Intracellular StationsAntibodyBotany Common Buffers Buffers Alphabetic Bookstore Cell Biology and Culture Gel Electrophoresis Histology Microarrays Immunology Mass Spectrometry Microbiology Molecular Imaging Peptide Protein Microarray Chips PCR Real-Time PCR RNAi and SiRNA Stem Cell Biology Transfection Western Blot |
User PanelMy PanelMy PanelBookmark Science Articles
|
||||
Table of Contents:
Sterilization Troubleshooting and Forum Topics
Sterilization is defined as the destruction of all forms of life. It may be effected by various agents. As applied to the practical requirements of the bacteriological laboratory many of those agents such as electricity, sunlight, etc., are of little value and are limited in their applications; others are so well suited to particular purposes that their use is almost entirely restricted to such applications. The principle methods used most often are autoclaving (heat, steam and pressure - a combination) and chemical, however autoclaving is most effective if performed properly
There are two major methods of sterilization.
A)
1) Concentrated solutions destroy microorganisms by withdrawing water from their cells (plasmolysis) , e.g., in the preservation of food by concentrated salt or sugar solutions. Microorganisms accustomed to a concentrated nutrient substrate may suffer plasmoplysis (bursting of the cell) if placed in a less concentrated medium.In either case, if they are subjected gradually to the changing conditions, death is delayed or prevented.
2) Desiccation is destructive to many microbes, espe cially those which do not form spores. For example, Ps. Radicicola is very sensitive to desiccation on the ordinary cover-glass or on cotton.
3).
This is usually used as a bunsen burner in the lab, however there are several heating methods:
1. In sterilization in a Naked Flame,
(a) The simplest means of sterilizing a metal instrument is to heat it to red ness in a flame. This method is always adopted for ster ilizing platinum, copper, etc., wires and iron and nickel spatulas, forceps, etc.
A platinum needle should always be carefully dried before sterilization, by holding it near the flame. This avoids sputtering, which scatters microorganisms, especially if moist material, e.g., fat or protein, on the needle is immediately thrust into the flame.
(b) An instrument may be sterilized by flaming it, i. e., by passing it rapidly through a hot flame. This method is useful for instruments, etc., having polished surfaces devoid of creases in which microorganisms might escape destruction, e.g., knives, glass rods, handles of platinum needles, mouths of test tubes, flasks, pipettes, etc.
(c) Deep wounds are sterilized by cautery with an instrument heated to a dull red heat.
2.
In an emergency, small instruments, needles, etc., may be sterilized by dipping them in ether or absolute alcohol and after removal lighting the adherent fluid and allowing it to burn off the surface of the instruments. Repeat the process. It may then be safely assumed that the apparatus so treated is sterile.
3.
Porcelain filter candles are sterilized by heating them to white heat in the muffle furnace. This method of sterilization cannot be applied to porcelain filters with metal fittings, such as Berkefeld filters.
The destruction of autopsied animals and accumulated wastes of the laboratory is also best accomplished in this manner.
4.
Exposure to hot air is the usual method of sterilizing all glassware, instruments with metal handles, etc., but it is not suitable for organic sub stances, with the exception of wool, cotton and paper.
To insure efficient sterilization, the prepared glassware, etc., must be placed in a gas or electrically heated oven (containing a thermometer registering over 200 C.) whose temperature is maintained at approximately 150 C. for one hour, or 180 C. for ten minutes. The oven must be allowed to cool down to 60 C. before opening the door to avoid the breaking of glassware by cold-air currents. Cotton, wool, and paper are slightly scorched at this temperature,
Apparatus must be absolutely clean and dry before being sterilized.
4.
Sterilization by moist heat may be effected in one of four ways:
1.
Some substances used as culture media, being rich in volatile or otherwise chemically unstable substances, cannot be heated to 100 C. without a marked alteration (e.g., coagulation) and to some extent a destruc tion of their properties; blood serum, for example.
Pasteur showed that such media can be better ster ilized by heating them at a low temperature (55-60 C.) for a long time than at a high temperature (70 C. or even 100 C.) for a short time. In this process, heat is not applied 'directly, as a rule. Control of the temperature is ordinarily accomplished by means of water heated to the degree desired.
Prolonged heating at a low temperature constitutes pasteurization. In practice, however, it is found that in order to kill all organisms pasteurization must be com bined with the method of discontinuous heating devised by Tyndall. Albuminous media subjected to the Tyndall method must be incubated finally at 37 C. for forty-eight hours to eliminate all specimens showing contamination.
2.
(a) Continuous Heating. Water at 100 C. destroys the vegetative forms of bacteria almost instantaneously, and spores in from five to fifteen minutes ordinarily, although many spores of resistant species are not killed by several hours' heating at 100 C. Water suspected of sewage contamination may thus be rendered safe for drinking purposes simply by boiling for a few minutes.
This method is applicable to metal instruments, syringes, rubber stoppers, rubber and glass tubing, and other small apparatus.
(b) Discontinuous Heating. (Tyndall method.) Tyndall observed that certain resistant forms found in an infusion made from hay were not destroyed by heating the infusion at 100 C., once, even when the temperature was sustained for a prolonged period, yet by boiling it for a short time on three successive days all living organisms were destroyed. His theory was that by heating at 100 C., the vege tative forms but not the spores were killed. The latter germinate as the fluid cools and are killed during the second heating. A few spores, however, escape de struction at the second heating;
these will have germinated by the time the third heating is due. After the third heating sterilization is accomplished.
The explanation now given, however, is that the resist ance of microorganisms is gradually lowered under the influence of repeated heatings. This principle of heating on three successive days, a medium to be sterilized is now known as the Tyndall method of sterilization. In general laboratory practice, steam is used instead of water at 100., but this necessitates special apparatus, whereas water lends itself readily to the means at hand.
The physical nature of the medium, the extraordinary resistance of the spores of certain species of bacteria or both in combination, may require that this intermittent heating be carried on over a longer period of time, i.e., four, five, six, etc., days in succession for the same or a longer period each time, or that the period between inter mittent heatings be lengthened from twenty-four hours to forty-eight hours.
Tyndall's method is valuable in that media of delicate composition may be sterilized without producing undesirable changes, such as are often produced by the high tempera ture of the autoclav.
3. Sterilization in Flowing Steam at 100 C. Continuous or Discontinuous, (a) Continuous Heating. Simple boiling or exposure to steam at 100 C., even though the exposure be prolonged, is not a reliable method of sterilization. When microorganisms have been dried, their resistance to the effects of heat is much enhanced, and especially is this the case when they are mixed with substances of a colloidal nature. Certain resistant forms of protoplasm known as spores may not be destroyed by one heating to 100 C., even when the temperature has been maintained for sev eral minutes.
(b) Discontinuous Heating. General use for the ster ilization of media.
This principle of sterilization advanced by Tyndall finds its widest application in bacteriological work with the use of flowing steam. High-pressure steam may be utilized to good advantage if a central heating station is available. The Arnold sterilizer makes use of steam for the sterilization process and lends itself readily to both the continuous and discontinuous method.
4. Sterilization by Superheated Steam (under pressure and therefore above 100 C.). Water, syringes, surgical dressings, bedding, india-rubber apparatus, filters, old cultivations, culture media, etc., not injured by high termperatures, may be more quickly sterilized by heating in steam under pressure.
Exposure to steam at a temperature of 115 C. for twenty minutes is in most cases sufficient to insure sterilization,
But some media, potato for instance, require a temperature of 120 C. for ten to fifteen minutes. It is now realized that media subjected to this high temperature undergo hydrolytic changes which render them unsuitable for the cultivation of more delicate microorganisms. Sterilization in the superheated steam is carried on in a special apparatus called an autoclave, which may be so constructed as to run by direct or indirect steam. The latter is the more desireable for the sterilization of media.
5)
Light seems to act by producing powerful chemical germicides, probably organic per oxides, in the medium surrounding the bacteria. Certain rays of light, the blue, violet and ultraviolet in particular are destructive to living cells. It is to these rays that sun light owes its disinfecting action. Practical use has been made of the ultraviolet rays in water sterilization by employing the Cooper-Hewitt mercury vapor lamp having a quartz instead of a glass tube, as these rays do not pass through glass.
6)
Sterilization may be effected by the filtration of gases or liquids through materials which will retain microorganisms.
The best example of the filtration of gases is the use of cotton plugs in flasks and tubes containing microorganisms. The cotton is porous enough to allow the necessary interchange of gases but will allow neither dust nor foreign microorganisms to enter. The sterilization of air or other gases if fore 3d through cotton would depend upon the thickness of the cotton layer and also upon the force which was exerted.
Certain fluids used in bacteriological work cannot be subjected even to a moderate amount of heat without profoundly altering their nature. In order to make such a fluid sterile, it is passed through a cylindrical vessel, closed at one end like a test tube, and made either of porous " biscuit" porcelain, hard burnt and unglazed (Chamber land filter) or of kieselguhr, a fine diatomaceous earth (Berkefeld filter) and termed a bougie or a candle.
The pores of the finer filters are so small that while liquids, and solids in solution pass through, microorganisms are retained and the liquid passes through in a germ-free condition.
Pasteur in his early work utilized plaster plates as the filtering medium, but as a result of Chamberland's researches, porous porcelain now supersedes plaster. Finely shredded asbestos packed tightly in a Gooch crucible will serve as a bacterial filter provided the layer of asbestos is sufficiently thick. The rate of filtration is usually very slow because the pores of the filter are so very minute; therefore to overcome this disadvantage either aspiration or pressure is generally employed to hasten the process. This method may not exclude filterable organisms.
7)
In one of the more recent methods devised for the preparation of antirabic vaccines the vaccine is prepared by placing the virus (spinal cord of a rabid rabbit) in a collodion sac and dialyzing it in running distilled water. The living virus is destroyed, yet its immunizing properties are retained unimpaired. Quite the opposite effect may be obtained under some what different circumstances. If a collodion sac containing a suspension of a pathogenic organism be placed in the body cavity of a susceptible animal the organisms within the sac thrive, being nourished by the body fluids which diffuse through the semi-permeable membrane.
GUMMING, J. G.: Rabies Hydrophobia. A study of fixed virus, determination of the M. L. D., vaccine treatment (Hogyes, Pasteur, and dialyzed vaccine), and immunity tests. Journal of Infectious Diseases, Vol. XIV (1914), pp. 33-52.
8)
Comminution or the actual crushing of the microbial cells is resorted to for demonstrating intracellular enzymes.
B.
1)
Sterilization by dis infectants has but limited use in bacteriological work. The amount of disinfectant necessary to destroy existing organisms in a nutrient medium is greater than the amount necessary to inhibit multiplication of an organism which may subsequently be used as an inoculum; the medium is therefore rendered useless.
As an example, 1-1000 mercuric chloride, 1.5% formalin, 5% phenol, 2% compound solution of cresol, etc., are cheap and adaptable in many cases. Tincture of iodin is valuable for painting wounds.
The common soaps, and more particularly green soap, have a plight germicidal value, and this in conjunction with their solvent action upon fats and protein, and the mechanical cleansing which accompanies their use, justifies assigning them an important place among the chemical disinfectants.
Disinfectants used for sterilizing the skin before col lecting pus, blood, etc., from the living subject must be carefully removed by washing the part well with alcohol before collecting material, otherwise the presence of the disinfectant would materially interfere with the subsequent growth of organisms in the culture.
3. Disinfectants are also added to sterile filtrates which are no longer required as culture media. For this purpose a small quantity of some disinfectant (such as thymol or camphor) which is without chemical action on the constituents of the fluid is selected.
An amount of carbolic acid (0.5%) or other chemical is frequently added to vaccines, bac terms, serums, etc., for preservative purposes.
4. Disinfectants are sometimes used to sterilize a culture when the products of the microorgansims are under inves tigation. Chloroform, ether, toluol, oil of garlic or mustard, etc., which may be driven off afterward by evaporation, are among the most useful in this connection.
2)
Chemical reagents such as belong to the class known as antiseptics, i.e., substances which inhibit the growth of, but do not destroy bacterial life, are obviously useless.
The mouths of test tubes, fermentation tubes, pipettes, etc., are ordinarily plugged with cotton before sterilization. For this purpose cotton is ideal as it is cheap and adaptable, serves to filter out microorganisms from the air, while allow ing the ready diffusion of gases, and after once used it may be burned.
Paper (ordinary newspaper) may be used to wrap glass ware as Petri dishes, deep-culture dishes, pipettes, etc., which one wishes to store in a sterile condition and for which cotton is not adaptable.
Glassware is sterilized for the purpose of destroying microorganisms present on its surface and in or on the cotton or paper used respectively for plugging or wrapping. After sterilization the cotton and paper serve to prevent micro organisms from entering and contaminating the sterile utensils.
Dry heat, though not as effective a germ destroyer as moist heat, is more adaptable to the sterilization of empty culture flasks, pipettes and other glassware. Hot-air steril ization not only accomplishes the sterilization of the glass ware, cotton plugs, etc., but " sets " the plugs so that they may be handled with greater facility.
All glassware must be absolutely clean and dry or contain traces of alcohol only before preparing for sterilization; otherwise sterilization cannot be accomplished. If consider able moisture is present in test tubes, flasks, etc., it will not evaporate during the hot-air sterilization process, and it is very evident that the temperature of such moist portions of the glassware will not reach or at leastwill not exceed 100 C.
Directions. Test tubes and flasks are plugged with cotton. The ordinary forceps are used for this purpose. (A glass rod may also be used.) A small piece of cotton is grasped on the edge with the forceps and inserted in the mouth of the test tube. Plugs should project into test tubes from 3 to 4 cms., and from 3 to 5 cms. Into the neck of flasks, according to the size of the flask. Only an amount of cotton should project out of the mouth that is sufficient to protect the outward turned portion (lip) of the test tubes or flasks from dust.
A " Christmas-tree " effect is to be avoided. Plugs should not be so tight as to be removed with difficulty, nor so loose as to offer no resistance to removal. A little experience will suffice to demonstrate the amount of cotton to use and the firmness with which the plug should fit.
Cotton plugs for test tubes, flasks, etc., may be rolled. This kind of plug is more stable and may be used several times. Have the instructor demonstrate the method of rolling.
For hot-air sterilization, test tubes plugged with cotton may be tied in large bundles or placed in wire baskets (never in agate cups), cotton plugs up. A few test tubes should not be placed in a large wire-basket or in a wire test-tube rack, as it is necessary to economize space in the hot-air sterilizer.
Petri dishes are wrapped separately in paper and tied together in sets of three, One sheet of newspaper makes four papers of proper size for wrapping Petri dishes and is inexpensive.
Three or more Petri dishes may be wrapped together if all are to be used at the same time. Mark each plainly with the desk number.
Pipettes. Place a piece of cotton in the bottom of a test tube, plug the top only of the pipette with cotton (not too tightly}, leaving but little of the cotton projecting out. Wrap a small portion of cotton around the lower third of the pipette, insert the pipette into the test tube until the tip rests on the cotton, making the cotton wrapping serve as a plug for the tube.
Wrap pipettes so prepared in paper (one layer) and tie and mark them plainly with the desk number.
A covered metal case is often used for holding pipettes to be sterilized. The upper end of the pipettes are plugged with cotton, the pipette inserted in the case, the open end of the case plugged with cotton, and the cover replaced. (This latter method is not recommended for the new student, as the necessity of careful technic in removing a sterile pipette from the case without contaminating those remaining is difficult to impress upon him) .
Fermentation tubes are plugged with cotton as directed for test tubes; the cotton plug should not project into the bulb.
Deep culture dishes are wrapped singly in paper as directed for Petri dishes.
Slides and cover-glasses are generally sterilized by flaming, but only as needed.
| Threads | |||
| Thread Title / Poster | Last Post | Replies | Views |
| 09-06-2006 11:02 AM | 3 | 883 | |
Research and Laboratory Products and Vendors Directory - organized by topic. Companies, list Your products and company in the product directory for FREE by Registering Your Company Here!
Mad Cow Disease Research
Living Cells as Nanotechnology Factories
Coral Reef Discovery
Protection Mechanism for Stressed Out Bacteria
Tropical Wetlands Hold More Carbon than Temperate Marshes
Notch Pathway may Contribute to Breast Cancer
Research Decodes Genome for Species of Malaria
Mechanism Regulating Neural Stem Cell Development
Balance Between Left and Right Brain for Walking
Behavioral Intervention for DepressionFor more click here:Science News
![[أربيك]](http://www.molecularstation.com/translate/images/flag_ar.gif){kind=small}
