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 محاضرة /د.محمد السكري/Control Of Microbial Growth

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مُساهمةموضوع: محاضرة /د.محمد السكري/Control Of Microbial Growth   الثلاثاء ديسمبر 14, 2010 9:13 pm

Controlof Microbial GrowthIntroductionWith the advent of the "Germ Theory" of Disease, themedical community gradually began to grow aware of the problem ofnosocomial infections and that there was a need to practice"asepsis" in order to prevent contamination of wounds,dressings and surgical instruments. Ignatz Semmelweis(1816-1865) and Joseph Lister (1827-1912), are consideredto be important pioneers for the promotion of "asepsis"; working in agerm free environment. Semmelweiss, an Hungarian obstetrician,recognized (and enforced) the need for hospital staff to frequentlywash their hands and take other precautions to minimize transmittinginfections to their patients. Semelweiss' interventions significantlyreduced deaths due to "childbirth fever;" a seriousstreptococcal infection. Lister, a British physician, pioneeredantiseptic surgery. He performed surgery while using a mist ofphenolic compounds and endorsed the treatment of dressings withcarbolic acid. Lister also promoted the heat sterilization ofsurgical instruments.Today we take "aseptic" practices for granted, especially in ahospital environment. However, many thousands of people die each yearin the U.S. and abroad of nosocomial (hospital-acquired)infections, many of which can be prevented!We are also living in an excessively "germ phobic" culture.Many over-the-counter antimicrobial products are being marketed andsold but prolonged and frequent use of these products can bedetrimental in several important ways:[وحدهم المديرون لديهم صلاحيات معاينة هذه الصورة]Antibacterialskin lotions and "sanitizers" destroy beneficial normal skin microbeswhich help out-compete pathogens. Overuse of such agents cantherefore create an opportunity for pathogens to cause infection.[وحدهم المديرون لديهم صلاحيات معاينة هذه الصورة]Chemicalpressure may "select" for the evolution of resistant microbes thatare no longer susceptible to the activity of the antimicrobialagent.[وحدهم المديرون لديهم صلاحيات معاينة هذه الصورة]Manyof the "antimicrobial" products are more costly than equivalentbrands.[وحدهم المديرون لديهم صلاحيات معاينة هذه الصورة]"Antimicrobial"chemicals are being applied to commonly used products such aschildren's toys. These chemicals may be ingested by young children orcause sensitivity responses.Although it is important and reasonable to expect a life free ofinfection and disease, the issues listed above stress that we shouldkeep our expectations realistic. Microbes also inhabit and createimportant ecosystem: if we upset the balance of microbial ecology wecan pay the price! Terminology related to control ofmicroorganisms
Germicide/Biocide A chemical agent that demonstrates killing power against various microbes
Antisepsis Refers to the killing or removal of microbes on living tissues
Disinfection Refers to the killing of microbes on inanimate objects or materials
Sterilization Kills or removes all forms of life, including bacterial endospores
Static Processes or chemical agents that inhibit bacterial growth but do not necessarily kill microbes
Sanitization Usually used by the food industry. Reduces microbes on eating utensils to safe, acceptable levels for public health.
Pasteurization A heating process that REDUCES the number of spoilage germs and eliminates pathogens in milk and other heat sensitive foods
Clean "Clean" has a very restricted meaning in microbiology. In this context, clean refers to the removal of VISIBLE dirt and debris from tissues or objects. Clean does not equal sterile!
[وحدهم المديرون لديهم صلاحيات معاينة هذا الرابط]Chemical Methods of MicrobialControlProperties of an idealantimicrobial agent

  • Fast-acting
  • Acts against many microbes without harming tissues (selective toxicity)
  • Penetrating power (improves if dirt and debris are first removed)
  • Inexpensive
  • Easy to prepare
  • Chemically stable
  • Inoffensive odor
  • Not harmful to the environment
Any one agent is unlikely topossess all of the above qualities but it is helpful to assess anagent for as many of these characteristics as possible. Germicidesare commonly used in hospitals, homes and elsewhere. Germicidalagents are also regularly used as preservatives of foods,cosmetics, vaccines and medical supplies.Many antiseptics and disinfectantsact at multiple sites and targets on microbial cells and therefore donot show a great deal of selective toxicity as do manyantibiotics.There is also broad spectrum insusceptibility to germicides among different microbes:

  • Microbial susceptibility to Germicides in descending order (from high susceptibility to lowest) Examples
    Comments
    Enveloped or medium-sized viruses

    • HIV*
    • Hepatitis B virus
    • Cytomegalovirus
    • Herpes simplex virus
    * Recent studies demonstrated viable HIV could be found in contaminated syringes for up to 4 weeks, emphasizing the need for "clean" needles
    Nonspore-forming gram positive bacteria

    • Staphylococci
    • Streptococci
    usually very susceptible
    Nonlipid (nonenveloped) viruses

    • Poliovirus
    • Rhinovirus

    Fungi

    • Trichophyton
    • Candida
    • Cryptococcus
    Spores may be resistant to germicides
    Nonspore-forming gram negative bacteria

    • Proteus spp
    • Pseudomonas aeruginosa*
    * Pseudomonads can grow in some disinfectants
    Protozoan cysts

    • Giardia
    • Cryptosporidium
    Both organsisms are highly resistant to standard levels of water chlorination
    Helminth ova

    • Ascaris lumbricoides
    Eggs may remain viable for many years, even in some preservatives
    Mycobacteria

    • Mycobacterium tuberculosis

    Bacterial endospores

    • Bacillus spp
    • Clostridium spp

    Prions

    • Transmissible spongiform encephalopathies
    Thought to be the most resistant of all agents
    </li>
[وحدهم المديرون لديهم صلاحيات معاينة هذا الرابط]A Summary of Mechanisms ofInactivation by Biocides
Microbial Targets Chemical(s)
Vegetative bacterium:
Cell wall

  • Formaldehyde
  • CRAs *
  • Mercury
  • Phenols
Cytoplasmic coagulation

  • Chlorhexidine
  • Glutaraldehyde
  • Hexachlorophene
  • Mercurial compounds
  • Silver salts
  • QACS*
Cell membrane: membrane potential or electron transport

  • Hexachlorophene
  • Phenols
  • Parabens
  • Weak acids used as food preservatives such as benzoic, sorbic and proprionic acids
Leakage of cell components

  • Phenols
  • Chlorhexidine
  • Alcohols
  • QACs
Nucleic acids

  • Alkylating agents such as ethylene oxide gas
Bacterial endospores:
Spore core

  • Glutaraldehyde
  • Formaldehyde
Spore cortex

  • CRAs
  • Glutaraldehyde
  • Nitrous acid
  • Nitrates/nitrates act as food preservatives by preventing germination of endospores
Virus
Envelopes

  • Alcohols
  • CRAs
  • QACs
  • Chlorhexidine
Viral nucleic acid

  • CRAs
Capsid

  • Glutaraldehyde
  • QACS
  • CRAs
  • Iodine
  • Phenols
  • Alcohols
Fungus
Cell membrane

  • Chlorhexidine
  • Alcohols
  • QACS
Cell wall

  • Glutaraldehyde
Nucleic acid

  • Acridine dyes
Source: Russell et al. 1997. ASM News. 63 (9): 481-487.*CRAs = Chlorine releasing agentsQACs = Quaternary ammonium compounds[وحدهم المديرون لديهم صلاحيات معاينة هذه الصورة][وحدهم المديرون لديهم صلاحيات معاينة هذه الصورة] [وحدهم المديرون لديهم صلاحيات معاينة هذا الرابط]Physical Methods ofmicrobial control

  • Heat
  • Filtration
  • Radiation
  • Refrigeration
  • Desiccation
Heat SterilizationHeating is the most frequently used means todestroy microbes, being both economical and easily controlled.Successful heat sterilization must consider the degree of heatresistance demonstrated by a microorganism. Death from heating is anexponential function and occurs more rapidly as temperatureincreases. The nature of heat is also important: moist heatpenetrates better than dry heat.Decimal reduction time (DRT) is a conceptemployed by the canning industry to determine heat sterilization andis defined as the time it takes for 90% of microbes will be killed ata particular temperature.Moist heatBoiling will kill most vegetative bacteriaand viruses within 10 minutes. Bacterial endospores can surviveboiling temperatures. Certain bacterial toxins such as Staphylococcalenterotoxin are also heat resistant.AutoclavingUses steam heat under pressure to penetrate andkill microorganisms. Steam produced at 15 psi heats to 121 C and willkill endospores after 15 minutes. Denser materials or large objectswill need to be autoclaved for longer periods.The USDA recommends that all low-acid foods shouldbe processed by pressure cooking. This is to prevent the persistenceof endospores in foods such as canned tomatoes. Jellies and jamsshould also be stored with sealed lids and not wax seals in order toprevent growth of molds and toxin production.PasteurizationPasteurization is named for a process developed byLouis Pasteur as he looked for ways to prevent wine spoilage. It isimportant to note that Pasteurization is not synonymous withsterilization. This process employs heat to destroy pathogens andreduce the number of spoilage microbes in foods. Before this processwas developed, milk was a common source of diseases such astuberculosis, typhoid fever and brucellosis. Today, pasteurization isprimarily used to prolong the shelf-life of various foods.Pasteurization employs the concept ofequivalent treatments. As temperature increases less time isneeded to kill a certain number of microbes that would take more timeto kill at a lower temperature. Classical (bulk)pasteurization heated foods at 63 C for 30 minutes. Today, flashpasteurization or high temperature, short-time (HTST) methods arefavored as they kill heat-resistant organisms more effectively andare less likely to alter the flavor of foods. The HTST methodsinvolve continuos passage of foods past a heat exchanger.Pasteurization methods include:

  • 72 C for 15 sec (HTST)
  • 140 C for 15 sec (Ultra-High Temp)
  • 149 C for 0.5 sec (UHT)
Dry heatsterilizationDry heat takes more time to kill microbes as itdoes not penetrate as well in the absence of steam.. Common uses ofdry heat sterilization are flaming of inoculating loops and thesterilization of glassware in hot air drying ovens.FiltrationFilter sterilization is commonly employed forsubstances that can not tolerate heat. Membrane filters with poresizes between 0.2-0.45 um are commonly used to remove particles fromsolutions that can't be autoclaved. Membrane filtration of beereliminates spoilage germs and pasteurization is no longer needed.Filtered beer is permitted to be sold as "draft beer." Sub-micronfilters are also being marketed for removal of protozoan cysts fromdrinking water.RadiationOne of the most controversial areas of microbialcontrol involves the use of radiation. The controversy largelyresults from a lack of understanding of the different types and usesof radiation.The effects of types of radiation depend on threeimportant factors:

  • Time (of exposure)
  • Distance (from the source)
  • Shielding (how penetrating is the radiation?)
Irradiation of various food has been used in theU.S since the 1960's and has been used to sterilize foods such asherbs and spices.Nonionizing radiation Includes microwaves and ultra violet radiation.Microwaves are not particularly antimicrobial in and of themselves,rather the killing effect of microwaves are largely due to the heatthat they generate. Microwaves are not recommended for cooking largevolumes or thick cuts of meat as the heat may not penetrate thefoodstuffs sufficiently.UV radiation is of short wavelength, between 220and 300 nm and is not very penetrating. UV can be stopped by glass, asheet of paper, or the top layers of your skin! UV rays can killexposed microbes by causing damage to their DNA. UV radiation isuseful for the disinfection of exposed surfaces such as laboratoryhoods. However, the usefulness of UV radiation is limited by the factthat certain microbes possess DNA repair mechanisms and can recoverafter exposure to this kind of radiation. In addition, UV light doesnot penetrate organisms well that are protected in mucus ordebris. Ionizing radiationIncludes gamma rays and X rays which arehighly penetrating to cells and tissues and have potent antimicrobialeffects. After colliding with a target, ionizing radiation generatesions and other reactive species from molecules including hydroxyl(free) OH- radicals. These free radicals can cause irreversiblebreaks in DNA, proteins and enzymes.Radiation is currently used for sterilization bythe medical supply and food industries. The FDA has approvedirradiation for sterilization of surgical supplies, vaccines anddrugs. Irradiation of spices and seasonings has grown over the use ofethylene oxide gas. This gas must be expelled from products beforeuse, and is mutagenic (possible cancer-causing agent).Many individuals and groups have voiced concernagainst the irradiation of foods. However, to date no study hasdemonstrated any long-term ill effects from this practice and thenutritional quality of the foods seem largely unaltered. Theirradiation of foods can be likened to your bag going through an Xray machine at an airport. When you pick up your bag at the other endyour baggage is not "glowing" with radiation and will do you no harm.You get a lot more exposure to natural forms of radiation (sunlight)than you do from your foodstuffs! You should be more concerned aboutyour risk of skin cancer from sunbathing!The USDA approved the irradiation of redmeat this February. The USDA rule would permit, but not require,irradiation for refrigerated or frozen uncooked meat and some meatproducts. Irradiation is the only effective means known to eliminateE. coli 0157 from meat. This process can also eliminate the foodpathogens, Listeria (likely to a 16 deaths in the US late98/early 99) Campylobacter and SalmonellaVegetarians look out! What about alfalfasprouts?Consumption of alfalfa sprouts carries a high riskof infection with Salmonella.Over 20,000 cases of Salmonella were linked toeating alfalfa sprouts in North America in 1995. The fundamentalproblem was linked to the fact that there is no "kill step" used toeliminate the pathogens during the germination of the seeds. The CDChas advised people at high risk of complications fromSalmonella (young, elderly, pregnant women, immunecompromised) to avoid eating these sprouts.Despite opposition, the only thing that may savethe alfalfa sprout industry is to irradiate their plants!RefrigerationRefrigeration will slow down and inhibit thegrowth of most microbes but it will not kill them! Note: somespoilage germs and psychrophiles can continue to replicate at coolertemperatures. Organisms can be maintained viable at -80 C ifsuspended in glycerol or DMSO.DesiccationDesiccation of microbes is a very useful means offood preservation and to control the growth of spoilage germs andpathogens. Foods that have a high water activity are mostsubject to spoilage and typically must be refrigerated or frozen.Numerous foods are preserved by adding salt or sugar to decrease thewater activity of the foods. This process creates hypertonicconditions and causes water to leave bacterial cells (plasmolyze).Salting of foods does not protect against all potential pathogens.Many fungi are halophilic as is Staphylococcus aureus, acommon source of bacterial food poisoning.
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محاضرة /د.محمد السكري/Control Of Microbial Growth
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