Describe the Construction and Operation of a Laboratory Autoclave Giving the Respective Sterilization Controls

DISCUSS THE CONSTRUCTION AND OPERATION OF AN AUTOCLAVE IN THE FOLLOWING ASPECTS:

A.    Types of Autoclaves

An autoclave is a pressure chamber that is used to sterilize equipments and supllies by subjecting them to high pressure saturated steam at 121 degrees Celsius for around 15-20 minutes depending on the size of the load and contents.

Three different types of autoclaves are available;

·         Gravity

·         Pre-vacuum

·         Isotherm

i.                    Gravity autoclaves are used in the sterilization of empty and or non-porous containers which are stable to both heat and moisture. Common examples of these items include; liquids, media and solids (i.e. biomedical waste)

ii.                  Pre-vacuum autoclaves draw out a significant portion of the air within the autoclave chamber improving the speed and efficiency of the sterilization process. The removal of air from the chamber allows for the penetration of porous materials which are both heat and moisture stable. Common examples of these items include bench-coat and animal bedding.

iii.                An isothermal autoclave uses a lower temperature in the sterilization of materials which are either heat sensitive or congealable. This sterilization process is commonly used in the pasteurization of items such as media.

B.     Articles  sterilized and precautions

The vast majority of items which are used to store liquids within a typical molecular or microbiology laboratory are comprised of autoclavable materials. However, some materials are manufactured out of materials which cannot be autoclaved either due to their inability to withstand high temperatures or their release of toxic gases when sterilized. Therefore, you must determine if the vessel can be autoclaved or not prior to treatment.

Articles sterilized include;

·         Culture media – bacterial, viral and fungal cultures

·         Contaminated items such as petri dishes, tips, tubes, gloves etc

·         Glassware, media, aqueous solutions and specific equipment

·         Culture dishes and related items

Precautions

i.                    Articles should not be tightly packed, do not overload the autoclave

ii.                  Air discharge must be complete leaving no residual air trapped inside.

iii.                Caps of bottles and flasks should not be tightly screwed on.

iv.                The autoclave must not be opened until the pressure has fallen or else the contents will boil over.

v.                  Articles must be paper to prevent drenching, bottles must not be overfilled.

C.    The Sterilization Process

A simple autoclave has vertical or horizontal cylindrical body with a heating element, a perforated try to keep the articles, a lid that can be fastened by screw clamps, a pressure gauge, a safety valve and a discharge tap. The articles to be sterilized must not be tightly packed. The screw caps and cotton plugs must be loosely fitted. The lid is closed but the discharge tap is kept open and the water heated. As the water starts boiling, the steam drives air out of the discharge tap. When all the air is displaced and steam start appearing through the discharge cap, the tap is closed.

The pressure inside is allowed to rise up to 15 lbs per square inch. At this pressure the articles are held for 15 minutes, after which the heating is stopped and the autoclave is allowed to cool. Once the pressure gauge shows the pressure equal to atmospheric pressure, the discharge tap is opened to let the air in. The lid is then opened and articles removed.

D.    Sterilization Control

I.                   Physical method includes automatic process control, thermocouple and temperature chart recorder.

II.                Chemical method includes Browne’s tube No. 1 (black spot) and succinic acid (whose melting point is 121̊C) and Browie Dick tape is applied to articles being autoclaved. If the process has been satisfactory, dark brown stripes will appear across the tape.

III.             Biological method includes a paper strip containing 106 spores of Geobacillus stearothermophilus.

E.     Advantages

1.      Very effective way of sterilization.

2.      Quicker than hot air oven.

F.     Disadvantages

1.      Drenching and wetting of articles may occur

2.      Takes long time to cool

3.      Trapped air may reduce the efficacy

References;

1.      Seymour Stanton Block (2001). Disinfection, Sterilization and Preservation. Lippincott Williams & wilkins. ISBN 978-0-683-30740-5. Retrieved 19 January 2013

2.      Le, R.N., et al (2005), Autoclave Testing in a University setting. Applied Biosafety, 10(4), 248-252.

Role of Pharmacist in BREAST CANCER

BREAST CANCER
INTRODUCTION

Breast cancer is a malignant (cancerous) growth that begins in the tissues of the breast. Cancer is a disease in which abnormal cells grow in an uncontrolled way. Breast cancer is the most common cancer in women, but it can also appear in men.Breast cancer usually begins with the formation of a small, confined tumor (lump), or as calcium deposits (microcalcifications) and then spreads through channels within the breast to the lymph nodes or through the blood stream to other organs. The tumor may grow and invade tissue around the breast, such as the skin or chest wall. Different types of breast cancer grow and spread at different rates -- some take years to spread beyond the breast while others grow and spread quickly.
Some lumps are benign (not cancerous), however these can be premalignant. The only safe way to distinguish between a benign lump and cancer is to have the tissue examined by a doctor through a biopsy

Men can get breast cancer, too, but they account for one percent of all breast cancer cases. Breast cancer in men is uncommon. This is possibly due to their smaller amount of breast tissue and the fact that men produce less hormones such as estrogen that are known to affect breast cancers in women

 Among women, breast cancer is the most common cancer and the second leading cause of cancer deaths after lung cancer.

Prognosis

 Although the precise causes of breast cancer are unclear, we know the main risk factors. Among the most significant factors are advancing age and a family history of breast cancer. 

 

 A link between breast cancer and hormones is gradually becoming clearer. Researchers think that the greater a woman's exposure to the hormone estrogen, the more susceptible she is to breast cancer.

The link between diet and breast cancer is debated. Obesity is a noteworthy risk factor, and drinking alcohol regularly

Symptoms & Types

Breast lumps aren’t the only possible sign of breast cancer, and most breast lumps aren’t cancerous.

In its early stages, breast cancer usually has no symptoms. As a tumor develops, you may note the following signs:

• A lump in the breast or underarm that persists after your menstrual cycle. This is often the first apparent symptom of breast cancer. Lumps associated with breast cancer are usually painless, although some may cause a prickly sensation. Lumps are usually visible on a mammogram long before they can be seen or felt.
• Swelling in the armpit.
•  Pain or tenderness in the breast. Although lumps are usually painless,pain or tenderness can be a sign of breast cancer.
• A noticeable flattening or indentation on the breast, which may indicate a tumor that cannot be seen or felt.
• Any change in the size, contour, texture, or temperature of the breast. A reddish, pitted surface like the skin of an orange could be a sign of advanced breast cancer.
• A change in the nipple, such as a nipple retraction,  dimpling, itching, a burning sensation, or ulceration. A scaly rash  of the nipple is symptomatic of Paget's disease, which may be associated with an underlying breast cancer.
• Unusual discharge from the nipple that may be clear, bloody, or another color. It's usually caused by benign conditions but could be due to cancer in some cases.
• A marble-like area under the skin.
• An area that is distinctly different from any other area on either breast.

 Diagnosis

Breast Cancer Detection

The earlier breast cancer is detected, the better it may be for the patient’s long-term health. Get a brief overview of the tests that can help detect breast cancer.

Breast Cancer Screening:

For women at normal risk of breast cancer, self-exams, clinical exams, and mammography starting at 40 may screen for breast cancer. Abnormal results or high-risk women may need earlier screening or additional tests.

Tests

Breast Self-Exam

Do you do regular breast self-exams? While some cancers are too tiny to feel, and most lumps aren’t cancer, self-exams are a proactive way to help take care of yourself.

Clinical Breast Exam

A clinical breast exam is a breast exam performed by a health care professional. It’s a basic part of women’s check-ups, starting at age 20.

Mammogram

A mammogram is a special type of X-ray taken to look for abnormal growths or changes in breast tissue. It’s a key tool in breast cancer detection, though no test is perfect.

Understanding the Mammogram Results

Most abnormal mammogram results aren’t breast cancer. But more testing is needed to make sure. The bottom line: Don’t panic, but do get the follow-up tests.

 Surviving Mammography

Do you find mammograms uncomfortable? Don’t skip the test; just learn how to handle it better.

Breast Ultrasound

Doctors sometimes use ultrasound images to check whether a breast lump is a cyst (a fluid-filled sac that is not cancer) or a solid mass.

Breast MRI

MRI stands for magnetic resonance imaging.

Breast Biopsy

When doctors perform a biopsy, they remove cells from a suspicious mass to see if it’s cancer.

Minimally Invasive Breast Biopsy

This type of breast biopsy generally uses a needle, not surgery.

Sentinel Node Biopsy

In a sentinal node biopsy, doctors check a few lymph nodes under the arm to see if cancer has spread into the lymph system.

Ductal Lavage

Ductal lavage checks cells from the milk ducts for precancerous cells.

Treatment

There are two major goals of breast cancer treatment:
1) To rid the body of the cancer as completely as possible.
2) To prevent cancer from returning.

How Is the Type of Breast Cancer Treatment Determined?

The type of breast cancer treatment recommended for you will depend on the size of your tumor, the extent of disease in your lymph nodes and/or throughout your body (the stage), and the presence of the HER2 oncogene and endocrine receptors (estrogen and progesterone receptors). Age, menstrual status, underlying health issues, and personal preferences play a role in this decision making process as well.

What Are the Types of Breast Cancer Treatment?

Breast cancer treatments are local or systemic.
Local treatments are used to remove or destroy the disease within the breast and surrounding regions, such as lymph nodes. These include:

• Surgery, either mastectomy or lumpectomy -- also called breast-conserving therapy. There are different types of mastectomies and lumpectomies.
• Radiation therapy

Systemic treatments are used to destroy or control cancer cells all over the body and include:

• Chemotherapy uses drugs to kill cancer cells. Side effects can include nausea, hair loss, early menopause, hot flashes, fatigue, and temporarily lowered blood counts.
•  
• estrogen, from promoting the growth of breast cancer cells that may remain after breast cancer surgery. Side effects can include hot flashes and vaginal dryness.
• Biological Therapy such as Herceptin, Perjeta, or Tykerb, which work by using the body's immune system to destroy cancer cells. These drugs target breast cancer cells that have high levels of a protein called HER2.

Systemic therapy may be given after local treatment (adjuvant therapy) or before (neoadjuvant therapy). Adjuvant therapy is used after local treatments to kill any cancer cells that may remain in the body, but are undetectable.
You may have just one form of breast cancer treatment or a combination of treatments, depending on your needs.
ROLE OF PHARMACIST

Pharmacists may want to encourage patients to talk about their treatment and side effects. Someone outside the family is the individual to whom they will likely tell the real truth. Patients are busy putting up a front to ease the distress of their family and close friends. Pharmacists are in an ideal position to encourage them to get problems out for discussion.

Pharmacists also can reassure their cancer patients that they have or will order any medication the patients need. In addition, patients can benefit from knowing they can depend on their pharmacist when they need to ask medical questions; often they feel they are imposing on their physician’s time if they ask during their appointments. Pharmacists should be ready with knowledgeable information

Pharmacists should always encourage their cancer patients to be treated by an oncologist as their primary physician. This is a time when no one but the most well-trained will do. When every decision can be a matter of life and death, it is necessary to have a physician who spends all day, every day, working with this disease state. An oncologist can help the patient make the best decisions.

Pharmacists can also help patients find the best treatment centers in their area. Institutions with the highest certification offered by NIH (National Institutes of Health) are known as comprehensive cancer centers.

PRESERVATION OF STERILITY

INTRODUCTION

Employing good manufacturing practices to limit microbial contamination in the first place is the preferred strategy, rather than, for example permitting contaminants to enter and proliferate in the product. This include proper design, cleansing and microbial monitoring of building and equipment. Personnel to observe health, hygiene, proper clothing and training. Proper storage of product and raw materials. Another factor utilized mostly in preservation of sterility include use of preservatives.

Characteristics of an ideal preservative

      It should have a broad spectrum of activity against gram negative and positive bacteria.

      It should be effective and stable over the range of pH values encountered in pharmaceutical products. In addition, it should be chemically stable so that there is no loss of preservative efficacy during the expected shelf life of the product.

      It should be compatible with other ingredients in the formulation and with packaging materials. This attribute would prevent loss of preservative potency as a result of interactions with formula components or packaging materials.

      It should not affect the physical properties of the product (i.e., color, clarity, odor, flavor, viscosity, texture).

      It should have a suitable O/W partition coefficient to ensure an effective concentration of the preservative in the aqueous phase of the product. Biological reactions take place in aqueous systems or at the interface of O/W systems; consequently, it is necessary to have sufficient preservative in the water phase to ensure adequate preservation of the product.

      It should inactivate microorganisms quickly enough to prevent microbial adaptation to the preservative system.

      It should be safe to use i.e. non-irritating, non-sensitizing. Safety includes handling of pure or concentrated materials in the manufacturing plant as well as the effect of preservatives in the finished formulation on the consumer.

      It should be cost-effective to use. From a commercial perspective, an effective concentration should add little to the cost of the formulated product.

Sterile products contain preservatives that designed to kill or limit the growth of micro-organisms that may gain entry. Two basic types of preservative system exist: chemical agents of either natural or synthetic origin; or approaches based on altering the products physical conditions to limit microbial growth.

Physical preservative system 

Microorganisms require certain physical conditions to survive; the deliberate adjustment of these to kill or suppress microorganisms can be used as a mode of preserving sterility. They include the following

a)      Available Water

Most organisms require over 70% water to grow, so dry products are unlikely to suffer colonization unless water is added. The amount of water available to an organism in a product is given by:

A_w=Vapour pressure of product/ vapour pressure of water (always less than 1) 

Certain organisms will only survive and grow at specific A_w levels. If A_w is lowered by adding solutes microbial growth can be prevented thus a variable level of preservation can therefore be achieved by a simple manipulation of a products A_W value. For example, a product with an A_w of 0.9 is unlikely to be affected by Pseudomonas species but could be colonized by other bacteria or fungi and moulds. This technique is limited by the large quantities of solute required to reduce A_w sufficiently.

b)      pH value

Majority of organisms grow best at pH 7 but survival is known at pH values from 3-11. Lowering or raising the product pH from neutrality provides a degree of preservation. However the scope for pH variation in pharmaceutical products is limited by physiological acceptability and formulation stability.

c)      Temperature

Microbial growth and majority of contaminants in pharmaceutical products are mesophilic organisms that grow best at ambient temperatures (15-45 degrees Celsius). A reduced storage temperature can be used as a means of inhibiting growth but has the disadvantage that the conditions are not integral to the product.

Chemical preservative systems

Include both synthetic and natural preservative agents.

1.      Natural preservative agents

i)        Essential oils

The antimicrobial properties of essential oils can be ascribed to their chemical constituents; alcohols, aldehydes, esters, ketones and terpenes. Sage oil for example is bactericidal and fungicidal; thyme oil which has been used as an antimicrobial contains 40% of phenolic compounds. Use of essential oils is limited due to high concentrations required for activity, which impart unusual organoleptic properties to the product.

ii)      Enzymes and proteins

The iron binding proteins lactoferrin and ovotransferrin reduce free iron concentration to around 10^-18M which inhibits the growth of many organisms with exception of Pseudomonas aeruginosa. These types of agents have been used primarily in the food industry but have not found common use in pharmaceuticals. Their protein nature precludes them from use in parenteral products.

2.      Synthetic preservatives

Biggest and the most common used group in the preservation of pharmaceutical products. They are classified based on their chemical structure. Within an individual chemical group several agents may be used as preservatives at low concentration and as disinfectants or antiseptics at higher concentrations.

Examples of the various groups include:

      Organic acids and salts: Include weak carboxylic acids such asbenzoic or sorbicacid and are used as preservatives in oral products. They are uncoupling agents that prevent the uptake of substrates requiring a proton motive force to enter the cell. Sorbic acid and benzoic acid have sufficiently low toxicity thus find application for oral use. Their main disadvantage-: Their activity is greatest at acid pH values thus the use of these preservatives is limited to products with pH less than 5.

      Parabens or hydoxybenzoates: Include methyl, ethyl, butyl, propyl, benzyl parabens and their salts. Preservatives used principally in topical and oral products and in some injections. They have relatively good activity against fungi and low toxicity. Unlike benzoic acid, the parabens retain their activity at raised pH values (pH 7 to 9). Disadvantages-: They have poor water solubility and a tendency to partition into the oily phase of emulsions. Relatively weak activity against gram-negative bacteria.

      Quaternary ammonium compounds: Include Benzalkonium chloride, cetrimide. Mode of action is by cell membrane damage resulting in loss of essential chemicals from the cell. They find use as preservatives in ophthalmic (in contact lens cleansing and soaking solutions), topical and some injectable products. They are very water soluble and effective at neutral and alkaline pH. Good stability, non-corrosive and generally non-hazardous. Their disadvantages is that they are usually incompatible with many negatively charged materials. Also, Benzalkonium chloride causes skin and ophthalmic sensitization.

      Alcohols: Only the arylalkyl and the substituted aliphatic alcohols are used as preservatives and include benzyl alcohol, phenoxyethanol, phenylethanol, chlorbutol, bronopol. The effect of aromatic substitution is to produce a range of compounds which are less volatile and less rapidly active and find use as preservatives as eye drops and contact lens solutions. Propylene glycol has preservative activity at concentrations of 10% in syrups and 20% in gelatin capsules. Bronopol find use in medicated shampoo solutions. They act by disrupting the bacterial cytoplasmic membrane, protein denaturation and cell lysis.

      Phenols: Include phenol and chlorocresol. The activity of phenolic compounds is pH dependent and is greatest below pH 9. Their mechanism of antimicrobial activity is by cell lysis. Phenolics find use as preservatives in topical and parenteral products. They have relatively low toxicity. Phenolics are usually absorbed by rubber and plastics.

      Biguanides: The only agent in this series is chlorhexidine. The Biguanides act on the cytoplasmic membrane causing leakage of cell contents Chlorhexidine is mainly used as preservatives in eye drops. Chlorhexidine is more active in the pH range 5 to 8. It has good activity against gram positive bacteria but less activity against gram negative bacteria and fungi. Biguanides are usually incompatible with many negatively charged materials.

      Mercurials: They include the phenylmercuric salts (phenylmercuric acetate, nitrate and borate) and thiomersal. They act through cell wall disruption, cytoplasmic coagulation and bind to thiol groups of enzymes and proteins. Thiomersal is used as a preservative in biological products and certain eye drops. Because of the toxicity of mercury, they are not recommended where prolonged administration is necessary.

Preservative choice is controlled by the physicochemical properties of the formulation and the processes involved during manufacture. Factors affecting activity of antimicrobial activity of preservatives-:

      Effect of concentration-: The activity of a preservative depends on the free concentration of the active form of the molecule in the aqueous phase. Partitioning between water and oil phase, micellar solubilization, and dissociation may all decrease the efficacy of a preservative.

      Effect of pH-: The pH of the formulation may affect the efficacy of the preservative system in a number of ways: pH affects the activity of preservatives, for example, sorbic acid is only active as the undissociated molecule. pH affects the oil–water partition coefficient of the preservative, the micellar solubilization, and the interaction with cyclodextrins, because the undissociated form is more hydrophobic than the dissociated form. Some preservative-component interactions may be pH-dependent because of ionization effects on components.

      Effect of temperature-: An increase in temperature enhances the activity of a preservative. Temperature affects the interaction of preservatives with other formulation ingredients for example, adsorption of preservatives by solid ingredients decreases with increasing temperature.

      Sorption of the preservative to packaging For example hydoxybenzoates, benzyl alcohol and phenol interact with olefin based plastics such as polyethylene and polypropylene. These interactions reduce free preservative concentration in a product

      Interaction between preservative and other ingredients. Surfactants that are present in concentration above their critical micelle concentration can solubilize lipophilic preservatives resulting in reduced free aqueous concentration and reduced antimicrobial activity, for example cyclodextrins form inclusion complexes with hydoxybenzoates

In conclusion, preservatives, either singly or in synergistic combinations remain necessary to prevent microbial contamination of multi-use liquid or semi-solid medicinal products, particularly from opportunistic pathogens. Non-inclusion can result in serious patient health consequences.

REFERENCES

1)      Hodges, N.A. and Hanlon, G. (2000). Antimicrobial preservative efficacy testing. In Handbook of Microbiological Quality Control: Pharmaceuticals and Medical Devices. Baird, R.M., Hodges, N.A., and Denyer, S.P., Eds. Taylor & Francis, London, pp.168–189.

2)      Hodges, N.A. and Denyer, S.P. (1996). Preservative testing. In Encyclopedia of Pharmaceutical Technology, Swarbrick, J. and Boylan, J.C., Eds. Vol. 13. Marcel Dekker, NewYork, pp. 21–37.

3)      Chapman, D.G. (1987). Preservatives available for use. In Preservatives in the Food, Pharmaceutical and Environmental Industries. Board, R.G., Allwood, M.C., and Banks, J.G., Eds. SAB Technical Series 22. Blackwell Scientific Publications, Oxford, pp.177–195.

4)      Denyer, S.P., Hugo, W.B., and Harding, V.D. (1985). Synergy in preservative combinations. Int. J Pharm., 25, 245–253.

PHYSICAL METHODS OF STERILIZATION:

PHYSICAL METHODS OF STERILIZATION:

Sunlight: The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays in it. It is responsible for spontaneous sterilization in natural conditions. In tropical countries, the sunlight is more effective in killing germs due to combination of ultraviolet rays and heat. By killing bacteria suspended in water, sunlight provides natural method of disinfection of water bodies such as tanks and lakes. Sunlight is not sporicidal, hence it does not sterilize.

Heat: Heat is considered to be most reliable method of sterilization of articles that can withstand heat. Heat acts by oxidative effects as well as denaturation and coagulation of proteins. Those articles that cannot withstand high temperatures can still be sterilized at lower temperature by prolonging the duration of exposure.

Factors affecting sterilization by heat are:

o Nature of heat: Moist heat is more effective than dry heat

o Temperature and time: temperature and time are inversely proportional. As temperature increases the time taken decreases.

o Number of microorganisms: More the number of microorganisms, higher the temperature or longer the

duration required.

o Nature of microorganism: Depends on species and strain of microorganism, sensitivity to heat may vary. Spores are highly resistant to heat.

o Type of material: Articles that are heavily contaminated require higher temperature or prolonged exposure. Certain heat sensitive articles must be sterilized at lower temperature.

o Presence of organic material: Organic materials such as protein, sugars, oils and fats increase the time

required.

Action of heat:

Dry heat acts by protein denaturation, oxidative damage and toxic effects of elevated levels of electrolytes. The moist heat acts by coagulation and denaturation of proteins. Moist heat is superior to dry heat in action. Temperature required to kill microbe by dry heat is more than the moist heat. Thermal death time is the minimum time required to kill a suspension of organisms at a predetermined temperature in a specified environment.

DRY HEAT:

Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing spatulas are sterilized by holding them in Bunsen flame till they become red hot. This is a simple method for effective sterilization of such articles, but is limited to those articles that can be heated to redness in flame.

Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are passed through the flame a few times. Even though most vegetative cells are killed, there is no guarantee that spores too would die on such short exposure. This method too is limited to those articles that can be exposed to flame. Cracking of the glassware may occur.

Incineration: This is a method of destroying contaminated material by burning them in incinerator. Articles such as soiled dressings; animal carcasses, pathological material and bedding etc should be subjected to incineration. This technique results in the loss of the article, hence is suitable only for those articles that have to be disposed. Burning of polystyrene materials emits dense smoke, and hence they should not be incinerated.

Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are exposed to high temperature (160o C) for duration of one hour in an electrically heated oven. Since air is poor conductor of heat, even distribution of heat throughout the chamber is achieved by a fan. The heat is transferred to the article by radiation, conduction and convection. The oven should be fitted with a thermostat control, temperature indicator, meshed shelves and must have adequate insulation.

Articles sterilized: Metallic instruments (like forceps, scalpels, scissors), glasswares (such as petri-dishes, pipettes, flasks, all-glass syringes), swabs, oils, grease, petroleum jelly and some pharmaceutical products.

Sterilization process: Articles to be sterilized must be perfectly dry before placing them inside to avoid breakage.

Articles must be placed at sufficient distance so as to allow free circulation of air in between. Mouths of flasks, test tubes and both ends of pipettes must be plugged with cotton wool. Articles such as petri dishes and pipettes may be arranged inside metal canisters and then placed. Individual glass articles must be wrapped in kraft paper or aluminum foils.

Sterilization cycle: This takes into consideration the time taken for the articles to reach the sterilizing temperature, maintenance of the sterilizing temperature for a defined period (holding time) and the time taken for the articles to cool down. Different temperature-time relations for holding time are 60 minutes at 160oC, 40 minutes at 170oC and 20 minutes at 180oC. Increasing temperature by 10 degrees shortens the sterilizing time by 50 percent. The hot air oven must not be opened until the temperature inside has fallen below 60oC to prevent breakage of glasswares.

Sterilization control: Three methods exist to check the efficacy of sterilization process, namely physical, chemical and biological.

􀂃 Physical: Temperature chart recorder and thermocouple.

􀂃 Chemical: Browne’s tube No.3 (green spot, color changes from red to green)

􀂃 Biological: 106 spores of Bacillus subtilis var niger or Clostridium tetani on paper strips are placed inside envelopes and then placed inside the hot air oven. Upon completion of sterilization cycle, the strips are removed and inoculated into thioglycollate broth or cooked meat medium and incubated at 37oC for 3-5 days. Proper sterilization should kill the spores and there should not be any growth.

Advantages: It is an effective method of sterilization of heat stable articles. The articles remain dry after sterilization. This is the only method of sterilizing oils and powders.

Disadvantages:

􀂃 Since air is poor conductor of heat, hot air has poor penetration.

􀂃 Cotton wool and paper may get slightly charred.

􀂃 Glasses may become smoky.

􀂃 Takes longer time compared to autoclave.

Infra red rays: Infrared rays bring about sterilization by generation of heat. Articles to be sterilized are placed in a moving conveyer belt and passed through a tunnel that is heated by infrared radiators to a temperature of 180oC.

The articles are exposed to that temperature for a period of 7.5 minutes. Articles sterilized included metallic instruments and glassware. It is mainly used in central sterile supply department. It requires special equipments, hence is not applicable in diagnostic laboratory. Efficiency can be checked using Browne’s tube No.4 (blue spot).

MOIST HEAT:

Moist heat acts by coagulation and denaturation of proteins.

At temperature below 100oC:

􀂃 Pasteurization: This process was originally employed by Louis Pasteur. Currently this procedure is employed in food and dairy industry. There are two methods of pasteurization, the holder method (heated at 63oC for 30 minutes) and flash method (heated at 72oC for 15 seconds) followed by quickly cooling to 13oC.

Other pasteurization methods include Ultra-High Temperature (UHT), 140oC for 15 sec and 149oC for 0.5 sec. This method is suitable to destroy most milk borne pathogens like Salmonella, Mycobacteria, Streptococci, Staphylococci and Brucella, however Coxiella may survive pasteurization. Efficacy is tested by phosphatase test and methylene blue test.

􀂃 Vaccine bath: The contaminating bacteria in a vaccine preparation can be inactivated by heating in a water bath at 60oC for one hour. Only vegetative bacteria are killed and spores survive.

􀂃 Serum bath: The contaminating bacteria in a serum preparation can be inactivated by heating in a water bath at 56oC for one hour on several successive days. Proteins in the serum will coagulate at higher temperature. Only vegetative bacteria are killed and spores survive.

􀂃 Inspissation: This is a technique to solidify as well as disinfect egg and serum containing media. The medium containing serum or egg are placed in the slopes of an inspissator and heated at 80-85oC for 30 minutes on three successive days. On the first day, the vegetative bacteria would die and those spores that germinate by next day are then killed the following day. The process depends on germination of spores in between inspissation. If the spores fail to germinate then this technique cannot be considered sterilization.

At temperature 100oC:

􀂃 Boiling: Boiling water (100oC) kills most vegetative bacteria and viruses immediately. Certain bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Some bacterial spores are resistant to boiling and survive; hence this is not a substitute for sterilization. The killing activity can be enhanced by addition of 2% sodium bicarbonate. When absolute sterility is not required, certain metal articles and

glasswares can be disinfected by placing them in boiling water for 10-20 minutes. The lid of the boiler must not be opened during the period.

􀂃 Steam at 100oC: Instead of keeping the articles in boiling water, they are subjected to free steam at 100oC.

Traditionally Arnold’s and Koch’s steamers were used. An autoclave (with discharge tap open) can also serve the same purpose. A steamer is a metal cabinet with perforated trays to hold the articles and a conical lid. The bottom of steamer is filled with water and heated. The steam that is generated sterilizes the articles when exposed for a period of 90 minutes. Media such as TCBS, DCA and selenite broth are sterilized by steaming. Sugar and gelatin in medium may get decomposed on autoclaving, hence they are exposed to free steaming for 20 minutes for three successive days. This process is known as tyndallisation (after John Tyndall) or fractional sterilization or intermittent sterilization. The vegetative bacteria are killed in the first exposure and the spores that germinate by next day are killed in subsequent days. The success of process depends on the germination of spores.

At temperature above 100oC:

Autoclave: Sterilization can be effectively achieved at a temperature above 100oC using an autoclave. Water boils at 100oC at atmospheric pressure, but if pressure is raised, the temperature at which the water boils also increases. In an autoclave the water is boiled in a closed chamber. As the pressure rises, the boiling point of water also raises.

At a pressure of 15 lbs inside the autoclave, the temperature is said to be 121oC. Exposure of articles to this temperature for 15 minutes sterilizes them. To destroy the infective agents associated with spongiform encephalopathies (prions), higher temperatures or longer times are used; 135oC or 121oC for at least one hour are recommended.

Advantages of steam: It has more penetrative power than dry air, it moistens the spores (moisture is essential for coagulation of proteins), condensation of steam on cooler surface releases latent heat, condensation of steam draws in fresh steam.

Different types of autoclave:

Simple “pressure-cooker type” laboratory autoclave, Steam jacketed downward displacement laboratory autoclave and high pressure pre-vacuum autoclave

Construction And Operation Of Autoclave:

A simple autoclave has vertical or horizontal cylindrical body with a heating element, a perforated try to keep the articles, a lid that can be fastened by screw clamps, a pressure gauge, a safety valve and a discharge tap. The articles to be sterilized must not be tightly packed. The screw caps and cotton plugs must be loosely fitted. The lid is closed but the discharge tap is kept open and the water is heated. As the water starts boiling, the steam drives air out of the discharge tap. When all the air is displaced and steam start appearing through the discharge tap, the tap is closed. The pressure inside is allowed to rise upto 15 lbs per square inch. At this pressure the articles are held for 15 minutes, after which the heating is stopped and the autoclave is allowed to cool. Once the pressure gauge shows the pressure equal to atmospheric pressure, the discharge tap is opened to let the air in. The lid is then opened and articles removed.Articles sterilized: Culture media, dressings, certain equipment, linen etc. Precautions: Articles should not be tightly packed, the autoclave must not be overloaded, air discharge must be complete and there should not be any residual air trapped inside, caps of bottles and flasks should not be tight, autoclave must not be opened until the pressure has fallen or else the contents will boil over, articles must be wrapped in paper to prevent drenching, bottles must not be overfilled. Advantage: Very effective way of sterilization, quicker than hot air oven. Disadvantages: Drenching and wetting or articles may occur, trapped air may reduce the efficacy, takes long time to cool

Sterilization control: Physical method includes automatic process control, thermocouple and temperature chart recorder. Chemical method includes Browne’s tube No.1 (black spot) and succinic acid (whose melting point is 121oC) and Bowie Dick tape. Bowie Dick tape is applied to articles being autoclaved. If the process has been satisfactory, dark brown stripes will appear across the tape. Biological method includes a paper strip containing 106 spores of Geobacillus stearothermophilus.

RADIATION:

Two types of radiation are used, ionizing and non-ionizing. Non-ionizing rays are low energy rays with poor penetrative power while ionizing rays are high-energy rays with good penetrative power. Since adiation does not generate heat, it is termed "cold sterilization". In some parts of Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent.

􀂃 Non-ionizing rays: Rays of wavelength longer than the visible light are non-ionizing. Microbicidal wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being most effective. UV rays are generated using a high-pressure mercury vapor lamp. It is at this wavelength that the absorption by the microorganisms is at its maximum, which results in the germicidal effect. UV rays induce formation of thymine-thymine dimers, which ultimately inhibits DNA replication. UV readily induces mutations in cells irradiated with a non-lethal dose. Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to the effective UV radiation are inactivated within seconds. Since UV rays don’t kill spores, they are considered to be of use in surface disinfection. UV rays are employed to disinfect hospital wards, operation theatres, virus laboratories, corridors, etc. Disadvantages of using uv rays include low penetrative power, limited life of the uv bulb, some bacteria have DNA repair enzymes that can overcome damage caused by uv rays, organic matter and dust prevents its reach, rays are harmful to skin and eyes. It doesn't penetrate glass, paper or plastic.

􀂃 Ionizing rays: Ionizing rays are of two types, particulate and electromagnetic rays.

o Electron beams are particulate in nature while gamma rays are electromagnetic in nature. Highspeed electrons are produced by a linear accelerator from a heated cathode. Electron beams are employed to sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticals.

Sterilization is accomplished in few seconds. Unlike electromagnetic rays, the instruments can be switched off. Disadvantage includes poor penetrative power and requirement of sophisticated equipment.

o Electromagnetic rays such as gamma rays emanate from nuclear disintegration of certain radioactive isotopes (Co60, Cs137). They have more penetrative power than electron beam but require longer time of exposure. These high-energy radiations damage the nucleic acid of the microorganism. A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores. It is used commercially to sterilize disposable petri dishes, plastic syringes, antibiotics, vitamins, hormones, glasswares and fabrics. Disadvantages include; unlike electron beams, they can’t be switched off, glasswares tend to become brownish, loss of tensile strength in fabric. Gamma irradiation impairs the flavour of certain foods. Bacillus pumilus E601 is used to evaluate sterilization process.