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 شرح/control of microbial growth

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تاريخ التسجيل : 26/03/2010

مُساهمةموضوع: شرح/control of microbial growth   الثلاثاء ديسمبر 14, 2010 9:16 pm

1. Controlling Microbial growth is necessary


  • Decline in Salmonella typhi deathsin the US from 1 in 1000 (1900) to 4 in 200 million (1970) due to controlmeasures

    • Water chlorination (1908)
    • Milk pasteurization (1909)
    • Sewage treatment plant design improvements
    • Transmission control

      • Fly populations (vectors)
      • Detection of diseased persons

        • carriers in the milk industry
        • patients


    • preventive vaccination
    • antibiotic therapy



  • Lister (1827 - 1912)

    • Washing hands prior to surgery leadto decline in infection rates
    • heat sterilization of surgical instruments
    • application of phenol (carbolicacid) to wounds

  • Todays control measures are moresophisticated and include a wide array of methods but not all methods donot kill microbes eg filtration = removal


  • Terminology related to the controlof microbial growth
    TERM DEFINITION EXAMPLE
    SterilizationComplete destruction121oC/15 min; 170oC/2h
    DisinfectionApplication of chemicals to objectsChlorination of water; kill pathogens
    AntisepsisApplication of chemicals to living tissueTreatment of wounds
    Bacteriostasis Halts growth but not killedRefrigeration, dyes in food
    AsepsisAbsence of pathogens; aseptic techniquesAir filtration, uv light, gloves, gowns
    SanitizationPublic health; mechanical / chemical cleansingPalatability of food

2. Factors influencing the effectiveness of control methods

A number of factors affect the usefulness (efficacy) ofcontrol methods & all factorsshould be considered to devise control measures

  • Size of microbial population:

    • Death is exponential ie more microbes = more time to killthe population
    • Plot the log of the no. of surving microbes vs time =straight line, the slope = killing rate
    • Initial microbe concentrations one can predict the killtime

  • Exposure time of the agent:

    • Increasing exposure time increases kill rates; kill timeused is usually well past the required minimal contact time
    • large volumes / vessels require more time for completedestruction
      Container Size* Liquid Volume (ml) Sterilization time (min)
      Test tubes / Erlenmeyer flask 10 - 900 15
      Erlenmeyer flask 1000 - 2000 20
      Bottle 6750 70
      * A container is not usually filled past 75% ofits capacity.

  • Effect of concentration, temperature & pH:

    • Increasing concentration is similar to increasing exposuretime
    • Temperature, pH & concentration at which the agentworks best
    • Stability of the agent at various pHs & temepratures
    • Legionella pnuemophila (Legionnaires disease) -the effect of the microbe suspended in tap water & exposed todifferent temperatures, pH & chlorine concentrations



  • Protective features of the microbes (refer to microbialstructures section)

    • Endospore formers such as C. tetani
    • Cysts -- Protozoa
    • Porins -- Pseudomonas
    • Cell wall -- Mycobacterium tuberculosis (opportunisticpathogen)
    • Active growth vs resting cells - antibiotics (penicillin- transpeptidase)



  • Interactions & protective features afforded by theenvironment:

    • sewage is rich in organics - polio virus protected
    • Disinfecting in hospitals - selection of ressistance
    • Lipids & fats in dairy industry - protect spoilagemicrobes
    • Remember that lab conditions used in testing are differentto field conditions

Methods for controlling microbial growth


  • Many methods to choose from:

    • Physical

      • Heat (Dry, Moist, Pasteurization)
      • Filtration
      • Low Temperatures (fridge, freezer)
      • Desication & osmotic pressure
      • Radiation (ionizing and nonionizing)

    • Chemical

      • Phenol & phenolics
      • Halogens
      • Alcohol
      • Heavy metals & their compounds
      • Surface active agents ( anionic & cationic detergents)
      • organic acids
      • gaseous sterilants
      • oxidising agents
      • chemotherapeutic agents target

        • cell wall
        • protein synthesis
        • nucleic acids
        • cell membrane
        • essential metabolites (antimetabolites)



  • The method of choice depends on the situation & practicalityof the approac


  • The material to be treated eg heat labile or heat stable???


  • Laboratory conditons differ from field conditions


  • Factors affecting growth eg growing or resting cells,ressistance factors
Physical Methods of controlling microbial growth

Method Mechanism of Action Comments Preferred Use
1. Moist Heat(A)


a. BoilingDenaturationKills vegetative cells but not sporesEquipment, dishes
b. AutoclavingDenaturation Sterilization (autoclaves, pressure cookers,retorts)Media, linens, equipment, dressings
c. UHTDenaturationSterilization; 141oC / 2secsMilk falls in a thin film thro a chamber of superheatedsteam
2. Dry Heat(A)


a. FlamingBurning to ashesSterilizationInoculating loops
b. IncinerationBurning to ashesSterilizationanimal cacasses, dressings, wipes
c. Hot-air
sterilization
Oxidation170oC / 2hrsGlassware, needles, glass syringes
3 Pasteurization(A)
a. Low temp
long time
(LTLT)
Denaturation63oC/30minsMilk: batch process in tanks
b. High temp,
shorttime
(HTST)
Denaturation72oC / 15secsMilk: Flash method thro continous winding pipe
4. Filtration(B)SeparationLiquid thro screenHeat labile material
5. Low Temp


a. FridgeGrowth slowsBacteriostatic Drug, Food
b. Deep FreezingGrowth slowsPreservation -70oCDrug, food & culture
6. Desiccation(C)


a. LyophilizationGrowth arrestedLong term preservation of microbesFood, Drug & culture
b. Osmotic PressurePlasmolysisLoss of waterFood preservation
8. Radiation(D)


a. IonizingDNA destructionNot commonly usedSterilizing medical & dental supplies
b. Nonionizing DNA py-py dimers (eg UV)Not very penetrating radiation UV (germicidal) lamp

(A) Moist Heat is used extensivelyby the food (canning & milk) industry

  • Remember: microbe properties are important & so arethe environmental conditions
  • Improve shelf life (sporeformers, anaerobes - hermeticsealing, thermodurics are a problem why?)
  • Kill pathogens to break the route of transmission

    • Non-spore formers in milk (M. bovis - TB, Coxiellaburnetii - Q fever, Brucella - brucellosis); S. typhi& carriers eg the milk industry
    • C. botulinum in canning industry

  • Thermal Death Point (TDP): lowest temp required to killall microbes in liquid suspension in 10 mins.
  • Thermal Death Time (TDT): Minimal time required to killall microbes keeping temperature (or bacteriocidal agent) ) constant.
  • Decimal Reduction Time (DRT or D value): Used for heatressistant bacteria; Time required to kill 90% of the population at a giventemp. (Log growth and log death concept)

    • B. stearothermophilus & / or C. sporogenesis used to determine acceptable D values
    • C. botulinum endspore D value = 0.21 min at 121oC(heating food at 121oC for 2.52 mins means 10-12chances of endospore survival ie 1 in trillion chance
    • Accidic canned foods (tomatoe, pineapples, orange juices)require lower temps. Eg 100oC for 10mins. Why?
    • Milk industry uses lower temps. Eg pasteurization preferred.Why?

(B) Filtration is used for sterilizing heat-labileliquids and to sterilize air (gas) for creating aseptic environment (physicalremoval of microbes)(i) Liquids:

  • Filters for removing pathogens come in different types:

    • Cellulose acetate
    • Celulose nitrate
    • Polycarbonate
    • Teflon

  • Filters vary in pore size (0.22um, 0.45um , 0.8um) but0.2um are usually used but Mycoplasma and viruses may pass thro.Why?
(ii) Air:

  • To create an aseptic environment for microbiological proceduresin semi contained areas. Eg laminar flow hood

    • Directs sterile air from the outside into the confinedspace via a 0.3um HEPA filter (High-efficiency Particulate Air) & stopscontaminants coming into the space

  • Cotton wool used to "plug" test tubes, flasks, pipettesfor growing ro working with cultures, surgical masks act in a similarway
(C) Cells requirewater for metabolic activities. Desiccation is a process by which wateris removed and therefore growth is affected


  • Desciccation prevents microbial reproduction but is dependenton

    • Cell structure. Waxy material of cell wall protects M.tuberculosis, fungal spores (plant pathogens) disemmination
    • Environment. Nasal secretions can protect influenza virusto survive

  • Lyophilization (Freeze-Drying): Preserving microbial cultures

    • American Type Culture Collection (ATCC)
    • Australian Culture of Microorganisms (ACM)

  • Drying food prevents spoilage:

    • Aw < 0.9 inhibits bacteria
    • Aw < 0.65 inhibits fungi & most microbes
    • Examples: prunes, dates, figs, rasins - natural, Evaporatedmilk - removal of 60% water from milk, powdered milk - removal of 85% water

(D) Electromagnetic radiation causes cellulardamage


Chemical methods of controlling microbial growth

Microbiology laboratory standards, design and safety

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