force used in ponds to control microbial populations, which is also used in the food processing industry and in medicine, is irradiation. What one needs to know about light, otherwise known as electromagnetic radiation, is that it exists at many wavelengths. Only a small fraction of light, or wavelengths, are visible. There are other forms of invisible light; this includes X-rays, gamma rays and ultra violet (UV) light. These forms of light have very short wavelengths and possess a lot of energy, and due to this, can be damaging to life forms (Figure 4).

Figure 4: The electromagnetic spectrum. The wavelengths of the electromagnetic radiation are represented by the wave on the top and the approximate size of the various waves is scaled to various objects on the bottom. Visible light makes up only a small fraction of electromagnetic radiation in the centre. The higher energy wavelengths, such as UV, gamma, and X-rays, are shorter and are situated on the right.

They exert their effect primarily by ionizing water molecules to form highly reactive hydroxyl radicals, which are compounds that can react and change the structures and compositions of other molecules present, some of which may be essential to normal biological function. UV light also causes damage by altering the structure of DNA, the genetic code of most organisms and the molecule essential for the replication of these organisms. UV light causes units in the code, called thymines, to dimerize or simply to bind together in an abnormal fashion (Figure 5). Once the thymines are dimerized, enzymes that would normally replicate the DNA when the cell replicates are unable to do so, thus cell replication is hindered. UV light is used in some pond filters. As water is passed through the filter, planktonic or free floating algae and bacteria come into contact with the UV light and are killed, should the conditions be ideal. However, algae and bacteria found in biofilms (as described in the second post “No Microbe is an Island: Biofilms”) are unaffected by the UV light since they grow on a surface in the pond and will not have a chance to pass through the filter.

Figure 5: Thymine dimerization. UV light, shown coming in on the left can dimerize thymines, which comprise part of the genetic code of DNA. On the left the thymines, shown in yellow, are bound to their normal counterparts in green. UV light can assist in binding these thymines together to form a dimer, seen on the right, which will alter the form of DNA and inhibit replication of the molecule when the cell divides.

In conclusion, there are many forces that can be used to sterilize or control microbial growth. Some, such as radiation and filtration, in addition to being used in the food processing industry, in laboratories, and in medicine, are also used to treat ponds. Ponds may have filters equipped with UV lights to aide in controlling microbial growth, however, not all ponds have filters, and not all filters have UV lights. Even if a pond is equipped with both, only regular maintenance will ensure the maximum efficiency of both. Filters may become plugged and dirty, and the UV light bulbs may also become dirty, reducing their efficaciousness, or burn out. If you, the pond enthusiast, is not certain whether or not your filter system with UV light is functioning best, or if you may want such a system installed, call us at Village Pond and Garden and we can discuss filter options with you.

In the next installment → Water, water everywhere! So what are we to think? Water quality and the pond.

Summary/Important Points

• The most common physical method used to kill microbes or control their replication is by heat (Ex: Incineration, boiling, autoclaving, and dry heat).
• Heat functions by drying out microbes and by denaturing their proteins. Denaturing proteins is when the configuration, or the three dimensional shape of the protein is lost. This shape is necessary to retain the biological activity of the protein.
• Cold is a physical method of controlling growth as seen commonly with refrigerators and freezers, but it does not kill microbes. It only slows or temporarily ceases their replication.
• Pressure and sonication, or sonic energy, are also used to kill microbes and burst them open.
• Osmotic pressure is caused by a concentration gradient across a semi-permeable membrane, as seen with cell membranes in living things. While the concentration gradient is being equalized, it can result in the cells bursting and dying due to an influx of water, or it can remove essential water and detriment the cells.
• Filtration and irradiation are used in ponds and in other applications, in some cases to control microbial populations. Filtration does not damage microbes but simply removes them from the solution, whereas the high energy electromagnetic radiation, such as UV light, actually does damage to the components of a cell.

References/Further Reading

Gould, GW. 2000. Preservation: past, present and future. British Medical Bulletin. 1:84-96.
Grabski, AC. 2009. Advances in Preparation of Biological Extracts for Protein Purification. Methods in Enzymolgy. 463:285-303.

Protein denaturation http://en.wikipedia.org/wiki/File:Protein_folding_schematic.png
French Press http://en.wikipedia.org/wiki/File:French_press.gif
Osmotic pressure http://en.wikipedia.org/wiki/File:Osmotic_pressure_on_blood_cells_diagram.svg
Electro-magnetic spectrum http://en.wikipedia.org/wiki/File:EM_Spectrum_Properties_edit.svg
Thymine dimerization http://upload.wikimedia.org/wikipedia/commons/f/fd/DNA_UV_mutation.svg

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