Clinical Bacteriology Module 3: Physical Methods of Microbial Control PDF

Figure 1.3 Sterilization by Filtration PORE SIZE OF MEMBRANE FILTERS & PARTICLES THAT PASS...

Figure 1.3 Sterilization by Filtration PORE SIZE OF MEMBRANE FILTERS & PARTICLES THAT PASS THROUGH THEM Figure 1.1 An Overview of Microbial Control Methods Pore Size in µm Particles that pass through them 10 Erythrocytes, yeast cells, bacteria, viruses, molecules 5 Yeast cells, bacteria, viruses, molecules 3 Some yeast cells, bacteria, viruses, molecules METHOD MECHANISM COMMENT PREFERRED USE 1.2 Most bacteria, viruses, molecules OF ACTION 0.45 A few bacteria, viruses, molecules Mechanic al Means / 0.22 Viruses, molecules Methods 0.10 Medium-sized to small viruses, molecules Physical Usually done with water Handwashing, 0.05 Small viruses, molecules separation of to which some chemical certain Scrubbing microorganis agent such as soap, equipment 0.025 Only the very smallest viruses, molecules m from the detergent or sodium Ultrafilter Molecules object to be carbonate has been sterilized. added. METHOD MECHANISM COMMENT PREFERRED Passage of liquid Used for OF ACTION USE or gas/air through sterilizing Pulled through a screen-like liquids small, material with pores and mechanically so small that traps culture introduced pores microbes. media w/ a vacuum. that Accomplished Organism larger Separation of cannot be through the use of than the size of Filtration bacteria from heated. thin membrane the pores are suspending filters composed of Separatin retained. liquid. plastic polymerase g toxins, 0.45-0.80µm pore Sterilization of enzymes or cellulose esters. size = bacteria, heat-sensitive and Materials used for proteins A. Liquid yeasts, molds solutions, such Filtration: 0.22µm = for as vaccines from the a. Unglazed critical sterilizing and antibiotic bacteria protein (e.g., Parental solutions. that b. Diatomaceo solutions) produce us earth 0.01µm = small them. c. Compressed viruses asbestos (0.2-0.14µm = size d. Sintered range of most glass bacteria) e. Cellulose (0.4-2µm = size membranes range of most pathogenic bacteria) Accomplished with the use of Laboratory HIGH EFFICIENCY hoods, rooms of B. Air AIR FILTERS immuno- (HEPA) compromised Removes patients. microorganisms larger than 0.3µm. Figure 1.2 Membrane Filtration  Selling of In nature, large particles Primary The usual procedure is to heat at 1.1 kg/𝒄𝒎𝟐 (15lb//𝒊𝒏𝟐 ) suspended and suspended bacteria sewage and Sedimentation particles to sink to the bottom of water steam pressure, which yields at a temperature of 121°C. the bottom of lakes, ponds and flowing treatment. At 121°C, the time of autoclaving to achieve sterilization is a liquid. streams. generally considered to be 15-20 min, depending on the volume of the load. To make sure, sterilization is successful METHOD MECHANISM OF COMMENT PREFERRED ACTION USE one should ensure: Heat a. Air should be evacuated so that the chamber fills with Moist Heat steam. Sterilization b. Articles should be placed in the autoclave so that steam Boiling or Kills vegetative Dishes, flowing bacterial and can easily penetrate them. basins, steam Denaturation fungal various - Important: Note that it is not the pressure of the (30mins. At pathogens and equipment autoclave that kills the microorganisms but the high 100°C) many viruses within 10 temperature that can be achieved when steam is minutes. placed under pressure. Less effective on endospores. If bulky objects are being sterilized, heat transfer to the Denaturation interior will be slow, and the heating time must be sufficiently of proteins long so that the object is at 121°C for 15 min. Steam under pressure Extended times are also required when large volumes of Principle: As liquids are being autoclaved because large volumes take heated water in Culture the vessel forms media, longer to reach sterilization temperature. Autoclaving steam, the steam solutions, Very effective (121°C, causes the method of linens, 15mins., 15 pressure in the utensils, psi) sterilization. Autoclave comprises of three parts: a pressure chamber, a vessel to increase All vegetative dressings, (135°C, 3mins beyond equipment lid and an electrical heater. = Flash atmospheric cells and autoclaving) pressure. The endospores and other higher the are killed in items that pressure in turn, about can Consists of: increases the 15mins. withstand temperature at 1. Large cylinder (vertical or horizontal) in which the 132°C, temperature which steam forms. 4.5mins = for and materials to be sterilized are placed. It is made up of Whereas steam produced at PRIONS. pressure. gunmetal or stainless steel and placed in a supporting atmospheric iron case. pressure never 2. A steam jacket (water compartment). exceeds 100°, steam produced at an additional 15 psi can reach 121°C, a Is fastened by screw clamps and rendered airtight by an temperature that asbestos washer. can kill even the The lid bears the following: endospores. The pressure itself 1. A discharge taps for air and steam discharge plays no direct role 2. A pressure gauge (sets the pressure at a particular in killing. level) 3. A safety valve (to remove the excess steam) The autoclave is a sealed device (similar to a pressure cooker) that kills microorganisms using saturated steam It is attached to the jacket that heats the water to produce under pressure. steam. The use of moist heat facilitates the killing of all microorganisms, including heat-resistant endospores which is achieved by heating the materials inside the device at temperatures above the boiling point of water. According to the principle of gas laws, this can be achieved by raising It is the most common type used in laboratories and is the pressure inside the device. available in various sizes and dimensions. The boiling point (vapor pressure equals that of the a. Vertical type (small volume capacity) surrounding atmosphere) of water varies depending upon b. Horizontal autoclave (large volume capacity) the surrounding environmental pressure. - Example: Water boils at 100°C at sea level (higher pressure), but at 93.4°C at 1,905 meters altitude (lower pressure). So, in an enclosed device, if we raise the pressure, the temperature at which water boils also increases.  The Centers for Disease Control (CDC) recommends weekly autoclaving of a culture containing heat resistant endospores of Geobacillus stearothermophilus, to check autoclave performance. Adhesive-backed paper tape with heat-sensitive, chemical indicator marking that change color or display-diagonal stripes, the words “sterile” or “autoclaved” when exposed to effective sterilization temperature (121°C) are used to check the efficacy of autoclave. Figure 3.1 Types of Autoclaves Thermocouple is a temperature measuring device that records the temperature by a potentiometer. Browne’s tube (invented by Albert Browne in 1930) contains a heat-sensitive red dye which turns green after being exposed to certain temperature for a definite period of time. Conversion of dye color gives information about the duration of time and temperature. Figure 3.2 Gravity Displacement Type Autoclave ❖ Steam is heated in the jacket, enters the sterilization Figure 3.3 Sterility Controls chamber through an opening and is exhausted through a vent. Checking for Sterility - To check if an autoclave is operating properly, a commercially prepared spore test ampule is placed in the autoclave and run with the rest of the load. Several methods are available to ensure that autoclaving - Afterward, the vial is crushed to release the medium achieve sterility. onto the strip containing spores. If the load was truly The effectiveness of the sterilization done by autoclave can sterilized, the spores will have been killed, and growth be monitored by: will not occur in the medium. - Sometimes an indicator dye is added to the medium, which will turn color if microbial growth occurs due to Spores of Geobacillus stearothermophilus (formerly called the accumulation of acid by-products. This is faster Bacillus stearothermophilus) are the best indicator because than waiting for sufficient growth to turn the medium they are resistant to steaming. Their spores are killed in 12 cloudy. minutes at 121°C. The vial is placed in the center of the material to be sterilized and is autoclaved. Then the inner ampule is broken, releasing the medium, and the whole container is incubated. If no growth appears in the autoclaved culture, sterilization is deemed effective.  ORGANISM TEMPERATURE TIME EXPOSURE TO KILL SPORES DRY HEAT Bacillus subtilis 121°C 1 min. Bacillus 121°C 12 mins. steathermophilus Clostridium 120°C 10 mins. botulinum Clostridium tetani 105°C 10 mins. MOIST HEAT Bacillus subtilis 121°C 120 mins. Bacillus 140°C 5 mins. steathermophilus Clostridium 120°C 120 mins. botulinum Figure 4.1 Checking for Sterility Clostridium tetani 100°C 60 mins. The following precautions should be taken while using an autoclave: METHOD MECHANISM COMMENT PREFERRED USE 1. Autoclave should not be used for sterilizing waterproof OF ACTION Dry Heat materials, such as oil and grease or dry materials, such Sterilization as glove powder. A. Direct Buring to Very effective Innoculating 2. Materials are loaded in, such a way that it allows loops & needles. Flaming ashes method of efficient steam penetration (do not overfill the chamber). sterilization. It is more efficient and safer to run two separate, B. Incinerat Burning to Very effective Paper cups, uncrowded loads than one crowded one. ion ashes method of dressings, sterilization. animal 3. Wrapping objects in aluminum foil is not recommended carcasses, because it may interfere with steam penetration. bags and Articles should be wrapped in materials that allow wipes steam penetration. C. Hot Air Denaturati Very effective Empty 4. Materials should not touch the sides or top of the Sterilizati on method of glasswar on sterilization. es, chamber. (170°C, 120°C-130°C, instrume 5. The clean items and the wastes should be autoclaved 1.5 hrs= nts, 2hrs – separately. Vegetative cell needles Hot air 6. Polyethylene trays should not be used as they may melt 160°C= and oven) and cause damage to the autoclave. Spores syringes Petrolatu ❖ Modern autoclaves have devices to maintain proper m, oils & pressure and record internal temperature during operation. fats (all Regardless of the presence of such a device, autoclave that pressure should be checked periodically and maintained. resist penetrati Never autoclave any liquid in a sealed container!!! on by steam or water) An autoclave is used to sterilize surgical equipment, Pasteurization Denaturati Heat Milk, creams laboratory instruments, pharmaceutical items, and other on treatment for and certain (63-65°C, 30 milk and alcoholic materials. mins.) heat- beverages It can sterilize solids, liquids, hollows, and instruments of (72°C, 15 secs.) sensitive (beers and various shapes and sizes. Autoclaves vary in size, shape materials & wine). and functionality solutions w/o changing the composition and food value of the 121°C 15 mins. material MOIST HEAT 125°C 10 mins. itself. 134°C 3 mins. Kills all pathogens and 121°C 600 mins. nonpathogen DRY HEAT 140°C 180 mins. s. 160°C 120 mins. Presently 170°C 60 mins. employs the HIGH  TEMPERATU RE-SHORT TIME METHOD (HTST) Milk = 72°C- 15 secs. Ice cream= 82°C- 20 secs. ULTRA HIGH TEMP. METHOD- designed to render a Figure 5.1 Applications Using Heat for Sterilization and Disinfection product free of all microorganis ms that can grow under normal storage conditions 140°C-150°C held for several seconds and then rapidly cooled. Fractional Denaturati Kills vegetative For heat- Sterilization / on cells and sensitive Tyndallization endospores. materials. Figure 5.2 Applications Using Heat for Sterilization and Disinfection LOW TEMPERATURE Refrigeration Decreased Has Food, drug METHOD MECHANIS COMMENT PREFFERED chemical bacteriostatic and culture M OF USE reactions effect preservation. ACTION and Dessication Disruption of Involves Food possible metabolism removing Preservation changes in water from proteins. microbes Deep freezing Decreased An effective Food, drug Primarily (-50°C- -95°C) chemical method for and culture bacteriostati reactions preserving preservation. c and microbial Osmotic Plasmolysis Plasmolysis Food possible cultures in Pressure (Hypertoni results in cell Preservation (in changes in which cultures c Sol’n) losing water sugar or salt) proteins. are quick Plasmoptysis (cell frozen. (Hypotinic membrane Lyophilization Decreased Water Food, drug Sol’n) and chemical removed by and culture cytoplasm reactions high preservation shrink away and vacuum at from the cell possible low wall). changes in temperatur Plasmoptysis proteins. e. results in cell Most rupture due effective to increase in method for fluid preserving pressure). microbial RADIATION cultures. 1. Ionizing Destruction of Not usually Used for radiatio DNA by X- done in routine sterilizing n rays, Gamma sterilization. pharmaceutical rays & High s, medical and electron dental supplies. beam energy. 2. Non- Damage to Radiation is not UV Lamp ionizing DNA by UV very mainly radiatio light. penetrating. germicidal. n  LASERS Split-second Sterilize procedure. medical It must reach equipment. all parts of Clear the air the item to in operating be sterilized. rooms. Pick organisms off a wound surface. Figure 6.3 Cellular effects of irradiation (a.) Ionizing radiation can penetrate a solid barrier, Figure 6.1 UV Water Purification Process bombard a cell, enter it, and dislodge electrons from molecules. Breakage of DNA creates massive mutations. The UV light disrupts the DNA by destroying the nucleic (b.) Nonionizing radiation enters a cell, strikes molecules acids and puts a serious damper on cellular functions which and excites them. The effect on DNA is mutation by ultimately leads to the germs dying. Water flowing past the formation of abnormal bonds. UV light in the supply pipe is exposed to 254 nm of radiation. (c.) A solid barrier cannot be penetrated by nonionizing This system can be used as alternative to Chlorination. radiation. METHOD MECHANISM COMMENT PREFFERED OF ACTION USE ULTRASONICS Waves Used at a Sterilization coagulate range of of proteins. vibration mechanical where they equipment are no longer heated as sound (supersonic/ ultrasonic) 18,000 hertz or more (hertz=cycle/ min.) Figure 6.2 Formation of pyrimidine dimers by the action of ultraviolet (UV) radiation This shows what occurs when two adjacent thymine bases on one strand of DNA are induced by UV rays to bond laterally with each other. The result is a thymine dimer shown in greater detail. Dimers can also occur between adjacent cytosines and thymine and cytosine bases. If they are not repaired, dimers can prevent that segment of DNA from being correctly replicated or transcribed. Massive dimerization is lethal to cells.