Can I sterilize the equipment for experiments without an autoclave?

Can I sterilize the equipment for experiments without an autoclave?

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If I don't have access to an autoclave, can I sterilize my equipment in a regular pot. If so, what time is needed at about 110°C (I measured inside) for a proper sterilization?

It certainly can be done. Tyndallisation may as discussed on labmate may work well for your situation. If not, there are many other viable alternatives suggested.

**Tyndallisation: **
Developed partway through the 19th century by English physicist, John Tyndall, the eponymous Tyndallisation was a commonly used technique by the microbiologists of the 1800s. Tyndall experimented with boiling beef broths to develop a method to sterilise liquids in a safe and comprehensive manner.

His research led him to the basic principles for Tyndallisation, a process by which medias are subjected to relatively short boils at a regular atmospheric pressure. This relatively straightforward method is still suitable for small labs, or research facilities which only require sterilised equipment part of the time.

It is not recommended to attempt to sterilise closed glass containers using this method, without lining them with cotton and capping them with foil - allowing for air to escape without being subjected to contaminants.

The process involves boiling fluids for 10-15 minutes before leaving to cool to room temperature and leaving it to sit for 24 hours. Repeat this process another three or four times, by which time sterilisation should have occurred.

Naturally this method has reduced in popularity due to the requirement to continue the process for up to five days to achieve sterilisation.

1: Media Preparation

Bacteria and fungi are grown on or in microbiological media of various types. The medium that is used to culture the microorganism depends on the microorganism that one is trying to isolate or identify. Different nutrients may be added to the medium, making it higher in protein or in sugar. Various pH indicators are often added for differentiation of microbes based on their biochemical reactions: the indicators may turn one color when slightly acidic, another color when slightly basic. Other added ingredients may be growth factors, (ce), and pH buffers which keep the medium from straying too far from neutral as the microbes metabolize.

In this exercise, you will make all-purpose media called trypticase soy broth and trypticase soy agar. These 2 media----one a liquid and the other a solid---are the exact same formula save for the addition of agar agar (really- agar agar), an extract from the cell walls of red algae.

The old way to make media was by the cookbook method--- adding every ingredient bit by bit. The only time that is done today is when making a special medium to grow a certain finicky organism, where particular growth factors, nutrients, vitamins, and so on, have to be added in certain amounts. This medium is called a chemically defined medium (synthetic). Fortunately, the most common bacteria that we want to grow will do nicely with media that we commonly use in lab. Some of our media is bought, but most is produced in the prep area behind the lab. Since this type of medium has some unknown ingredients, or sometimes unknown quantities it is called complex media.

It is really very simple to make complex media these days:

  • rehydrate the powder form of the medium
  • stir and boil the agar medium to get the agar powder dissolved (if making an agar medium rather than a broth medium)
  • distribute the medium into tubes
  • autoclave to sterilize the tube media
  • autoclave the agar medium for plate production and then pour into sterile petri dishes

Autoclave Compatible and Incompatible Material

The following are examples of compatible and incompatible materials. This is not an exhaustive list.

Important Notice: Autoclaving hazardous materials may generate toxic vapors or explosive environments.

Compatible Materials

Incompatible Materials

Biological cultures and stocks

Materials containing solvents, volatile or corrosive, or flammable chemicals

Culture dishes and related materials

Material contaminated with chemotherapeutic agents or cytotoxic drugs

Contaminated solid items (i.e. pipette tips, gloves, Petri dishes, etc.)

Material containing Bleach*

Discarded live (including attenuated) viruses/vaccines

Carcinogens or mutagens (i.e. ethidium bromide)

Polypropylene (PP) and polycarbonate (PC) plastics

Polystyrene (PS), polyethylene (PE), and high-density polyethylene (HDPE) plastics

* Neutralize waste containing bleach with equal amounts of 1% sodium thiosulfate in water prior to autoclaving

  • Every autoclave and sterilizer should be inspected and serviced on a regular basis. This will help ensure the equipment is functioning properly.
  • Each unit should have a standard operating procedure written in sufficient detail to ensure that operators will use the equipment properly controls vary between brands, with each having unique loading characteristics, load-sizing requirements, and cycle setting and types. Principal Investigators and/or lab managers should ensure users are properly trained on the autoclave in use.
  • Units should be tested regularly with a commercial preparation containing Geobacillus stearothermophilus spores (a biological indicator), in particular, any unit in a BSL3 facility.
  • Tape indicators (autoclave tape) with heat sensitive, chemical indicators should be used in every autoclave load. Note: the indicators only verify that the autoclave has reached normal operating temperatures they do not indicate that the contents were heated for the appropriate length of time or at the proper pressure. Therefore, tape indicators cannot be used to prove organisms are actually killed during an autoclave run.
  • Keep detailed records on biological tests, recording thermometers, and service work performed on the unit.
  • High density wastes or materials that insulate the agents from heat and steam penetration are not suitable for steam sterilization. Items that are covered with dirt or film require additional retention times. The importance of properly cleaning items to be sterilized cannot be over emphasized.
  • Place all autoclaved infectious waste into red biohazard bags for disposal.
  • An online training video was developed by Arizona State University that offers safety information, examples of waste to be autoclaved, and procedures for spore testing that may be helpful to any user. The video is available at:
  • Autoclaved waste can cause odors, the use of autoclave deodorizers may assist if there is a general problem in the area.

The following PPE should be used during loading and unloading:

  • Standard laboratory clothing including long pants and closed-toed shoes
  • Eye/face protection
  • Gloves (including heat resistant gloves)
  • Laboratory coat

The proper packaging and containment of infectious materials are crucial to achieve effective sterilization. The most frequent reason for sterilization failure is the lack of contact between the steam and microorganisms. Dry material should be separated from liquid material to achieve proper sterilization.

  • For liquids, always choose the liquid or "slow exhaust" cycle.
  • Ask your lab manager which cycle is recommended for sterilizing dry goods or equipment.
  • For descriptions of the 2 basic autoclave cycles, read Autoclave Overview.

These guidelines contain recommended sterilization times. Always follow your lab's written operating procedures.

  • Nonhazardous dry goods: 30 minutes of sterilization plus 20 minutes of drying time. Dry time may need to be increased for enclosed items such as pipette tips or bottles with lids.
  • Liquids (add 10&ndash20 minutes for crowded items):
    • Less than 500 milliliters (ml): 30 minutes
    • 500 ml &ndash 1 liter: 40 minutes
    • 2&ndash4 liters: 55 minutes
    • More than 4 liters: 60 minutes

    Note: Autoclaving new glassware for 90 minutes will partially temper it, increasing its strength.

    Steam Sterilization

    Of all the methods available for sterilization, moist heat in the form of saturated steam under pressure is the most widely used and the most dependable. Steam sterilization is nontoxic, inexpensive 826 , rapidly microbicidal, sporicidal, and rapidly heats and penetrates fabrics (Table 6) 827 . Like all sterilization processes, steam sterilization has some deleterious effects on some materials, including corrosion and combustion of lubricants associated with dental handpieces 212 reduction in ability to transmit light associated with laryngoscopes 828 and increased hardening time (5.6 fold) with plaster-cast 829 .

    The basic principle of steam sterilization, as accomplished in an autoclave, is to expose each item to direct steam contact at the required temperature and pressure for the specified time. Thus, there are four parameters of steam sterilization: steam, pressure, temperature, and time. The ideal steam for sterilization is dry saturated steam and entrained water (dryness fraction &ge97%). 813, 819 Pressure serves as a means to obtain the high temperatures necessary to quickly kill microorganisms. Specific temperatures must be obtained to ensure the microbicidal activity. The two common steam-sterilizing temperatures are 121°C (250°F) and 132°C (270°F). These temperatures (and other high temperatures) 830 must be maintained for a minimal time to kill microorganisms. Recognized minimum exposure periods for sterilization of wrapped healthcare supplies are 30 minutes at 121°C (250°F) in a gravity displacement sterilizer or 4 minutes at 132°C (270°F) in a prevacuum sterilizer (Table 7). At constant temperatures, sterilization times vary depending on the type of item (e.g., metal versus rubber, plastic, items with lumens), whether the item is wrapped or unwrapped, and the sterilizer type.

    The two basic types of steam sterilizers (autoclaves) are the gravity displacement autoclave and the high-speed prevacuum sterilizer. In the former, steam is admitted at the top or the sides of the sterilizing chamber and, because the steam is lighter than air, forces air out the bottom of the chamber through the drain vent. The gravity displacement autoclaves are primarily used to process laboratory media, water, pharmaceutical products, regulated medical waste, and nonporous articles whose surfaces have direct steam contact. For gravity displacement sterilizers the penetration time into porous items is prolonged because of incomplete air elimination. This point is illustrated with the decontamination of 10 lbs of microbiological waste, which requires at least 45 minutes at 121°C because the entrapped air remaining in a load of waste greatly retards steam permeation and heating efficiency. 831, 832 The high-speed prevacuum sterilizers are similar to the gravity displacement sterilizers except they are fitted with a vacuum pump (or ejector) to ensure air removal from the sterilizing chamber and load before the steam is admitted. The advantage of using a vacuum pump is that there is nearly instantaneous steam penetration even into porous loads. The Bowie-Dick test is used to detect air leaks and inadequate air removal and consists of folded 100% cotton surgical towels that are clean and preconditioned. A commercially available Bowie-Dick-type test sheet should be placed in the center of the pack. The test pack should be placed horizontally in the front, bottom section of the sterilizer rack, near the door and over the drain, in an otherwise empty chamber and run at 134°C for 3.5 minutes. 813, 819 The test is used each day the vacuum-type steam sterilizer is used, before the first processed load. Air that is not removed from the chamber will interfere with steam contact. Smaller disposable test packs (or process challenge devices) have been devised to replace the stack of folded surgical towels for testing the efficacy of the vacuum system in a prevacuum sterilizer. 833 These devices are &ldquodesigned to simulate product to be sterilized and to constitute a defined challenge to the sterilization process.&rdquo 819, 834 They should be representative of the load and simulate the greatest challenge to the load. 835 Sterilizer vacuum performance is acceptable if the sheet inside the test pack shows a uniform color change. Entrapped air will cause a spot to appear on the test sheet, due to the inability of the steam to reach the chemical indicator. If the sterilizer fails the Bowie-Dick test, do not use the sterilizer until it is inspected by the sterilizer maintenance personnel and passes the Bowie-Dick test. 813, 819, 836

    Another design in steam sterilization is a steam flush-pressure pulsing process, which removes air rapidly by repeatedly alternating a steam flush and a pressure pulse above atmospheric pressure. Air is rapidly removed from the load as with the prevacuum sterilizer, but air leaks do not affect this process because the steam in the sterilizing chamber is always above atmospheric pressure. Typical sterilization temperatures and times are 132°C to 135°C with 3 to 4 minutes exposure time for porous loads and instruments. 827, 837

    Like other sterilization systems, the steam cycle is monitored by mechanical, chemical, and biological monitors. Steam sterilizers usually are monitored using a printout (or graphically) by measuring temperature, the time at the temperature, and pressure. Typically, chemical indicators are affixed to the outside and incorporated into the pack to monitor the temperature or time and temperature. The effectiveness of steam sterilization is monitored with a biological indicator containing spores of Geobacillus stearothermophilus (formerly Bacillus stearothermophilus). Positive spore test results are a relatively rare event 838 and can be attributed to operator error, inadequate steam delivery, 839 or equipment malfunction.

    Portable (table-top) steam sterilizers are used in outpatient, dental, and rural clinics. 840 These sterilizers are designed for small instruments, such as hypodermic syringes and needles and dental instruments. The ability of the sterilizer to reach physical parameters necessary to achieve sterilization should be monitored by mechanical, chemical, and biological indicators.

    The oldest and most recognized agent for inactivation of microorganisms is heat. D-values (time to reduce the surviving population by 90% or 1 log10) allow a direct comparison of the heat resistance of microorganisms. Because a D-value can be determined at various temperatures, a subscript is used to designate the exposure temperature (i.e., D121C). D121C-values for Geobacillus stearothermophilus used to monitor the steam sterilization process range from 1 to 2 minutes. Heat-resistant nonspore-forming bacteria, yeasts, and fungi have such low D121C values that they cannot be experimentally measured. 841

    Moist heat destroys microorganisms by the irreversible coagulation and denaturation of enzymes and structural proteins. In support of this fact, it has been found that the presence of moisture significantly affects the coagulation temperature of proteins and the temperature at which microorganisms are destroyed.

    Different sterilization methods used in the laboratory

    Sterilization can be achieved by a combination of heat, chemicals, irradiation, high pressure and filtration like steam under pressure, dry heat, ultraviolet radiation, gas vapor sterilants, chlorine dioxide gas etc. Effective sterilization techniques are essential for working in a lab and negligence of this could lead to severe consequences, it could even cost a life.

    So what are the most commonly used methods of sterilization in the laboratory, and how do they work? Read on…

    Heat Method: This is the most common method of sterilization. The heat is used to kill the microbes in the substance. The extent of sterilization is affected by the temperature of the heat and duration of heating. On the basis of type of heat used, heat methods are categorized into-

    (i) Wet Heat/Steam Sterilization- In most labs, this is a widely used method which is done in autoclaves.. Autoclaves use steam heated to 121–134 °C under pressure. This is a very effective method that kills/deactivates all microbes, bacterial spores and viruses. Autoclaving kills microbes by hydrolysis and coagulation of cellular proteins, which is efficiently achieved by intense heat in the presence of water. The intense heat comes from the steam. Pressurized steam has a high latent heat and at 100°C it holds 7 times more heat than water at the same temperature. In general, Autoclaves can be compared with a typical pressure cooker used for cooking except in the trait that almost all the air is removed from the autoclave before the heating process starts. Wet heat sterilization techniques also include boiling and pasteurization.

    (ii) Dry heat sterilization- In this method, specimens containing bacteria are exposed to high temperatures either by flaming, incineration or a hot air oven. Flaming is used for metallic devices like needles, scalpels, scissors, etc. Incineration is used especially for inoculating loops used in microbe cultures. The metallic end of the loop is heated to red hot on the flame. The hot air oven is suitable for dry material like powders, some metal devices, glassware, etc.

    Filtration is the quickest way to sterilize solutions without heating. This method involves filtering with a pore size that is too small for microbes to pass through. Generally filters with a pore diameter of 0.2 um are used for the removal of bacteria. Membrane filters are more commonly used filters over sintered or seitz or candle filters. It may be noted that viruses and phage are much smaller than bacteria, so the filtration method is not applicable if these are the prime concern.

    Radiation sterilization:This method involves exposing the packed materials to radiation (UV, X-rays, gamma rays) for sterilization. The main difference between different radiation types is their penetration and hence their effectiveness. UV rays have low penetration and thus are less effective, but it is relatively safe and can be used for small area sterilization. X-rays and gamma rays have far more penetrating power and thus are more effective for sterilization on a large scale. It is, however, more dangerous and thus needs special attention. UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between uses. X-rays are used for sterilizing large packages and pallet loads of medical devices. Gamma radiation is commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets, and food.

    Chemical method of sterilization: Heating provides a reliable way to get rid of all microbes, but it is not always appropriate as it can damage the material to be sterilized. In that case, chemical methods for sterilization is used which involves the use of harmful liquids and toxic gases without affecting the material. Sterilization is effective using gases because they penetrate quickly into the material like steam. There are a few risks, and the chances of explosion and cost factors are to be considered.

    The commonly used gases for sterilization are a combination of ethylene oxide and carbon-dioxide. Here Carbon dioxide is added to minimize the chances of an explosion. Ozone gas is another option which oxidize most organic matter. Hydrogen peroxide, Nitrogen dioxide, Glutaraldehyde and formaldehyde solutions, Phthalaldehyde, and Peracetic acid are other examples of chemicals used for sterilization. Ethanol and IPA are good at killing microbial cells, but they have no effect on spores.

    Ask an Expert: Sterilizing Equipment

    I will be growing the bacteria found in yogurt, and I'm wondering how I can sterilize the filter disks I'm using to test antimicrobial effectiveness without using an autoclave. For the metal utensils, I'm planning to simply clean them with rubbing alcohol, but I'm not sure if that could be done with filter paper. Please note that I do not have access to a fancy lab at the moment.

    I'm also wondering if the bacteria grown directly from yogurt would grow in a uniform fashion and not in several small colonies? Here is the link to the method I'm using: . p072.shtml

    Re: Sterilizing Equipment

    Post by jcschrandt » Tue Mar 15, 2016 12:39 pm

    Hi Mad_Scientist! The easiest way to solve your problem would be to buy sterile filter disks. Otherwise, you could soak with 70% isopropyl alcohol or 70% ethanol and then let it dry. However, the problem with this approach is that you need a sterile environment for the disks to dry. Otherwise the disks could get contaminated with bacteria by the time they dry. So, if you decide to take this approach, you should wipe everything in the vicinity of the filter disks with 70% alcohol as well.

    To answer your second question, the density of colonies depends on the concentration of bacteria. If the bacteria is present at a low concentration in the yogurt, then you may see a few colonies that grow in size over time. Each colony is produced by a single bacterium. If the yogurt has a high concentration of bacteria, you may see a film of bacteria.

    Re: Sterilizing Equipment

    Post by Mad_Scientist » Tue Mar 15, 2016 1:04 pm

    Thanks for the quick reply!

    I just did some research and apparently yogurt contains from 3 billion cfu/ml to 10 billion cfu/ml. Would this be considered enough bacteria to grow a nice film? If not, is there anyway to make the bacteria grow in a film? This would be important since I'm using the diffusion method to determine disinfectant ability.

    Re: Sterilizing Equipment

    Post by deleted-284605 » Tue Mar 15, 2016 9:20 pm

    I think as long as you coat the entire plate with at least a bit of yogurt, you'll get a so-called "lawn" of bacteria (continuous growth in two dimensions rather than many small colonies).

    If you can't find sterile disks, you could also try microwaving them for 30-60 seconds (be careful though- I'm not sure how flammable they might be)!

    Re: Sterilizing Equipment

    Post by Mad_Scientist » Tue Mar 15, 2016 10:03 pm

    Thanks! I'll consider using either the ethanol or microwave method. If I do use the microwave method, should I soak the disks in water first or should I wrap them in aluminum foil? (I'm kind of worried that the disks will burn) I'm also wondering how far apart I should place each filter disk from each other. I'm thinking about placing them about 3 cm away from each other but I'm not exactly sure since I couldn't find an exact procedure for that.

    Another quick question: should I dilute the yogurt before spreading them on the dishes? The initial procedure said to make a 50:50 dilution.

    Thanks for the help!

    Re: Sterilizing Equipment

    Post by deleted-284605 » Tue Mar 15, 2016 11:34 pm

    Don't put foil in the microwave. That will start a fire!

    Wetting the disks and nuking for 30 seconds should be fine, though whatever you buy will probably be sterile anyway! Dousing with ethanol and then allowing the disks to dry under a bowl or some other clean space should be fine too.

    What sort of anti-microbial agents are you using? Placing

    3 disks/plate in a triangle shape (to maximize distance between them) will probably be okay, but the safest bet would be to put one disk in the center of each plate. Just depends on how many plates you have.

    The same goes for the yogurt dilution. I would test a range of dilutions (e.g. undiluted, 75%, 50%, and 25% yogurt) to see which gives the best bacterial lawn before adding the disks. If you don't have enough plates to do all this, I'd just try the suggested 50% yogurt mix. These experiments should have been tested, so that ratio was suggested for a reason!

    Re: Sterilizing Equipment

    Post by Mad_Scientist » Wed Mar 16, 2016 9:05 pm

    Thanks for the advice! I sterilized the filter disks today! Now I'm just wondering, if I were to sterilize glass and metal equipment, should I boil them, bake them, or just simply wipe them down with some alcohol? I want to get rid of as much bacteria, and I'm not sure which method is most effective.

    By the way, I'm going to go with the 75% yogurt dilution.

    Re: Sterilizing Equipment

    Post by ncarter79 » Thu Mar 17, 2016 7:11 am

    If I were you, I would wipe them down with alcohol. I work in a microbiology lab and wiping with alcohol is exactly how I clean my spatulas.

    Re: Sterilizing Equipment

    Post by Mad_Scientist » Thu Mar 17, 2016 10:39 pm

    I'm just wondering if there is a way to speed up the growth of the yogurt bacteria because I just read that it takes about 6 days. I just tried incubating the plates at high temperatures, but since I used gelatine plates, the medium simply melted. Thanks for the help!

    Re: Sterilizing Equipment

    Post by PharmaMan » Sun Mar 20, 2016 6:26 pm

    For the growth of the bacteria, I would just be patient! Raising the temperature too far will likely kill the cells, and will melt the medium upon which they are growing. Other than giving them optimal temperature and a nutrient-rich medium, there isn't much you can do to accelerate their growth.

    Re: Sterilizing Equipment

    Post by ChristineJensen » Mon Mar 21, 2016 12:40 am

    Methods for Sterilization of Media and Air (With Diagram)

    A bioreactor can be sterilized by destroying the organisms by heat/chemicals/radiation or sometimes by physical procedures such as filtration.

    Sterilization of media and air are discussed below:

    1. Sterilization of Culture Media:

    The constituents of culture media, water and containers contribute to the contamination by vegetative cells and spores. The media must be free from contamination before use in fermentation. Sterilization of the media is most commonly achieved by applying heat and to a lesser extent by other means (physical methods, chemical treatment, and radiation).

    Heat sterilization:

    Heat is the most widely used sterilization technique. The quality and quantity of contamination (i.e., the type and load of microorganisms), composition of the media and its pH and size of the suspended particles are the important factors that influence the success of heat sterilization.

    In general, vegetative cells are destroyed at lower temperature in a short time (around 60°C in 5-10 minutes). However, destruction of spores requires higher temperature and relatively longer time (around 80°C for 15-20 minutes). Spores of Bacillus stearothermophilus are the most heat resistant. In fact, this organism is exploited for testing the sterility of fermentation equipment.

    Physical methods:

    The physical methods such as filtration, centrifugation, and adsorption (to ion-exchangers or activated carbon) are in use. Among these, filtration is most widely used. Certain constituents (vitamins, blood components, antibiotics) of culture media are heat labile and therefore, are destroyed by heat sterilization. Such components of the medium are completely dissolved (absolutely essential or else they will be removed along with microorganisms) and then subjected to filter sterilization.

    There are a couple of limitations of filtration technique:

    1. Application of high pressure in filtration is unsuitable for industries.

    2. Some of the media components may be lost form the media during filtration.

    Sometimes, a combination of filtration and heat sterilization are applied. For instance, the water used for media preparation is filtered while concentrated nutrient solution is subjected to heat sterilization. The filtered water is now added for appropriate dilution of the media. The chemical methods (by using disinfectants) and radiation procedures (by using UV rays, y rays, X-rays) are not commonly used for media sterilization.

    The culture media are subjected to sterilization at 121°C in batch volumes, in the bioreactor. Batch sterilization can be done by injecting the steam into the medium (direct method) or injecting the steam into interior coils (indirect method). For the direct batch sterilization, the steam should be pure, and free from all chemical additives (that usually come from steam manufacturing process).

    There are two disadvantages of batch sterilization:

    1. Damage to culture media:

    Alteration in nutrients, change in pH and discolouration of the culture media are common.

    2. High energy consumption:

    It takes a few hours (2-4 hrs.) for the entire contents of the bioreactor to attain the requisite temperature (i.e. 120°C). Another 20-60 minutes for the actual process of sterilization, followed by cooling for 1-2 hours. All this process involves wastage of energy, and therefore batch sterilization is quite costly.

    Continuous sterilization:

    Continuous sterilization is carried out at 140°C for a very short period of time ranging from 30 to 120 seconds. (This is in contrast to the batch fermentation done at 121°C for 20-60 minutes). This is based on the principle that the time required for killing microorganisms is much shorter at higher temperature. Continuous sterilization is carried out by directly injecting the steam or by means of heat exchangers.

    In either case, the temperature is very quickly raised to 140°C, and maintained for 30- 120 seconds. The stages of continuous sterilization process and the corresponding temperatures are depicted in Fig. 19.7. The different stages are— exchanger, heater, heat maintenance unit, recovery of residual heat, cooling and fermenter.

    In the continuous sterilization process, 3 types of heat exchangers are used. The first heat exchanger raises temperature to 90-1 20°C within 20-30 seconds. The second exchanger further raises temperature to 140°C and maintains for 30-120 seconds. The third heat exchanger brings down the temperature by cooling in the next 20-30 seconds. The actual time required for sterilization depends on the size of the suspended particles. The bigger is the size, the more is the time required.

    The main advantage with continuous sterilization is that about 80-90% of the energy is conserved. The limitation however, is that certain compounds in the medium precipitate (e.g., calcium phosphate, calcium oxalate) due to very high temperature differences that occur in a very short time between sterilization and cooling. The starch-containing culture media becomes viscous in continuous sterilization and therefore is not used.

    2. Sterilization of Air:

    In general, the industrial fermentations are carried out under vigorous and continuous aeration. For an effective fermentation, the air should be completely sterile, and free from all micro­organisms and suspended particles. There is a wide variation in the quantity of suspended particles and microbes in the atmospheric outdoor air.

    The microorganisms may range from 10-2,000/m 3 while the suspended particles may be 20-100,00/ m 3 . Among the microorganisms present in the air, the fungal spores (50%) and Gram-negative bacteria (40%) dominate. Air or other gases can be sterilized by filtration, heat, UV radiation and gas scrubbing. Among these, heat and filtration are most commonly used.

    (a) Air sterilization by heat:

    In the early years, air was passed over electrically heated elements and sterilized. But this is quite expense, hence not in use these days.

    (b) Air sterilization by filtration:

    Filtration of air is the most commonly used sterilization in fermentation industries.

    When the air is passed through a glass wool containing depth filters the particles are trapped and removed (Fig. 19.8). This filtration technique primarily involves physical effects such as inertia, blocking, gravity, electrostatic attraction and diffusion. Glass wool filters can be subjected to steam sterilization and reused. But there is a limitation in their reuse since glass wool shrinks and solidifies on steam sterilization. In recent years, glass fiber filter cartridges (that do not have the limitations of glass wool filter) are being used.

    4 Main Methods of Sterilization | Organisms | Microbiology

    Among the various methods followed for controlling microbial activity, the best by far is sterilization as it eliminates all the microbes. Sterilization is achieved by the following methods: 1. Physical Methods 2. Radiation Methods 3. Ultrasonic Methods 4. Chemical Methods.

    1. Physical Methods:

    Physical methods of sterilization include killing of microbes by applying moist heat as in steaming or dry heat as in a hot air oven or by various methods of filtration to free the medium of microbes. We shall study each one of them.

    i. Physical Control with Heat:

    The Citadel is novel by A.J. Cronin that follows the life of a young British physician, beginning in the 1920s. Early in the story the physician, Andrew Manson, begins his practice in a small coal­mining town in Wales. Almost immediately, he encounters an epidemic of typhoid fever.

    When his first patient dies of the disease, Manson becomes terribly distraught. However, he realizes that the epidemic can be halted, and in the next scene, he is tossing all of the patient’s bed-sheets, clothing, and personal effects into a huge bonfire.

    The killing effect of heat on microorganisms has long been known. Heat is fast, reliable, and relatively inexpensive, and it does not introduce chemicals to a substance, as disinfectants sometimes do. Above maximum growth temperatures, biochemical changes in the cell’s organic molecules result in its death.

    These changes arise from alterations in enzyme molecules or chemical breakdowns of structural molecules, especially in the cell membranes. Heat also drives off water, and since all organisms depend on water, this loss may be lethal.

    The killing rate of heat may be expressed as a function of time and temperature. For example, tubercle bacilli are destroyed in 30 minutes at 58°C, but in only 2 minutes at 65°C, and in a few seconds at 72°C. Each microbial species has a thermal death time (TDT), the time necessary for killing it at a given temperature. Each species also has a thermal death point (TDT), the temperature at which it dies in a given time.

    In this method temperature is kept constant and time necessary to kill the cells is determined. The term thermal death point is no more practice. Since a particular temperature cannot be lethal all the times and also for all kinds of microorganisms.

    Recently the heat sensitivity is defined using the term D value. D value is the exposure time at a given temperature required to reduce the number of viable organisms by 90%. Mathematically, it is equal to the reciprocal of the slope of the survivor curve or survivor curve to traverse one log cycle. D values can be used to determine the relative heat sensitivity of a microorganism to different temperature by calculation.

    The z value is the temperature change needed to reduce the D value by one log cycle when log D is plotted against temperature. The F value is the D value at 250°F. These measurements are particularly important in the food industry, where heat is used for preservation.

    In determining the time and temperature for microbial destruction with heat, certain factors bear consideration. One factor is the type of organism to be killed. For example, if materials are to be sterilized, the physical method must be directed at bacterial spores. Milk, however, need not be sterile for consumption, and heat is therefore aimed at the most resistant vegetative cells of pathogens.

    Another factor is the type of material to be treated. Powder is subjected to dry heat rather than moist heat, because moist heat will leave it soggy. Saline solutions, by contrast, can be sterilized with moist heat but are not easily treated with dry heat.

    Other factors are the presence of organic matter and the acidic or basic nature of the material. Organic matter may prevent heat from reaching microorganisms, while acidity or alkalinity may encourage the lethal action of heat.

    ii. Direct Flame:

    Perhaps the most rapid sterilization method is the direct flame method used in the process of incineration. The flame of the Bunsen burner is employed to sterilize the bacteriological loop before removing a sample from a culture tube and after preparing a smear. Flaming the tip of the tube also destroys organisms that happen to contact the tip, while burning away lint and dust.

    In general, objects must be disposable if a flame is used for sterilization. Disposable hospital gowns and certain plastic apparatus are examples of materials that may be incinerated. In past centuries, the bodies of disease victims were burned to prevent spread of the pestilence.

    It is still common practice to incinerate the carcasses of cattle that have died of anthrax and to put the contaminated field to the torch because anthrax spores cannot adequately be destroyed by other means. British law even stipulates that anthrax-contaminated animals may not be autopsied before burning.

    iii. Hot-Air Sterilizer:

    The hot-air-sterilizer utilizes radiating dry heat for sterilization. It is also called hot air oven. It is constructed with three walls and two air spaces. The outer walls are covered with thick asbestos to reduce the radiation of heat. A burner manifold runs along both sides and rear between the outside and the intermediate walls. Convection currents travel a complete circuit through the wall space and interior of the oven, and the products of combustion escape through an opening in the top.

    The hot-air sterilizer is operated at a temperature of 160 to 180°C. (320 to 356°F.) for a period of 1½ hr. If the temperature goes above 180°C., there will be danger of the cotton stoppers charring. Therefore, the thermometer must be watched closely at first until the sterilizer is regulated to the desired temperature. The necessity of watching the sterilizer may be avoided by having the oven equipped with a temperature regulator.

    The effect of dry heat on microorganisms is equivalent to that of baking. The heat changes microbial proteins by oxidation reactions and creates an arid internal environment, thereby burning microorganisms slowly. It is essential that organic matter such as oil or grease films be removed from the materials, because organic matter insulates against dry heat. Moreover, the time required for heat to reach sterilizing temperatures varies among materials. Thus this factor must be considered in determining the total exposure time.

    The hot-air sterilizer is used for sterilizing all kinds of laboratory glassware, such as test tubes, pipettes, Petri dishes, and flasks. In addition, it may be used to sterilize other laboratory materials and equipment that are not burned by the high temperature of the sterilizer. Under no conditions should the hot-air sterilizer be used to sterilize culture media, as the liquids would boil to dryness.

    iv. Arnold Sterilizer (Boiling Water):

    Immersion in boiling water is the first of several moist-heat methods that we shall consider. Moist heat penetrates materials much more rapidly than dry heat because water molecules conduct heat better than air. Lower temperatures and less time of exposure are therefore required than for dry heat.

    The Arnold makes use of streaming steam as the sterilizing agent. The sterilizer is built with a quick-steaming base that is automatically supplied with water from an open reservoir. The water passes from the open reservoir, through small apertures, into the steaming base, to which the heat is applied. Since the base contains only a thin layer of water, steam is produced very rapidly. The steam rises through a funnel in the center of the apparatus and passes into the sterilizing chamber.

    Moist heat kills microorganisms by denaturing their proteins. Denaturation involves changes in the chemical or physical properties of proteins. It includes structural alterations due to destruction of the chemical bonds holding proteins in a three-dimensional form.

    As proteins revert to a two-dimensional structure, they coagulate (denature) and become nonfunctional. Egg protein undergoes a similar transformation when it is boiled. You might find a review of the chemical structure of proteins, helpful to your understanding of this process. The coagulation and denaturing of proteins require less energy than oxidation, and therefore , less heat need be applied.

    Sterilization is effected by employing streaming steam at a temperature of approximately 100°C. (212°F.) for a period of 20 min or longer on three consecutive days. The length of the heating period will depend upon the nature of the materials to be treated and the size of the container. Agar, for example, must be first completely melted before recording the beginning of the heating period.

    It must be remembered that a temperature of 100°C. for 20 min. is not sufficient to destroy spores. A much higher temperature is required to effect a complete sterilization in one operation over a relatively short exposure period.

    The principle underlying this method is that the first heating period kills all the vegetative cells present. After a-lapse of 24 hr. in a favourable medium and at a warm temperature, the spores, if present, will germinate into vegetative cells. The second heating will again destroy all vegetative cells.

    It sometimes happens that all spores do not pass into vegetative forms before the second heating period. Therefore, an additional 24-hr. period is allowed to elapse to make sure that all spores have germinated into vegetative cells. It may be seen that unless the spores germinate the method will fail to sterilize.

    v. Fractional Sterilization:

    In the years before development of the autoclave, liquids and other objects were sterilized by exposure to free-flowing steam at 100°C for 30 minutes on each of three successive days. The method was called fractional sterilization because a fraction was accomplished on each day. It was also called tyndallization after its developer, John Tyndall and intermittent sterilization because it was a stop-and-start operation.

    Sterilization by the 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 spores 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 that spores be in a suitable medium for germination, such as a broth.

    Fractional sterilization has assumed 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.

    vi. Pasteurization:

    Pasteurization is not the same as sterilization. Its purpose is to reduce the bacterial population of a liquid such as milk and to destroy organisms that may cause spoilage and human disease. Spores are not affected by pasteurization.

    One method for milk pasteurization, called the holding method, involves heating at 62.9°C for 30 minutes. Although thermophilic bacteria thrive at this temperature, they are of little consequence because they cannot grow at body temperature.

    For decades, pasteurization has been aimed at destroying Mycobacterium tuberculosis, long considered the most heat-resistance bacterium. More recently, however, attention has shifted to destruction of Coxiella burnetii, the agent of Q fever, because this organism has a higher resistance to heat.

    Two other methods of pasteurization are the flash pasteurization method at 71.6°C for 15 seconds, and the ultra-pasteurization method at 82°C for 3 seconds.

    vii. Desiccation:

    In addition to freezing, many foods are preserved by desiccation. Water is required for microbial growth. Although lack of available water prevents microbial growth, it does not necessarily accelerate the death rate of microorganisms. Some microorganisms, therefore, can be preserved by drying.

    One can readily purchase active dried yeast for baking purposes and after the addition of water, the yeasts begin to carry out active metabolism. Freeze-drying or lyophilization is a common means of removing water that can be used for preserving microbial cultures. During freeze-drying, water is removed by sublimation. This process generally eliminates damage to microbial cells from the expansion of ice crystals.

    Whereas some microorganisms are relatively resistant to drying, other microorganisms are unable to survive desiccating conditions for even a short period of time. For example, Treponema pallidum, the bacterium that causes syphilis, is extremely sensitive to drying and dies almost instantly in the air or on a dry surface.

    The fact that microorganisms are unable to grow at low water activities can be used for the preservation of many products. Salting was one of the early means of preserving foods and is still employed today. By adding high concentrations of salt, the Aw is lowered sufficiently to prevent the growth of most microorganisms.

    Canvas and other textiles are preserved in temperate zones by the lack of water in the air, but in tropical zones these same materials are subject to bio-deterioration because the humidity is sufficiently high to permit microbial growth. Exposed wood surfaces are often painted to keep the wood dry enough to preclude microbial growth. Many food products are also preserved by drying.

    This preservation method depends on maintaining the product in a dry state, and exposure to high humidity can negate the factor limiting microbial growth and promote microbial spoilage of food products preserved in this manner. If food can be maintained at an Aw value of 0.65 or less, spoilage is unlikely for several years. Products preserved by drying include fruits, vegetables, eggs, cereals, grains, meat, and milk.

    Physical Control by Other Methods:

    Heat is valuable physical agent for controlling microorganisms but sometimes it is important to use. For example, no one would suggest removing the microbial population from a tabletop by using a Bunsen burner, nor can heat-sensitive solutions be subjected to an autoclave. In instances such as these and numerous others, a heat-free method must be used. This section describes some examples.

    Filters came into prominent use in microbiology as interest in viruses grew in the 1890s. Previous to that time, filters had been utilized to trap airborne organisms and sterilize bacteriological media, but now they became essential of filter technology was Charles Chamberland, an associate of Pasteur. His porcelain filter was important to early virus research. Another pioneer was Julius Petri (inventor of the Petri dish), who developed a sand filter to separate bacteria from the air.

    The filter is a mechanical device for removing microorganisms from a solution. As fluid passes through the filter, organisms are trapped in the pores of the filtering material. The solution that drips into the receiving container is decontaminated or, in some cases, sterilized. Filters are used to purify such things as intravenous solutions, bacteriological media, many pharmaceuticals, and beverages.

    Several types of filters are available for use in the microbiology laboratory.

    i. Porcelain or Chamberland Filters:

    Porcelain filters are hollow, unglazed cylinders, closed at one end. They are composed of hydrous aluminium silicate or kaolin with the addition of quartz sand and are heated to a temperature sufficiently low to avoid sintering. These filters are prepared in graduated degrees of porosity, from LI to LI3.

    Cylinders having the largest pores are marked LI those having the smallest pores are designated LI3. The finer the pores, the slower will be the rate of filtration. The LI and L2 cylinders are preliminary filters intended for the removal of coarse particles and large bacteria. The L3 filter is probably satisfactory for all types of bacterial filtrations.

    ii. Berkefeld Filters:

    Kieselguhr is a deposit of fine, usually white siliceous powder composed chiefly or wholly of the remains of diatoms. It is also called diatomaceous earth and infusorial earth.

    Berkefeld filters are manufactured in Germany. They are prepared by mixing carefully purified diatomaceous earth with asbestos and organic matter, pressing into cylinder form, and drying. The dried cylinders are heated in an oven to a temperature of about 2000°C. to bind the materials together. The burned cylinders are then machined into the desired shapes and sizes.

    The cylinders are graded as W (dense), N (normal), and V (coarse), depending upon the sizes of the pores. The grading depends upon the rate of flow of pure filtered water under a certain constant pressure.

    iii. Mandler Filters:

    These filters are similar to the Berkefeld type but are manufactured in this country. They are composed of 60 to 80 per cent diatomaceous earth, 10 to 30 per cent asbestos, and 10 to 15 per cent plaster of Paris. The proportions vary, depending upon the sizes of the pores desired. The ingredients are mixed with water, subjected to high pressure, and then baked in ovens to a temperature of 980 to 1650°C. to bind the materials together.

    The finished cylinders are tested by connecting a tube to the nipple of the filter, submerging in water, and passing compressed air to the inside. A gauge records the pressure when air bubbles first appear on the outside of the cylinder in the water. Each cylinder is marked with the air pressure obtained in actual test.

    A convenient arrangement of apparatus for filtering liquids through a Mandler or Berkefeld filter is shown in Fig.3.10. The reduced pressure is indicated by the manometer. The liquid to be filtered is poured into the mantle, and the filtrate is collected in a graduated vessel, from which it may be withdrawn aseptically. Filtration may be interrupted at any time by stopping the vacuum pump and opening the stopcock on the trap bottle to equalize the pressure.

    iv. Fritted-Glass Filters:

    Filters of this type are prepared by fritting finely pulverized glass into disk form in a suitable mold. The pulverized glass is heated to a temperature just high enough to cause the particles to become a coherent solid mass, without thoroughly melting, and leaving the disk porous.

    The disk is then carefully fused into a glass funnel and the whole assembled into a filter flask by means of a rubber stopper. Another arrangement is the coupling of the filter to the flask through a ground-glass joint thus eliminating the use of a rubber stopper.

    The filters are marketed in five degrees of porosity as follows: EC (extra coarse), C (coarse), M (medium), F (fine), and UF (ultrafine).

    Bacteriological filters are generally employed under conditions of reduced pressure Bush (1946) recommended filtration through glass filters by the use of positive pressure. Positive pressure not only reduces or eliminates evaporation of the filtrate, but greatly facilitates the interchange of receivers— particularly important in bacteriological filtrations which must be handled aseptically.

    A convenient arrangement is shown in Fig. 3.13. The main body of the filter B contains a fritted-glass disk. A shield A protects the receiver from dust, and a pressure head carries a stopcock. An alternate pressure permits the retention of pressure head C’ contains a built-in mercury manometer. The stopcock on C (C’) permits the retention of pressure after the apparatus is detached from the source of compressed air.

    An ordinary rubber pressure bulb is satisfactory for producing pressures up to at least 450 mm. Mercury. If the ground-glass joints are well lubricated and the parts held together with strong rubber bands or springs, the apparatus should hold this pressure for days.

    v. Asbestos Filters:

    The best-known filter employing asbestos as the filtering medium is the Seitz filter. The asbestos is pressed together into thin disks and tightly clamped between two smooth metal rims by means of three screw clamps. The liquid to be filtered is poured into the metal apparatus, in which the asbestos disk is clamped, and the solution drawn through by vaccum, the filtering disks are capable of effectively retaining bacteria and other particulate matter.

    At the end of the operation, the asbestos disk is removed, a new one inserted, and the assembled filter sterilized. This feature makes the Seitz filter very convenient to use, since no preliminary cleaning is necessary.

    A modification of the Seitz filter, utilizing centrifugal force instead of suction or pressure, has been suggested by Boerner. The filter consists of a cylinder and a funnel-shaped part with stem, which holds the filter pad supported on a wire gauze disk. The cylinder screws into the funnel with the filter disk pressed between them.

    The assembled filter fits closely into the top of a 15-ml. metal centrifuge tube, with the knurled collar of the funnel portion resting on the top of the metal tube. The filtrate is collected in a glass tube inside the cup. The filter can also be used for vacuum filtration in the conventional manner by inserting the stem through a rubber stopper fitted to a filter flask.

    vi. Jenkins Filter:

    This filter consists of a metal mantle holding a soft rubber sleeve and a porcelain filter block. The porcelain block is held in the rubber sleeve and made watertight by screwing together two metal parts. The filter block is not fragile. It is washed after each use, dried, and inserted in the mantle. The mantle is fitted with a rubber stopper, wrapped in paper, and sterilized in an autoclave.

    The filter is designed to be used for the sterilization of small quantities of liquids.

    vii. Ultrafilter:

    Ultrafiltration generally means the separation of colloidal particles from their solvents and from crystalloids by means of jelly filters known a ultrafilter.

    The early jelly filters were composed of gelatin and of silicic acid, but these have been replaced by collodion in membrane or sac form, or collodion deposited in a porous supporting structure. The supporting structure may be filter paper in sheet and thimble form, unglazed porcelain dishes and crucibles, Buchner funnels, filter cylinders, etc.

    viii. Membrane Filter:

    The membrane filter is a third type of filter that has received broad acceptance. It consists of a pad of organic compounds such as cellulose acetate or polycarbonate, mounted in a holding device. This filter is particularly valuable because bacteria multiply and form colonies on the filter pad when the pad is placed 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.

    ix. Cleaning Filters:

    Some filters are discarded after each use and new ones employed others are intended to be cleaned after each filtration and, with proper care, may be used repeatedly. Collodion membranes are easily prepared, and the Seitz asbestos disks are relatively low in cost. These filters are intended to be used once, then discarded. On the other hand, porcelain, diatomaceous earth, and fritted-glass filters are too expensive to be used only once, but are easily cleaned.

    Porcelain filters are cleaned by placing them in a muffle furnace and raising the temperatures to a red heat. This burns the organic matter in the pores and restores the filters to their original condition.

    Filters of the Berkefeld and Mandler types are cleaned by placing the cylinders in a special metal holder connected to a faucet. The flow of water is reversed by passing through the cylinder from within outward. This should be continued until all foreign matter has been washed away from the filter pores.

    Albuminous or similar materials remaining in the pores of the filters are likely to be coagulated by heat during the process of sterilization, with the result that the filters will be clogged. Filters in this condition are useless for further work.

    Clogged filters may be cleaned in various ways but probably most conveniently by continuous suction of full-strength Clorox, or similar solution, for 5 to 15 min. This treatment quickly dissolves the coagulated material and restores the usefulness of the filter. Thorough washing is necessary to remove the last traces of the oxidizing solution.

    Fritted-glass filters may be cleaned by treatment with concentrated sulfuric acid containing sodium nitrate. The strong acid quickly oxidizes and dissolves the organic matter. Thorough washing is necessary to remove the last traces of acid.

    2. Radiation Method:

    i. Sterilization by Ultraviolet Light:

    Visible light is a type of radiant energy detected by the sensitive cells of the eye. The wavelength of this energy is between 400 and 800 nanometers (nm). Other types of radiations have wavelengths longer or shorter than that of visible light and therefore, they cannot be detected by the human eye.

    One type of radiant energy, ultraviolet light, is useful for controlling microorganisms. Ultraviolet light has a wavelength between 100 and 400 nm, with the energy at about 265 nm most destructive to bacteria. When microorganisms are subjected to ultraviolet light, cellular DNA absorbs the energy, and adjacent thymine molecules link together.

    Linked thymine molecules are unable to position 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 dies quickly.

    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. It is noteworthy that ultraviolet light from the sun may be an important factor in controlling microorganisms in the air and upper layers of the soil, but it may not be effective against all bacterial spores. Ultraviolet light does not penetrate liquids or solids, and it may cause damage in human skin cells.

    ii. Ionizing Radiation:

    High-energy, short wavelength radiation disrupts DNA molecules, and exposure to short wavelength radiations may cause mutations, many of which are lethal. Exposure to gamma radiation (short wavelengths of 10 -3 – 10 -1 nanometers), X ray (wavelengths of 10 -3 – 10 2 nanometers), and ultraviolet radiation (ultraviolet light with wavelengths of 100-400 nanometers) increases the death rate of microorganisms and is used in various sterilization procedures to kill microorganisms. Viruses as well as other microorganisms are inactivated by exposure to ionizing radiation.

    Sensitivities to ionizing radiation vary. Resistance to ionizing radiation is based on the biochemical constituents of a given microorganism. Non-reproducing (dormant) stages of microorganisms tend to be more resistant to radiation than are growing organisms. For example, endospores are more resistant than are the vegetative cells of many bacterial species.

    Exposure to 0.3-0.4 Mrads (million units of radiation) is necessary to cause a tenfold reduction in the number of viable bacterial endospores. An exception is the bacterium Micrococcus radiodurans, which is particularly resistant to exposure to ionizing radiation.

    Vegetative cells of M. radiodurans tolerate as much as 1 Mrad of exposure to ionizing radiation with no reduction in viable count. It appears that efficient DNA repair mechanisms are responsible for the high degree of resistance to radiation exhibited by this bacterium.

    Ionizing radiation is used to pasteurize or sterilize some products. Some commercially produced plastic petri plates are sterilized by exposure to gamma radiation. Most sterilization procedures involving exposure to radiation employ gamma radiation from cobalt-60 or cesium-137.

    Bacon, for example, can be sterilized by radappertization, a process of sterilizing foods by exposure to radiation, using radiation doses of 4.5-5.6 Mrads. Hadurization, functionally equivalent to pasteurization, is used to kill non-spore-forming human pathogens that may be present in food. Radurization can be used to increase the shelf life of sea-foods, vegetables, and fruits.

    Unlike gamma radiation, ultraviolet light does not have high penetrating power and is useful for killing microorganisms only on or near the surface of clear solutions. The strongest germicidal wavelength of 260 nanometers coincides with the absorption maxima of DNA, suggesting that the principle mechanism by which ultraviolet light exerts its lethal effect is through the disruption of the DNA. In fact, ultraviolet light causes the formation of covalently linked thymine dimers within the DNA in place of the normal thymine-adenine hydrogen bonded base pairs.

    Microorganisms have enzymes that can repair the alterations in the DNA caused by exposure to ultraviolet light. The photo-reactivation enzymes require exposure to light in the visible spectrum. Exposure to ultraviolet light sometimes is used to maintain the sterility of some surfaces. In some hospitals bench-tops are maintained bacteria-free when not in use by using an ultraviolet lamp.

    The dangers involved in human exposure to excess ultraviolet radiation, which include blindness if an ultraviolet light is viewed directly, have led to the use of alternative methods for maintaining the sterility of such areas.

    Like ultraviolet radiation, long wavelength infrared radiation (103-105 nanometers) and microwave radiations (wavelengths greater than 106 nanometers) have poor penetrating power. Infrared and microwave radiations do not appear to kill microorganisms directly. Absorption of such long wavelength radiation, however, results in increased temperature.

    Exposure to infrared or microwave radiations can thus indirectly kill microorganisms by exposing them to temperatures that are higher than their maximal growth temperatures. Because microwaves generally do not kill microorganisms directly, there is some concern in the food industry that cooking with microwave ovens may not adequately kill microorganisms contaminating food products.

    3. Ultrasonic Method:

    Ultrasonic Vibrations:

    Ultrasonic vibrations are high-frequency sound waves beyond the range of the human ear. When directed against environment surfaces, they have little value because air particles deflect and disperse the vibrations. However, when propagated in fluids, ultrasonic vibrations cause the formation of microscopic bubbles, or cavities, and the water appears to boil. Some observers call this “cold boiling.”

    The cavities rapidly collapse, and send out shock waves. Microorganisms in the fluid are quickly disintegrated by the external pressures. The formation and implosion of the cavities is known as cavitation.

    Ultrasonic vibrations are valuable in research for breaking open tissue cells and obtaining their parts for study. A device called the cavitron is used by dentists to clean teeth, and ultrasonic machines are available for cleaning dental plates, jewelry, and coins. A major appliance company has also experimented with an ultrasonic washing machine.

    As a sterilizing agent, ultrasonic vibrations have received minimal attention because liquid is required and other methods are more efficient. However, many research laboratories use ultrasonic probes for cell disruption and hospitals use ultrasonic devices to clean their instruments. When used with an effective germicide, an ultrasonic device may achieve sterilization, but the current trend is to use ultrasonic vibrations as a cleaning agent and follow the process by sterilization in autoclave.

    4. Chemical Method:

    i. Preservation Methods:

    Over the course of many centuries, various physical methods have evolved for controlling microorganisms in food. Though valuable for preventing the spread of infectious agents, these procedures are used principally to retard spoilage and prolong the shelf life of foods, rather than for sterilization.

    Drying is useful in the preservation of various metals, fish, cereals, and other foods. Since water is a necessary requisite for life, it follows that where there is no water, there is virtually no life. Many of the foods in the kitchen pantry typify this principle. One example is discussed in MicroFocus.

    Preservation by salting is based upon the principle of osmotic pressure. When food is salted, water diffuses out of microorganisms to the higher salt concentration and lower water concentration in the surrounding environment. This flow of water, called osmosis, leaves microorganisms to shrivel and die.

    The same phenomenon occurs in highly sugared foods such as syrups, jams, and jellies. However, fungal contamination may remain at the surface because aerobic molds tolerate high sugar concentrations.

    Low temperatures found in the refrigerator and freezer retard spoilage by reducing the rate of metabolism in microorganisms and, consequently, reducing their rate of growth. Spoilage is not totally eliminated in cold foods, however, and many microorganisms remain alive, even at freezer temperatures. These organisms multiply rapidly when food thaws, which is why prompt cooking is recommended.

    Note in these examples that there are significant differences between killing microorganisms, holding them in check, and reducing their numbers. The preservation methods are described as bacteriostatic because they prevent the further multiplication of bacteria.

    ii. Gaseous Sterilization:

    Heat sterilization is mostly unstable for thermolabile solid medicament and thermolabile equipment including articles of plastics, delicate rubber items. Because of high capital cost and use of elaborate precautionary measures, the radiation method which is one of the methods of sterilization has become unpopular.

    Thus the sterilization of such materials with a chemical in gaseous state finds a greater application. Previously formaldehyde was widely used, but at present ethylene oxide is the only compound of outstanding importance in pharmaceutical and medical fields.

    Autoclave Procedure

    Wear personal protective equipment:

    • Lab coat
    • Eye protection
    • Closed-toe shoes
    • Heat-resistant gloves to remove items, especially hot glassware

    Packaging and Loading

    • Only designated individuals should be allowed to set and/or change parameters for the autoclaves.
    • Before using the autoclave, check inside for any items left by the previous user that could pose a hazard.
    • Clean the drain strainer before loading the autoclave.
    • Always place items in a secondary container.
    • Do not overload or package bags too tightly. Leave sufficient room for steam circulation. If necessary, place container on its side to maximize steam penetration and avoid entrapment of air.
    • Use only autoclavable bags to package waste.
    • Do not allow bags to touch the interior walls of the autoclave to avoid melting of plastic.
    • Ensure sufficient liquid is packed with contents of autoclave bags if dry.
    • Place soiled glassware and lab ware in secondary containers and autoclave them in the solids cycle. Do not fill containers more than 2/3 full with liquids. Loosen caps or use vented closures.
    • In case of clean glassware and wrapped instruments, lay them in a secondary container before autoclaving in wrapped goods cycle.
    • For secondary containment, use autoclave trays made out of polypropylene, polycarbonate or stainless steel. The trays should have a solid bottom and sides to contain the contents and catch spills.
    • Choose appropriate cycle for the material. Incorrect selection of cycle may damage the autoclave, cause liquid to boil over or bottles to break.
    • Start your cycle and fill out the autoclave user log. A completed cycle usually takes between 1 to 1.5 hours.
    • Check chamber/jacket pressure gauge for minimum pressure of 20 pounds per square inch (psi).
    • Close and lock door.
    • Check temperature for 250⁰F (121⁰C) every load.
    • Do not attempt to open the door while autoclave is operating.


    • Ensure cycle has completed and both temperature and pressure have returned to a safe range.
    • Wear PPE described above, plus an apron and face shield if removing liquids. Stand back from the door as a precaution and carefully open door no more than 1 inch. This will release residual steam and allow pressure within liquids and containers to normalize.
    • Allow the autoclaved load to stand for 10 minutes in the chamber. This will allow steam to clear and trapped air to escape from hot liquids, reducing risk to operator.
    • Do not agitate containers of super-heated liquids or remove caps before unloading.
    • Place liquids in an area which clearly indicates the items are “hot” until the items cool to room temp.
    • Allow autoclaved materials to cool to room temperature before transporting. Never transport superheated materials.
    • Place cooled autoclaved biohazard bag into regulated medical waste box. Autoclaved infectious liquids may be disposed of into the sanitary sewer.

    What is Sterile? Find Your Way around a Sterile Tissue Culture Hood

    You’ve been told that maintaining a sterile environment in a tissue culture hood is vital to preventing contamination of cell cultures. But what exactly is meant by sterile? The definition of sterile is ‘completely clean, sanitized, and free of all forms of life’. Obviously you still want your cells and/or any other organisms you are studying to live, but any reagents or equipment that are used for tissue culture should be sterile. A better word for how you want to work in the hood is asepsis, or the state of being free from biological contaminants. Aseptic technique will ensure that only the things you want to grow in your culture will. If you are mindful of sources of contamination and work carefully, then you will be able to work effectively in a tissue culture hood. Below are several tips for good aseptic technique.

    Plan your experiments. Plan your experiments ahead of time so you can make sure that you have time to sterilize all reagents and equipment. Make a list of everything you will need in the hood to minimize leaving the hood during your experiment. Set up your hood with everything needed for the experiment prior to starting your work.

    Be good go your hood. A clean, functioning tissue culture hood is critical for aseptic work. Make sure you know how to use the hood properly. If you have questions you can start by reading Bitesize Bio’s Quick Protocol and Ways to Abuse Biological Safety Cabinets. A qualified technician should certify the hood annually, and a sticker on the hood should indicate certification. Take apart and deep clean the hood about twice a year. Take out all removable parts, clean with disinfectant and sterilize in the autoclave. Every time you turn the hood on, make sure the fan is working properly by reading the magnahelic gauge (Figure 1). Clean the hood with disinfectant before and after every experiment. If you spill something, clean it up as soon as you can without compromising your experiment. This includes checking and cleaning the drip pans if large amounts of liquid are spilled. Do not store unnecessary equipment or reagents in the hood: this will make it harder to keep the hood clean.

    Figure 1. A Magnahelic Gauge

    Start clean. Make sure everything you bring into the hood is clean. Sterilize or sterile filter (with a 0.22micron filter) all media and reagents. Designate store bought reagents for tissue culture use only. Sterilize or disinfectant all equipment used in the hood. Remember to spray the outside of all bottles with disinfectant prior to placing them in the hood. Prior to starting your work, wash your hands well, put on clean gloves and make sure your clothing does not bring in unwanted organisms. Many labs designate lab coats for tissue culture use. If you use a lab coat, make sure it is cleaned on a regular basis.

    Work from clean to dirty. This concept should be used throughout your day as you move from experiment to experiment, and as you work on each experiment within the hood. Plan your day so that you do your clean experiments first and your dirty experiments later on. For example, I usually split cells for maintaining cell lines in the morning then do any work infecting tissue culture cells with viruses in the afternoon. Within the hood I usually designate the right side of the hood my “clean” side and the left side my “dirty” side. As much as possible I work from the clean side over to the dirty side.

    Don’t trap things with your trap. If you use a vacuum trap for removing large amounts of liquid, make sure you take care of the trap. On a regular basis inspect all hoses and lines to make sure nothing is growing in them. Clean the outside of all hoses and lines within the hood with disinfectant before and after every experiment. If the vacuum hose is stored in the hood, make sure liquid is not dripping out of the hose into the hood when the vacuum is turned off. Do not allow the trap to overfill. Empty and clean the collection flask on a regular basis, preferably at the end of your experiment. If the vacuum hoses are connected to a filter, check the filter when you empty the flask to make sure it is not wet replace the filter as needed.

    Knowing how to find your way around a tissue culture hood using aseptic technique will prevent contamination of your experiments with unwanted organisms. Follow these tips and you will have a good start!

    Watch the video: Dental Assisting - Disinfection, Sterilization and Bloodborne Pathogens: Part 2 - Sterilization (July 2022).


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