Fractional Sterilization
Introduction:
In the years before the development of the autoclave, liquids and other objects were sterilized by exposure to free flowing steam at 100oC for 30 minutes on each of 3 successive days, with incubation periods between the steaming. The method was called fractional sterilization because a fraction was accomplished on each day. It was also called tydallization after its developer, John Tyndall.
Procedure:
Sterilization by fractional method is achieved by an interesting series of events. During the first day's exposure, steam kills virtually all organisms except bacterial spores, and it stimulates spore to germinate to vegetative cells. During overnight incubation, the cells multiply and are killed on the second day. Again, the material is cooled and the few remaining spores germinate, only to be killed on the third day. Although the method usually results in sterilization, occasions arise when several spores fail to germinate. The method also requires the spores be in a suitable medium for germination, such as a broth.
Importance in modern Age:
Fractional sterilization has assumed renewed importance in modern microbiology with the development of high technology instrumentation and new chemical substances. Often, these materials cannot be sterilized at autoclave temperatures, or by long periods of boiling or baking, or with chemicals. An instrument that generates free flowing steam, such as the Arnold sterilizer, is used in these instances.
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.
clip_image002
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.
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.