MB - 351 Lecture 20 Flashcards
One of the most common physical methods of control of microbial growth. Ex?
use of heat (bacticinerator - uses infrared heat to sterilize inoculating loops or needles)
Physical methods of microbial control
heat (dry and moist), radiation (non-ionizing & ionizing), filtration
Types of Dry Heat, and how these things kill the organism?
flame/incinerate, hot air
“Dry” heat: provided by flame or incineration: like a Bunsen burner, or by infrared heat using a bacticinerator. Or use of hot air sterilization, which is forced ventilation of heated air: like that provided by a drying oven. Dry heat kills the organisms by destructive oxidation of essential cell constituents.
Killing endospores with dry heat, and what is dry heat most frequently used for?
Killing of the most resistant endospores by hot air sterilization requires a temperature of 170 °C for 2 hours. Dry heat is most often employed for glassware; syringes, metal instruments and paper wrapped goods, which are not spoiled by high temperatures. You will often see these in doctor’s offices. It is also used for anhydrous fats, oils and powders that are impermeable to moisture.
Moist heat, and types. How does moist heat kill proteins? What bonds are broken?
- requires a liquid
- moist heat kills by coagulation of proteins (denaturation), caused by breakage in Hydrogen bonds that hold proteins
Types: pasteurization, boil, autoclave
pasteurization
- heat treating of food or liquid to eliminate pathogents, will not achieve sterilization
- is a process of heating a food, usually a liquid, to a specific temperature for a definite length of time and then cooling it immediately. This process slows microbial growth in the food. Unlike sterilization, pasteurization is not intended to kill all microorganisms in the food. Instead, pasteurization aims to reduce the number of viable pathogens so they are unlikely to cause disease (distruction of pathogens most important thing). Commercial-scale sterilization of food is not common because it adversely affects the taste and quality of the product. Certain foods, like dairy products, may be pasteurized to ensure pathogenic microbes are destroyed.
Boil the liquid(moist heat)
- kills most pathogens, but not necessarily all endospores, so not always reliable
- boiling 100 degrees celcius for 10 minutes, kills everything except some endospores
- will kill most vegetative (actively growing/divinding) bacterial pathogens, viruses, and fungi within 10 minutes. Some endospores resist boiling for 20 hours! Therefore, boiling is not always a reliable sterilization procedure.
For reliable sterilization using moist heat, what is used? And what is it used for, and how does it work?
Autoclave: steam under pressure
Other important facts outside of powerpoint:
- used to sterilize equipment and supplies by subjecting them to high pressure steam
- Because the boiling point of water increases as the pressure increases, the pressure built up inside the cooker allows the liquid in the pot to rise to a higher temperature before boiling.
- Sterilization by moist heat of the most resistant endospores generally requires 121 °C for 15-20 minutes. By comparison, you could boil (100degC) For 20hrs and the endospores might still be in tact. Endospores of bacteria are considered the most thermoduric of all cells so their destruction guarantees sterility.
What is the best way to sterilize/disinfect microbes that are heat-liable (sensitive to heat, like plastic)
MEMBRANE FILTRATION
Paper and other products that may be damaged by steam must also be steralized in another way…? What way is this?
Dry heat (hot air oven): 170 degrees celcius for 2 hours
Ionizing radiation includes? And how does it work? What is it used for?
includes X-rays, gamma rays, or high energy electron beams. These types of radiation have high penetration and can ionize molecules; particularly ionizing H20 = forming highly reactive hydroxyl free radicals which results in DNA damage, killing the microbe.
Used for sterilizing pharmaceuticals and medical, dental, supplies.
Non-ionizing radiation
has a wavelength longer than that of ionizing radiation (>1nm). Ultraviolet light (UV) is a type of nonionizing radiation, it causes thymine dimers which inhibit correct DNA replication. UV is very effective but doesn’t get through glass or plastic very well, but we do use it in closed environments, like biosafety cabinets (an enclosed, ventilated lab workspace for safely working with materials contaminated with pathogens
Filtration
Filtration, this physically removes microbes by passage through a screen-like material with pores small enough to retain microbes. You can readily purchase disposable filters of various sizes- 0.22um or 0.45um, even some 0.01um, and these should capture most microbes, given that the average E. coli is ~1 um.
Other physical methods for destroying microbes that we won’t discuss:
Cold , High atmospheric pressure , Desiccation Osmotic pressure, etc.
Most common physical method for destruction of microbes
MOIST HEAT. Moist heat is more effective than dry heat - it takes lower temperatures and less time bc heat in water is more easily transferred to a cool object/body than heat in air
How does moist heat destroy organisms? And what is it most commonly used for?
Destroys oranisms by coagulating and denaturing their enzymes and structural proteins. Used for sterilization of culture media, and all other materials through which steam can penetrate.
Pasteurization of milk, and types
Pasteurization of milk typically uses temperatures below boiling (~63degC for 30minutes) since at very high temperatures the milk protein, casein, will irreversibly aggregate, or “curdle.” There are two main types of pasteurization used today: High Temperature/Short Time (HTST) and Ultra-high temperature (UHT or ultra-heat treated) is also used for milk treatment. In the HTST process, milk is forced between metal plates or through pipes heated on the outside by hot water, and is heated to 71.7 °C (161 °F) for 15–20 seconds. UHT processing holds the milk at a temperature of 135 °C (275 °F) for a minimum of one second. The result of pasteurization is lower microbial loads and elimination of most serious pathogens, but Thermoduric bacteria, which can survive, to varying extents, but don’t grow.
Phenolic compounds (derivatices of phenol). How do they work? Good for disinfecting what? And examples of phenolics?
- phenols act by disrupting plamsa membranes, inactivating enzymes, denaturing proteins.
- but are irritants and toxic to humans
- Good for disinfecting things like pus, saliva, and feces from surfaces and instruments. Phenolics are derivatives of phenol that have been altered to reduce irritating qualities or increase antimicrobial activity when combined with detergents. They are a large class of chemical agents that include O-phenylphenol (main ingredient of Lysol), a great surface disinfectant.
Bisphenols
Bisphenols: 2 phenolic groups connected by a bridge, such as hexachlorophene, it is an ingredient of a prescription lotion, pHisoHex used for surgical and hospital microbial control procedures. Particularly good against Gram-positive strep and staph (cause skin infections in infants) however, can cause neurological damage if exposure is high (bathing several times per day) . Triclosan, another Bisphenol, is widely used in soaps, toothpaste, and incorporated into plastic kitchenware. It inhibits fatty acid synthesis, is bacteriostatic at low concentrations and bactericidal at high concentrations. Often used as a disinfectant. Broad spectrum, especially against gram-positive bacteria and fungi, however, resistant strains of bacteria have arisen and triclosan may contribute to antibiotic resistance as well.
Alcohols. First examples of alcohols listed on slide, and details? How does it work to kill microbes? What is the best concentration of ethanol to use?
works by denaturing proteins, disrupting cell membranes, and dissolving lipids. It is bacteriocidal at certain concentrations, used as both an antiseptic and disinfectant, but they do not kill all microbes and certainly cannot get rid of endospores and non-enveloped viruses. Since alcohols exert their action by denaturing proteins and dissolving lipids, it is not good for wound disinfection because proteins coagulate and form a protective coat around bacteria.
Halogens. The two types and examples of each – see slide.
Chlorine. Types and what it does?
Chlorine, either as a gas or in combination with other chemicals is also widely used as a disinfectant. The germicidal action of chlorine is based on the formation of hypochlorous acid (HOCl) when chlorine is added to water. HOCl is a good oxidizing agent that prevents most cellular enzymes from functioning**. Chlorine is used as a disinfectant in **gaseous form (Cl2 ) or in the form of a compound, such as calcium hypochlorite (Semmelweis’s chlorinated lime water), sodium hypochlorite (NaOCl, Clorox), sodium dichloroisocyanurate, and chloramines. Used to disinfect drinking water and swimming pools
Biguanide compounds. What is an example? How do they destroy bacteria?
They are especially effective against gram positive bacteria. Also effective against gram-negatives, with the significant exception of most pseudomonads. Biguanides are not sporicidal but have some activity against enveloped viruses. The best known biguanide is chlorhexidine, which is frequently used for microbial control on skin and mucus membranes. Combined with a detergent or alcohol, chlrohexidine is very often used for surgical hand scrubs and preoperative skin preparation in patients.
Chlorhexidine digluconate (absorbed by tooth surfaces) is the salt of chlorhexidine and gluconic acid. Among the most common uses of chlorhexidine digluconate is as an oral antiseptic. Since the compound is an effective anti-microbial agent, use as a mouthwash reduces the amount of bacteria in the mouth.
Surface-Active Agents, or Surfactants
Disk-Diffusion Method
- used to evaluate the efficacy of a chemical agent
- A disk of filter paper is soaked with a chemical and placed on an agar plate that has been previously inoculated and then incubated. The size and appearance of the “zone of inhibition”, which is a clear zone representing inhibition of growth, will indicate that an organism is sensitive to that agent. Disks with antibiotics are commercially available and used to determine microbial susceptibility to antibiotics. In the plates shown here, we have 3 different bacteria exposed to four different chemical agents, which microbe is most sensitive to all chemical agents? S. aureus, it has a zone of inhibition around all the chemicals. Which is the most resistant? The Pseudomonas aeruginosa, it appears to only be sensitive to chlorine.
