Radiation: A Sterilization Method

Nonionizing Radiation:

Nonionizing radiation has a wavelength longer than that or ionizing radiation, usually greater than about 1 nm. The best example of non ionizing radiation is ultraviolet (UV) light.

Mode of Action:

When microorganisms are subjected to UV light, cellular DNA absorbs the energy by purines and pyrimidine bases, and adjacent thymine molecules link together, as figure illustrates. Linked thymine molecules are unable to encode adenine on messenger RNA molecules during the process of protein synthesis. Moreover, replication of the chromosome in binary fission is impaired. The damaged organism can no longer produce critical proteins or reproduce, and it quickly dies. Ultraviolet light is especially effective in inactivating viruses. However, it kills far fewer bacteria than one might expect because of DNA repair mechanisms. Once DNA is repaired, new molecules of RNA and protein can be synthesized to replace the damaged molecules.

Conditions for Microbe control:

Ultraviolet (UV) light consists of light of wavelengths between 40 to 390 nm, but wavelength in the 200 nm range are most effective in killing microorganisms But according to some books wavelength between 260 – 265 nm is most effective.

Uses of Ultraviolet:

Ultraviolet light effectively reduces the microbial population where direct exposure takes place. It is used to limit airborne or surface contamination in a hospital room, morgue, pharmacy, toilet facility, or food service operation. In some communities, ultraviolet light is replacing chlorine in sewage treatment. When chlorine-treated sewage effluent is discharged into streams or other bodies of water, carcinogenic compounds form and may enter the food chain. The cost of removing chlorine before discharging treated effluent could add as much as $100 per year to the sewage bills of the average American family, and very few sewage plants do this. Running the sewage effluent under ultraviolet light before discharging it can destroy microorganism without altering the odor, pH, or chemical composition of the water and without forming carcinogenic compounds.

Advantages:

It does penetrate air, effectively reducing the number of airborne microorganism and killing them on surfaces on operating rooms and rooms that will contain caged animals. To help sanitize the air without irradiation humans, these lights can be turned on when there rooms are not in use. . Hanging laundry outdoors on bright, sunny days takes advantage of the ultraviolet light present in sunlight. Although the quantity of UV rays in sunlight is small, these rays may help kill bacteria on clothing, especially diapers.

Disadvantage of UV Light:

A major disadvantage of UV light as a disinfectant is that the radiation is not very penetrating, so the organism to be killed must be directly exposed to the rays. It is noteworthy microorganisms in the air and upper layers of the soil, but it may not the effective against all bacterial spores. Organisms protected by solids and such coverings as paper, glass, and textiles are not affected. Another potential problem is that UV light can damage human eyes, and prolonged exposure can cause burns and skin cancer in humans. And it may cause damage in human skin cells and permanent damage the eyes.

SUN--Free Source of UV:

Sunlight contains some UV radiation, but the shorter wavelengths – those most effective against bacteria – are screened out by the ozone layer of the atmosphere. The antimicrobial effect of sunlight is due almost entirely to the formation of singlet oxygen on the cytoplasm. Many pigments produced by bacteria provide protection from sunlight.

Filtration: Types of Filters

Several types of filters are available for use in the microbiology laboratory. Inorganic filters are typified by the Seitz filter, which consists of a pad of porcelain or ground glass mounted in a filter flask.

Organic filters:

Organic filters are advantageous because the organic molecules of the filter attract organic components in microorganisms. They are given below:

1) Berkefeld filter:

One example, The Berkefeld filter, utilizes as substance called diatomaceous earth. This material contains the remains of marine algae known as diatoms. Diatoms are unicellular algae that abound in oceans and provide important foundations for the world's food chains. Their remains accumulate on the shoreline and are gathered for use in swimming pool aquarium filters, as well as for microbiological filters used in laboratories.

2) Membrane Filter:

The membrane filter is at third type of filter that has received broad acceptance. It consists of a pad of organic compounds such as cellulose acetate (cellulose esters) or polycarbonate (plastic polymers), mounted in a holding device. These filters are only 0.1 mm thick. The pores of membrane filters include, for example, 0.22μm and 0.45μm sizes, which are intended for bacteria. This filter is particularly valuable because bacteria multiply and for colonies on the filter pad when the pad is place on a plate of culture medium. Microbiologists can then count the colonies to determine the number of bacteria originally present. For example, if a 100-ml sample of liquid were filtered and 59 colonies appeared on the pad after incubation, it could be assumed that 59 bacteria were in the sample. However, Some very flexible bacteria, such as spirochetes, or the wall less mycoplasma, will sometimes pass through such filters.

Membrane filters used to trap bacteria form air and water samples can be transferred directly to agar plates, and the quantity of bacteria in the sample can be determined. Alternatively, the filters can be transferred from one medium to another, so organisms with different nutrient requirements can be detected. Filtration is also used to remove microorganisms and other small particles from public water supplies and in sewage treatment facilities. This technique, however, cannot sterilize; it merely reduces contamination.

3) HEPA filters:

Air can also be filtered to remove microorganisms. The filter generally used is a high-efficiency particulate air (HEPA) filter. This apparatus can remove over 99 percent of all particles, including microorganisms with a diameter larger than 0.3 μm. The air entering surgical units and specialized treatment facilities, such as burn units, is filtered to exclude microorganisms. In some hospital wards, such as for respiratory diseases, and in certain pharmaceutical filling rooms, the air is recirculated through HEPA filters to ensure its purity. Used filter are soaked in formalin before they are disposed of.

Industrial Fluid Filtration:

Two examples of filters used in conjunction with fluids. (a) A filter of woven mesh Dacron (arrow) is used to trap clumps of unwanted blood cells that might otherwise enter the recipient's circulation during a transfusion. (b) A cartridge filter removes contaminants from fluids to be used for intravenous injections or for other medical purposes.

The Membrane Filter Technique:

a) The membrane filter consists of a pad of cellulose acetate, or similar material, mounted in a holding device. (b) The holding device is secured by a clamp, and a measured amount of fluid is filtered by pouring it into the cup. The solution runs through to a flask beneath, and bacteria are trapped in the filter material. (c) The filter pad is place onto a plate of nutritious medium, and the plate is incubated. (d) After incubation, colonies appear on the surface of the filter pad. The colony count reflects the original number of bacteria in the fluid sample.

Suitable Selection of Filters --- By pore size

In the manufacture of vaccines that require the presence of live viruses, it is important to select a filter pore size that will allow viruses to pass but prevent bacteria from doing so. By selecting a filter with a proper pore size, scientists can separate polioviruses from the fluid and debris in tissue cultures in which they were grown. This procedure simplifies the manufacture of polio vaccine. Cellulose acetate filters with extremely tiny pores are now available and are capable of removing many viruses (although not the very smallest) from liquids. However, these filters are expensive and clog easily.

Filtration

Importance of Filter in Microbiology history:

In the early days of microbiology, hollow candle shaped filters of unglazed porcelain were used to filter liquids. The long and indirect passageways through the walls of the filter adsorbed the bacteria. Filters came into prominent use in microbiology as interest in viruses grew during the 1890s. Previous to that time, filters had been utilized to trap airborne organisms and sterilize bacteriological media, but now they became essential for separating viruses from other microorganisms. Among the early pioneers of filter technology was Charles Chamberland, as associate of Pasteur. His porcelain filter was important to early virus research. Another pioneer was Julius Petri (inventor of Petri dish), who developed a sand filter to separate bacteria from the air.

Introduction and Action:

Filtration is the passage of a liquid or gas through a screen like material with pores small enough to retain microorganisms (often the same apparatus used for counting. A vacuum that is created in the receiving flask helps gravity pull the liquid through the filter. As fluid passes through the filter, organisms are trapped in the pores of the filtering material, as (figure) shows.

The solution that drips into the receiving container is decontaminated or, in some cases, sterilized.They are usually made of nitrocellulose and have the great advantage that they can be manufactured with specific pore sizes from 25 µm to less than 0.025μm. Particles filtered by various pore sizes are summarized in table.

Table 4	Pore sizes of membrane filters and particles that pass through them
Pore Size in (µm)	Particles that pass through them
10	Erythrocytes, yeast cells, bacteria, viruses, molecules
5	Yeast cells, bacteria, viruses, molecules
3	Some yeast cells, bacteria, viruses, molecules
1.2	Most bacteria, viruses, molecules
0.45	A few bacteria, viruses, molecules
0.22	Viruses, molecules
0.10	Medium-sized to Small Viruses, molecules
0.05	Small viruses, molecules
0.025	Only the very smallest viruses, molecules
Ultra-filter	Small molecules

Uses of Filtration:

Membrane-filters are used to sterilize heat sensitive materials include media, special nutrients that might be added to media, enzymes, vaccines, and pharmaceutical products such as drugs, sera, and vitamins. They are also used to sterilize the things such things as beverages, intravenous solutions and bacteriological media. Some operating theaters and rooms occupied by burn patients receive filtered air to lower the numbers of air borne microbes.

Some filters can be attached to syringes so that materials can be forced through them relatively quickly. Filtration can also be used instead of pasteurization in the manufacture of beer. When using filters to sterilize materials, it is important to select a filter pore size that will prevent any infectious agent from passing into the product.

Advantages:

Membrane filters have certain advantages and disadvantages. Except for those with the smallest pore sizes, membrane filters are relatively inexpensive, do not clog easily, and can filter large volumes of fluid reasonably rapidly. They can be autoclaved or purchased already sterilized.

Disadvantages:

A disadvantage of membrane filters is that many of them allow viruses and some mycoplasmas to pass through. Other disadvantages are that they may absorb relatively large amounts of the filtrate and may introduce metallic ions into the filtrate.

Different types of filters will be discussed in some future post.