Sterilization:x Process of killing all microorganisms in a preparationx A physical or chemical process that completely destroys or removes all microbial life – including spores (remember ??)
Terms:x Bacteriostatic – having the property of inhibiting bacterial multiplication, once the agent is removed multiplication resumes.x Bactericidal – having the property of killing bacteriax Sterile – free of any life form.x Disinfectant – chemicals used to kill pathogenic microbes by applying to a surface but too toxic to apply to living tissues
x Septic – characterized by the presence of pathogenic microbes on living tissue x Septicemia – presence of microbes and are actively multiplying x Bacteremia – presence of microbes which are not actively multiplyingx Aseptic – characterized by absence of pathogenic microbesx Antiseptic – a biocide product that destroys/inhibits growth of microbes on living tissues.
x Biocide – general term describing a chemical agent, usually broad spectrum that inactivates microbesx Preservation – prevention of bacterial multiplication in formulated products like food or phamaceutical productsx Antibiotics – naturally or synthetic organic compounds that inhibit or destroy selective bacteria at low concentration.
Antimicrobials/ Antibiotics x Anti-microbial drugs x antibacterialx Minimum Inhibitory x antimycobacterial Concentration (MIC) x antifungalx Minimum x antihelminthic Bactericidal Concentration x Antiviral ( MBC) (refer to Pharmacology )
Modes of Action:x Damage to DNAx Protein Denaturationx Disruption of Cell Membranex Removal of free sulfhydryl groupx Chemical Antagonism
Damage to DNA:x Ionizing radiation: x Produce breaks in single and double strandsx UV light x Induces cross linking between adjacent pyrimidines on one or other of the 2 polynucleotide strands forming pyrimidine dimersx DNA- reactive chemicals x Alkylating agent and other compounds that react covalently with purine ( GA ) & pyrimidine bases (CUT) to form DNA adducts/interstrand cross links
Protein Denaturation:x The disruption of the tertiary structure of protein x Tertiary structure of protein means it exists in a folded 3 dimensional state determined by intramolecular covalent disulfide linkages and some non covalent linkages ( i.e. ionic, hydrophobic & hydrogen bonds )
Disruption of Cell membrane or wallx Lysozyme x Enzyme that destroys cell wall or prevents normal synthesis may bring about lysis of the cell wall. x Substances that concentrate at the cell surface may alter the physical and chemical properties of the membrane, preventing normal functions thereby killing or inhibiting cell function.
Other modes of action:x Removal of Free Sulfhydryl groups x Oxidizing agents and heavy metals do wide spread damagex Chemical antagonism x Isknown as the interference by a chemical agent with the normal reaction between a specific enzyme and its substrate
Treatment to control microbesx Physical Methods x Chemicals x Heat x Phenols x Cold x Phenolics x Radiation -UV , x Alcohols X-ray x Halogens x Filtration x Surfactants x Drying x Heavy metals x Ultrasonic x Alkylating waves agents
Physical methods:x Heat:kills by denaturation of enzymes x Moist heat x Kills microbe primarily by coagulation of proteins x Boiling – 100 degrees – 10-20 minutes x Free flowing unpressurized steam x Fractional sterilization, arnolds sterilizer x 100 degrees, 30 minutes, 3 consecutive days
Cont. moist heatx Autoclave x Steam under pressure x Temperature 121 degrees, 15-20minutes, 15psi x Most effective spore eliminator x Kilit ampule – contains the thermophile – Bacillus stearothermophilus – check autoclave efficiency
Moist heat pa din:x Inspissation x Thickening thru evaporation x Temperature, 75 – 80 degrees, 2hrs for 3 days x Usually used for high protein medium that cannot stand high temperature of the autoclave
Last na moist heat:x Pasteurization –partial sterilization by heat, used for milk & related products x Classic, Low temperature holding (LTH) x Also known as Batch method, 63 C, 30 min x High temperature short Time (HTST) x Flash process, temp 72 C, Time 15 sec x Ultra high Temp ( UHT ) treatment x Stored with refrigeration x 140 C, 3-5 sec ( less than 5 sec, from 74–140 -74
Dry Heat:x Kills by oxidation effects x Direct flaming – aseptic technique x Flaming of mouth of tubes, wires, needles, forceps, petridishes x Oven – hot air sterilization x Temp 160-180 C, 90-120 minutes x Incineration – burning materials to ashes x Temp 300-400 C x Cremation – prevent communicable diseases x Temp 300-400 C
Cold temperaturex Has bacteriostatic effectx Metabolic rate of microbe is so reduced that they cannot reproduce or synthesize toxinsx Slow freezing is most harmful to bacteriax Ice crystals that form and grow disrupt the cellular and molecular structure of the bacteria x i.e.Treponema pallidum in blood bags can be killed by 3 day refrigeration , 2-8 C
Filtration:x Passage of liquid or gas thru a screenlike material with pores small enough to retain microbes x High efficiency particular air (HEPA) filters x Remove almost all microbes larger that 3 um in diameter x Membrane filters – composed of substances like cellulose esters or plastic polymers x Used in laboratoy and industry x 0.1 mm thick x Pores of membrane filters include 0.22 to 0.45 um x i.e. Seitz filter, Sintered glass filter, Berkefield, Chamberland, Colloidon filter
Other methods:x Lyophilization – water is removed by high vacuum at low temperature x Most effective method for long term preservation of microbial culture x Freeze drying – in which cultures are quick frozen between -50 to -95 degrees Cx Ultracentrifugationx Mechanical Shaking – agitationx Grincing – Trituration
Other methods:x Dessication: microbes cannot grow or reproduce with moisture, but can remain viable for years. To grow and multiply microbes need waterx Osmotic pressure – the use of high conc. Of salts & sugars to preserve food is based on the effect of osmotic pressurex Radiation – UV light or Xrays x Ionizing– gamma, xrays, high energy e- beans x Non-ionizing – UV light – damage DNA
Chemical Methodsx Non-selective methods x Disinfectants – inanimate x Antiseptics – animate x E.g. alcohols, acids, alkali, halogens, salts of heavy metal, cresols, lysol, phenols & oxidizing agentsx Selective chemical agents x Antibiotics x Penicillin notatum – penicillin, Bacillus subtilis = bacitracin, Streptomyces venerulae = Chloramphenicol
Uses of common Germicidesx Sterilization - x Disinfection - x Ethylene oxide, x Chlorine compound, Gluteraldehyde, Alcohols, Phenolic Hydrogen peroxide compound, Iodophor Formaldehyde, compound, Chlorine dioxide, Quaternary Peracetic Acid Ammonium compd.x Antisepsis - x alcohol, Iodophors
Modes of Transmissionx Respiratory droplets x Vectorsx Fomites: x Mechanical x facial tissue x Biological x household surfaces x Airborne x eating utensils x Parenteral x contaminated x injections needlesx Direct contactx Fecal-oral
Portals of Entryx Skin: x Conjunctiva x abscess - blood x Mouth x tetanus - wound x primarily GIT x plague - insect bite pathogens x AIDS - injections x Urethrax Nose: x Vagina x primarily respiratory x Placenta Pertussis, colds, measles , small pox
Biological vectors of infectionx Flea - Plague x Mosquitoesx Anopholes x viral encephalitis mosquito - Malaria x Filariasisx Tick : x Body louse x Lyme disease x endemic typhus x Rocky Mountain x Tsetse fly spotted fever x African sleeping dis.x Aedes mosquito: x Reduviid bug x Yellow fever x Chaga’s disease
Exotoxins:x Proteins are main x variable effect on composition hostx produced by some x no fever Gram (-) & (+) x active immunex excreted into response extracellular space x toxoid used asx heat labile vaccine (sensitive) x many diseasesx high toxicity
Endotoxins:x Lipid portion main x similar effect for component all endotoxinsx produced by Gram x fever producing (-) only x poor immunex released only upon response death of bacterium x toxoid cannot bex relatively heat produced stable x variety of diseasesx low toxicity
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