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Sterilization Techniques in Laboratory

Heat: most important and widely used. For sterilization one must consider the type of heat, and most importantly, the time of application and temperature to ensure destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric of all cells so their destruction guarantees sterility.
Incineration: burns organisms and physically destroys them. Used for needles, inoculating wires, glassware, etc. and objects not destroyed in the incineration process.
Boiling: 100°C for 30 minutes. Kills everything except some endospores. To kill endospores, and therefore sterilize a solution, very long (>6 hours) boiling, or intermittent boiling is required (See Table 1 below).
Autoclaving (steam under pressure or pressure cooker) Autoclaving is the most effective and most efficient means of sterilization. All autoclaves operate on a time/temperature relationship. These two variables are extremely important. Higher temperatures ensure more rapid killing. The usual standard temperature/pressure employed is 121°C/15 psi for 15 minutes. Longer times are needed for larger loads, large volumes of liquid, and more dense materials. Autoclaving is ideal for sterilizing biohazardous waste, surgical dressings, glassware, many types of microbiologic media, liquids, and many other things. However, certain items, such as plastics and certain medical instruments (e.g. fiber-optic endoscopes), cannot withstand autoclaving and should be sterilized with chemical or gas sterilants. When proper conditions and time are employed, no living organisms will survive a trip through an autoclave.
Schematic diagram of a laboratory autoclave in use to sterilize microbiological culture medium. Sterilization of microbiological culture media is is often carried out with the autoclave. When microbiological media are prepared, they must be sterilized and rendered free of microbial contamination from air, glassware, hands, etc. The sterilization process is a 100% kill, and guarantees that the medium will stay sterile unless exposed to contaminants.
An autoclave for use in a laboratory or hospital setting. Why is an autoclave such an effective sterilizer? The autoclave is a large pressure cooker; it operates by using steam under pressure as the sterilizing agent. High pressures enable steam to reach high temperatures, thus increasing its heat content and killing power. Most of the heating power of steam comes from its latent heat of vaporization. This is the amount of heat required to convert boiling water to steam. This amount of heat is large compared to that required to make water hot. For example, it takes 80 calories to make 1 liter of water boil, but 540 calories to convert that boiling water to steam. Therefore, steam at 100°C has almost seven times more heat than boiling water.
Moist heat is thought to kill microorganisms by causing denaturation of essential proteins. Death rate is directly proportional to the concentration of microorganisms at any given time. The time required to kill a known population of microorganisms in a specific suspension at a particular temperature is referred to as thermal death time (TDT). Increasing the temperature decreases TDT, and lowering the temperature increases TDT. Processes conducted at high temperatures for short periods of time are preferred over lower temperatures for longer times.
Environmental conditions also influence TDT. Increased heat causes increased toxicity of metabolic products and toxins. TDT decreases with pronounced acidic or basic pHs. However, fats and oils slow heat penetration and increase TDT. It must be remembered that thermal death times are not precise values; they measure the effectiveness and rapidity of a sterilization process. Autoclaving 121°C/15 psi for 15 minutes exceeds the thermal death time for most organisms except some extraordinary sporeformers.
Dry heat (hot air oven): basically the cooking oven. The rules of relating time and temperature apply, but dry heat is not as effective as moist heat (i.e., higher temperatures are needed for longer periods of time). For example 160°/2hours or 170°/1hour is necessary for sterilization. The dry heat oven is used for glassware, metal, and objects that won’t melt.
Irradiation: usually destroys or distorts nucleic acids. Ultraviolet light is commonly used to sterilize the surfaces of objects, although x-rays, gamma radiation and electron beam radiation are also used.
Ultraviolet lamps are used to sterilize workspaces and tools used in microbiology laboratories and health care facilities. UV light at germicidal wavelengths (two peaks, 185 nm and 265 nm) causes adjacent thymine molecules on DNA to dimerize, thereby inhibiting DNA replication (even though the organism may not be killed outright, it will not be able to reproduce). However, since microorganisms can be shielded from ultraviolet light in fissures, cracks and shaded areas, UV lamps should only be used as a supplement to other sterilization techniques.
An ultraviolet sterilization cabinet Gamma radiation and electron beam radiation are forms of ionizing radiation used primarily in the health care industry. Gamma rays, emitted from cobalt-60, are similar in many ways to microwaves and x-rays. Gamma rays delivered during sterilization break chemical bonds by interacting with the electrons of atomic constituents. Gamma rays are highly effective in killing microorganisms and do not leave residues or have sufficient energy to impart radioactivity.
Electron beam (e-beam) radiation, a form of ionizing energy, is generally characterized by low penetration and high-dose rates. E-beam irradiation is similar to gamma radiation in that it alters various chemical and molecular bonds on contact. Beams produced for e-beam sterilization are concentrated, highly-charged streams of electrons generated by the acceleration and conversion of electricity.
e-beam and gamma radiation are for sterilization of items ranging from syringes to cardiothoracic devices.
Filtration involves the physical removal (exclusion) of all cells in a liquid or gas. It is especially important for sterilization of solutions which would be denatured by heat (e.g. antibiotics, injectable drugs, amino acids, vitamins, etc.). Portable units can be used in the field for water purification and industrial units can be used to “pasteurize” beverages. Essentially, solutions or gases are passed through a filter of sufficient pore diameter (generally 0.22 micron) to remove the smallest known bacterial cells.
This water filter for hikers and backpackers is advertised to “eliminate Giardia, Cryptosporidium and most bacteria.” The filter is made from 0.3 micron pleated glass fiber with a carbon core.
A typical set-up in a microbiology laboratory for filtration sterilization of medium components that would be denatured or changed by heat sterilization. The filter is placed (aseptically) on the glass platform, then the funnel is clamped and the fluid is drawn by vacuum into a previously sterilized flask. The recommended size filter that will exclude the smallest bacterial cells is 0.22 micron.
Chemical and gas Chemicals used for sterilization include the gases ethylene oxide and formaldehyde, and liquids such as glutaraldehyde. Ozone, hydrogen peroxide and peracetic acid are also examples of chemical sterilization techniques are based on oxidative capabilities of the chemical.
Ethylene oxide (ETO) is the most commonly used form of chemical sterilization. Due to its low boiling point of 10.4°C at atmospheric pressure, EtO) behaves as a gas at room temperature. EtO chemically reacts with amino acids, proteins, and DNA to prevent microbial reproduction. The sterilization process is carried out in a specialized gas chamber. After sterilization, products are transferred to an aeration cell, where they remain until the gas disperses and the product is safe to handle.
ETO is used for cellulose and plastics irradiation, usually in hermetically sealed packages. Ethylene oxide can be used with a wide range of plastics (e.g. petri dishes, pipettes, syringes, medical devices, etc.) and other materials without affecting their integrity.
An ethylene oxide sterilization gas chamber. Ozone sterilization has been recently approved for use in the U.S. It uses oxygen that is subjected to an intense electrical field that separates oxygen molecules into atomic oxygen, which then combines with other oxygen molecules to form ozone.
Ozone is used as a disinfectant for water and food. It is used in both gas and liquid forms as an antimicrobial agent in the treatment, storage and processing of foods, including meat, poultry and eggs. Many municipalities use ozone technology to purify their water and sewage. Los Angeles has one of the largest municipal ozone water treatment plants in the world. Ozone is used to disinfect swimming pools, and some companies selling bottled water use ozonated water to sterilize containers.
An ozone fogger for sterilization of egg surfaces. The system reacts ozone with water vapors to create powerful oxidizing radicals. This system is totally chemical free and is effective against bacteria, viruses and hazardous microorganisms which are deposited on egg shells.
An ozone sterilizer for use in the hospital or other medical environment. Low Temperature Gas Plasma (LTGP) is used as an alternative to ethylene oxide. It uses a small amount of liquid hydrogen peroxide (H2O2), which is energized with radio frequency waves into gas plasma. This leads to the generation of free radicals and other chemical species, which destroy organisms.
An LTGP sterilizer that pumps vaporized H2O2 into the chamber. We sterilize most media and supplies using a steam autoclave to produce moist heat. Other methods, including filtration, ethylene oxide, radiation, or ultraviolet light, may be necessary if components are heat-labile or materials are not heat-resistant.
An autoclave is designed to deliver steam into a pressure chamber, generating high heat and pressure at the same time. Heating media to above 121 degrees C for 4 to 20 min. destroys nearly all living cells and spores. High pressure (typically 20 lbs/sq. in) allows the temperature to exceed 100 degrees, which can’t be accomplished with steam at one atmosphere. We use an autoclave that starts timing when the temperature reaches 121 degrees, and exhausts the steam slowly after the prescribed time above 121 degrees (to prevent exploding bottles!). The autoclave is effectively a giant pressure cooker.
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To properly use an autoclave
Know the instrument – some are fully automatic, some are fully manual
Prepare supplies properly – the more layers or greater the volume, the longer it will take for the interior to heat up
Check the steam pressure and ensure that the instrument is set for slow exhaust if liquids are to be sterilized
Ensure that the door is closed properly and securely
Check that the time and/or automatic cycle are set properly
Ensure that the temperature is well below 100 degrees before attempting to open the door
Crack the door to allow steam to vent, keeping face and hands well away from the opening
***CAUTION*** Exposing tightly stoppered bottles to variable pressures invites explosion and injury. When heating any liquids using any method, take care disturbing the flask or bottle. Material near the bottom may be superheated and boil over when moved. Stoppers, caps, covers, must be vented – never make them fit tightly.

Plant Adaptation to their Environment

Hanin Al-geizi

Plants require four simple things to live; water, warm temperature, light, minerals and most places that consist partly of these vital requirements, will be hospitable environments for plants. The most important environmental factors to which plants must adapt are water availability, temperature change, sunlight, soil conditions and predation. For any plant to thrive and survive, each of these factors is vital, and plants that have adapted to extreme environments have undergone changes to acclimate and survive. Adaptations happen over time as a response to changing environments. Acclimations allow plants to reduce competition for space, nutrients, predation and increase reproduction. There are some factors which limit these changes: availability of water, light, predation and temperature.
Land plants have a different set of adaptations as compared to desert plants. Desert plants have adapted to the high temperatures and dryness by changing physically and modifying behavioral mechanisms. When land plants adapted to life on land, they had to face environmental challenges. The plants were used to a water environment and on land they were faced with drying out in the air; whereas desert plants have adjusted to the scarcity of water, land plants developed structural support to protect water, store nutrients and reproduce. Life on land for plants had some advantages: sunlight was abundant, water acts as a filter, altering the quality of light absorbed by the photosynthetic chlorophyll, carbon dioxide is more quickly available in air than water since it diffuses faster in air, land plants evolved before land animals; therefore, until dry land was also found by animals, no predators threatened plant life. These circumstances changed as animals appeared from the water and started feeding on the nutrients in the plants. In turn, plants developed strategies to deter predation: from spines and thorns to toxic chemicals. Early land plants did not thrive far from sources of water and developed survival strategies to fight the dryness, called desiccation tolerance. Many mosses can dry out to a brown and brittle mat, but as soon as rain or a flood makes water available, mosses will absorb it and are restored to their healthy green appearance. Another strategy is to colonize environments where droughts are uncommon, such as rainforests where ferns thrive in damp and cool places. As time went by, plants moved away from moist or aquatic environments and developed resistance to desiccation, rather than tolerance. Plants, such as cacti, reduce the loss of water to such an extent they can survive in extremely dry environments.
Cacti are plants that have adapted by changing their physical structure, called Xerophytes. Cacti have evolved a way of storing and conserving water. They altered their structure in order to resist the high temperatures and scarcity of water. They have fewer or no leaves which minimizes transpiration. Cacti are considered the most drought-resistant plants on the planet due to this adaptation as well as their root systems, and the ability to store water in their stems. Another cacti evolutionary adaptation is that they use spines for shade and have waxy skin to seal in moisture which is also a characteristic of land plants. These spines are a protective measure from animals while shading the plant from the sun and gather water. Extensive shallow root systems are spread in the ground, allowing for them to receive large quantities of water when it rains, they store water in the core of both stems and roots, thus allowing them to survive years of drought on the water collected from a single rainfall.
Phreatophytes are also a type of desert plant that has adapted to arid climates by growing extremely long roots, allowing them to collect moisture at or near the water table. The term phreatophyte means water-loving plant.
Other desert plants that acclimated, have evolved a lifecycle according to the seasons of moisture or cool temperatures. These plants are commonly called perennials meaning they live year after year rather than annuals which only survive a season. Desert perennials thrive by sleeping during dry periods of the year, then springing to life when water becomes available. Most annual desert plants grow only after heavy seasonal rain, then complete their reproductive cycle very quickly. They bloom very large for a few weeks in the spring, accounting for most of the annual wildflower explosions of the deserts. Their heat- and drought resistant seeds remain sleeping in the soil until next year’s rains. Some perennials survive by becoming dormant during dry periods, then springing to life when water becomes available. After rain falls, these plants quickly grow a new suit of leaves to photosynthesize food. Flowers bloom within a few weeks, and when seeds become ripe and fall, they lose their leaves again and re-enter dormancy. This process may occur as many as five times a year. They also have a waxy coating on stems, which serves to seal in moisture during periods of dormancy.
Other desert plants use a combination of many adaptations. Instead of thorns, they rely for protection on a smell and taste wildlife find unpleasant. They have tiny leaves that close their stomata (pores) during the day to avoid water loss and open them at night to absorb moisture.
Desert plants must act quickly when heat, moisture, and light goes inside of them, let’s just say it is time to bloom. Another plant adaptation important to the survival and early dominance of flowering plants is the production of secondary plant metabolites. These bad tasting and sometimes toxic compounds have been one of plants most powerful means of defense. These are the defensive mechanisms, which the desert plants use to check predators.
Desert plants have taken land plant adaptations to an extreme and survive due to these changes. Land plants still thrive but these desert plants will be around even after land plants are unable to acclimate. All in all, without water, sunlight, warm temperatures and nutrients neither type of plant has a chance of survival.
Works Cited
(2014) https://www.boundless.com/biology/seedless-plants/early-plant-life/introduction/
(2014) https://www.boundless.com/biology/seedless-plants/early-plant-life/plant-adaptations-to-life-on-land/
(2014) http://biology.about.com/od/landbiomes/a/aa041406a.htm
(2014) http://www.blueplanetbiomes.org/desert_plant_page.htm
(2002) http://biology.unm.edu/ccouncil/Biology_112/Summaries/Plant_Adaptations.html
(2011) http://www.itreeservice.com/pdfs/how_plants_survive_in_the_desert.pdf
(2014) http://library.thinkquest.org/C0113340/text/biomes/biomes.desert.plantadapt.html#
(2014) http://www.desertusa.com/du_plantsurv.html

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