Chapter 7 micro


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Chapter 7 micro

  1. 1. Chapter 7 The Control of Microbial Growth
  2. 2. Control of Microbial Growth <ul><li>Sterilization = the complete removal or killing of all microbes - they are unable to reproduce even in most favorable conditions – no partial sterilization </li></ul><ul><ul><li>Endospores are one of the most resistant forms of life </li></ul></ul><ul><ul><ul><li>Their destruction is the goal of sterilization because any process that kills endospores will kill the vegetative cell </li></ul></ul></ul><ul><ul><li>Used on inanimate objects or material ex. Liquid media </li></ul></ul>
  3. 3. Control of Microbial Growth <ul><li>Disinfection = the reduction or removal of vegetative pathogens but not endospores by chemicals, UV radiation, boiling H2O or steam </li></ul><ul><ul><li>Chemical agents used </li></ul></ul><ul><ul><ul><li>If object is inanimate (tabletop) = disinfectant </li></ul></ul></ul><ul><ul><ul><li>If object is living (animal tissue) = antiseptic </li></ul></ul></ul><ul><ul><ul><ul><li>Antiseptics are regulated by FDA </li></ul></ul></ul></ul>
  4. 4. Some definitions <ul><li>Sepsis = Greek for putrid </li></ul><ul><ul><li>Refers to growth of microbes in blood and other tissues </li></ul></ul><ul><ul><li>Is stem for septicemia (in blood), antiseptic, aseptic </li></ul></ul><ul><li>Degerming = mechanical removal (not killing) of microbes from surface of object </li></ul><ul><ul><li>Ex. ROH w/ shot, wash hands </li></ul></ul>
  5. 5. Some definitions
  6. 6. Physical Control of Microbes <ul><li>Thermal death point (TDP) = the lowest temp. required to kill all microbes in a liquid culture in 10 minutes </li></ul><ul><ul><li>For spore bearing organisms there are 2 TDPs – 1 for the vegetative cell and 1 for the spore form </li></ul></ul><ul><li>Thermal death time (TDT) = length of time to kill all microbes in a liquid culture at a given temperature </li></ul><ul><li>TDP and TDT are important in the food industry wh/ uses heat for preservation </li></ul>
  7. 7. Sterilization by Heat <ul><li>Heat is fast, reliable and cheap and does not introduce toxic substances into material being treated </li></ul><ul><li>Moist heat </li></ul><ul><ul><li>Kills microbes by denaturing proteins – breaks H-bonds thus chgs shape </li></ul></ul><ul><ul><li>Penetrates material faster than dry heat so need less time and lower temperatures </li></ul></ul><ul><ul><li>Boiling H2O – not a sterilizing agent because of spores and viruses </li></ul></ul>
  8. 8. Sterilization by Heat <ul><li>Autoclave (pix –pg 191) = pressure is used to raise temp of steam > 100 Deg. C </li></ul><ul><ul><li>Most rapid and efficient method of sterilization by heat </li></ul></ul><ul><ul><li>As pressure > temp. rises </li></ul></ul><ul><ul><li>At 15 psi pressure the temperature rises from 100 deg C to 121 deg C </li></ul></ul><ul><ul><li>15 psi at 121 deg C 15 – 30 min depending on what you are sterilizing </li></ul></ul>
  9. 9. Sterilization by Heat <ul><li>Autoclave continued: </li></ul><ul><ul><li>Used to sterilize culture media, instruments, dressings, glassware, equipment (transfusions) </li></ul></ul><ul><ul><ul><li>Steam can’t penetrate Al foil – use paper </li></ul></ul></ul><ul><ul><li>Sterilization check w/ Bacillus spores – incubate to check for growth, tape </li></ul></ul><ul><li>Pasteurization – destroys Mycobacterium tuberculosis (TB) and Coxiella burnetii (Q fever) </li></ul>
  10. 10. Sterilization by Heat <ul><li>Pasteurization continued: </li></ul><ul><ul><li>Purpose is to reduce bacterial populations of liquid (milk, juice) and to destroy organisms that cause spoilage and human disease without affecting flavor </li></ul></ul><ul><ul><li>Does not sterilize – spores aren’t affected </li></ul></ul><ul><ul><li>Methods </li></ul></ul><ul><ul><ul><li>Classic = heating at 63 deg C 30 min </li></ul></ul></ul><ul><ul><ul><li>High temp short time tx – 72 deg C 15 sec. – kills pathogens and lowers bacterial counts, >shelf life </li></ul></ul></ul>
  11. 11. Sterilization by Heat <ul><li>Pasteurization cont. </li></ul><ul><ul><li>Ultra – high temp tx </li></ul></ul><ul><ul><ul><li>– 140 deg. C for 1 sec </li></ul></ul></ul><ul><ul><ul><li>Holding tube 3 sec. </li></ul></ul></ul><ul><ul><ul><li>Vacuum chamber 1 sec to cool </li></ul></ul></ul><ul><ul><ul><li>Can then store milk without refrigerating </li></ul></ul></ul><ul><ul><ul><ul><li>Actually sterilizes milk </li></ul></ul></ul></ul>
  12. 12. Sterilization by Heat <ul><li>Dry heat – Direct flame – incinerates microbes rapidly </li></ul><ul><ul><li>Bunsen burner – inoculation loop, culture tube – organisms, dust, lint </li></ul></ul><ul><ul><li>England 4 million cows in 2001 – infected with hoof and mouth disease </li></ul></ul><ul><ul><li>Used to burn diseased bodies centuries ago </li></ul></ul><ul><ul><li>Cows and fields exposed to Anthrax (spores) </li></ul></ul>
  13. 13. Sterilization by Heat <ul><li>Dry heat – hot air ovens </li></ul><ul><ul><li>Does not penetrate material easy so needs long periods of time at high temps – 170 deg C at least 2 hrs </li></ul></ul><ul><ul><li>Used to sterilize dry powders, water free oily substances, glassware </li></ul></ul><ul><ul><li>Changes microbial proteins by oxidation rxs </li></ul></ul>
  14. 14. Filtration <ul><li>Used in food industries, laboratories (medical, environmental, industrial) </li></ul><ul><li>A filter is a mechanical device used to remove microbes from a solution or gas </li></ul><ul><li>Several types of filters used in a laboratory </li></ul><ul><ul><li>Membrane filter – cellulose acetate or polycarbonate – 0.22um, 0.45um, .01um – viruses, lg protein molecules </li></ul></ul>
  15. 15. Filtration <ul><li>Membrane filtration cont. </li></ul><ul><ul><li>Can use to sterilize media or any liquid that can’t tolerate heat </li></ul></ul><ul><ul><ul><li>Serum, blood products, drugs, IV fluids, enzymes </li></ul></ul></ul><ul><ul><li>Can use for a bacterial count – traps bacteria on filter which is place on media in a petri dish and incubated </li></ul></ul>
  16. 16. Membrane Filtration of Liquids Bacteria removal is easier than removal of viruses. Bacteria removal is not quite equivalent to sterilization.
  17. 17. Filtration <ul><li>HEPA filters (high efficiency particulate air) – removes microbes from air </li></ul><ul><ul><li>Removes 99 % of particles > 0.3um </li></ul></ul><ul><ul><li>Used in hospitals – whole rooms, labs – hoods, home air filters </li></ul></ul>
  18. 18. HEPA Filtration of Air
  19. 19. Control of Microbial Growth <ul><li>Low temperatures – refrig, freezer </li></ul><ul><ul><li>Slows growth but does not kill </li></ul></ul><ul><ul><li>< enzyme activity, lowers rate of chemical rxs in cytoplasm </li></ul></ul><ul><li>Desiccation = drying </li></ul><ul><ul><li>Viruses, endospores resistant </li></ul></ul><ul><li>Lyophilization = freeze drying </li></ul><ul><ul><li>Coffee, fruit additives for cereals, used to preserve microbes in laboratory </li></ul></ul>
  20. 20. Control of Microbial Growth <ul><li>Osmotic pressure </li></ul><ul><ul><li>High salt, sugar or spice external environments ( hypertonic environment ) – higher water concentration in cell than outside cell causing H2O to flow from the cytoplasm thru cell membrane out to the environment </li></ul></ul><ul><ul><li>Causes cell to shrivel and die – cell membrane pulls away from cell wall = plasmolysis </li></ul></ul><ul><ul><li>Preserves foods – ex. Ham, bacon </li></ul></ul>
  21. 21. Radiation <ul><li>Ionizing radiation = high energy radiation that induces electrons to jump out of molecules they strike and create ions = atoms or molecules that lack 1 or more electrons </li></ul><ul><ul><li>ex. X-rays, gamma rays, high energy electron beams </li></ul></ul><ul><ul><li>X-rays and gamma rays enter cells and break bonds in DNA causing cell mutations and death </li></ul></ul>
  22. 22. Radiation <ul><li>Ionizing radiation cont. </li></ul><ul><ul><li>Some foods have been irradiated for more than 50 years </li></ul></ul><ul><ul><ul><li>Radiation is used to kill bacterial pathogens, insects, worms and to inhibit the sprouting of potatoes ( > shelf life of food) </li></ul></ul></ul><ul><ul><li>Used to sterilize heat sensitive vitamins, hormones, Abs, plastics, suture material </li></ul></ul><ul><ul><ul><li>Used to sterilize mail after Anthrax scare </li></ul></ul></ul><ul><ul><ul><li>Used to preserve food </li></ul></ul></ul><ul><ul><ul><li>Bone, skin and heart valves for grafting - sterilized </li></ul></ul></ul>
  23. 23. Radiation <ul><li>Non-ionizing radiation - has a longer wavelength and less energy </li></ul><ul><ul><li>Ultraviolet Light (UV) – has a wavelength of 100 - 400 nm </li></ul></ul><ul><ul><ul><li>Used for airborne or surface contamination in hospital rooms, food industry, toilets </li></ul></ul></ul><ul><ul><ul><li>Bacteria are destroyed at approx. 260 nm – causes thymine dimers (bonds form between adjacent bases on the same DNA strand) – bacteria can no longer produce proteins or reproduce DRAW </li></ul></ul></ul><ul><ul><li>Disadvantage = does not penetrate liquids or solids </li></ul></ul>
  24. 24. Chemical control of Microbes tables pg 207-08 <ul><li>Most chemical methods are unreliable for sterilization but are effective as disinfectants and preservatives </li></ul><ul><li>Evaluating a disinfectant </li></ul><ul><ul><li>Phenol coefficient test – compare the activity of a disinfectant with that of phenol </li></ul></ul><ul><ul><li>American official Analytical Chemist’s use – dilution test – current standard </li></ul></ul>
  25. 25. Evaluating a Disinfectant <ul><li>Use – dilution test </li></ul><ul><ul><li>Use Salmonella, Staphylococcus , and Pseudomonas as test microbes </li></ul></ul><ul><ul><li>A series of tubes containing concentrations of the test disinfectants are inoculated and incubated </li></ul></ul><ul><ul><li>The more the chemical can be diluted and still be effective the higher its rating </li></ul></ul>
  26. 26. Evaluating a Disinfectant <ul><li>Disk-diffusion method – pg 199 </li></ul><ul><ul><li>Filter paper disk is soaked with a chemical and placed on an agar plate that has been inoculated with a test organism and then incubated </li></ul></ul><ul><ul><li>After incubation if the chemical is effective will see a clear zone of inhibition around the disk </li></ul></ul><ul><ul><ul><li>Also used to evaluate microbial susceptibility to Abs </li></ul></ul></ul>
  27. 27. Types of Disinfectants <ul><li>Phenol and phenolics (phenol derivatives)- disrupt plasma membranes resulting in leakage of cellular contents, denature proteins </li></ul><ul><li>1860’s – Joseph Lister established the principles of aseptic surgery by using carbolic acid (phenol ) on wounds and instruments </li></ul><ul><li>Phenol is caustic to skin and has been replaced by phenol derivatives wh/ are better germicidals and have lower toxicity </li></ul>
  28. 28. Types of Disinfectants <ul><li>Phenol derivatives </li></ul><ul><ul><li>Cresols – derived from coal tar </li></ul></ul><ul><ul><ul><li>O-phenylphenol used in Lysol, Amphyll </li></ul></ul></ul><ul><ul><li>Bisphenols = 2 phenol molecules – prominent in modern disinfection </li></ul></ul><ul><ul><ul><li>Hexachlorophene = pHisoHex – combined with detergent to bath infants to retard Staph infections of scalp and umbilical cord – found could be absorbed through the skin and cause neurological damage – removed from OTC products </li></ul></ul></ul>
  29. 29. Types of Disinfectants <ul><li>Bisphenols cont. </li></ul><ul><ul><li>Triclosan – broad spectrum antimicrobial agent – destroys plasma membrane by blocking lipid synthesis </li></ul></ul><ul><ul><ul><li>Found in soap </li></ul></ul></ul><ul><li>Halogens oxidize proteins, disrupts membranes </li></ul><ul><ul><li>Chlorine – as gas or in combination with other chemicals </li></ul></ul><ul><ul><ul><li>Germicidal action caused by hypochlorous acid (HOCL) that forms when chlorine is added to H2O </li></ul></ul></ul><ul><ul><ul><li>Sodium hypochlorite 5% - Clorox bleach , disinfectant in dairies, food processing plants, hemodialysis systems </li></ul></ul></ul><ul><ul><ul><ul><li>In emergency CDC recommend 2 drops/liter (clear H2O) for 30 min. </li></ul></ul></ul></ul><ul><ul><ul><li>Chloramines (Cl + ammonia) release free chlorine slower than hypochlorite solution and are more stable </li></ul></ul></ul><ul><ul><ul><ul><li>Used for disinfectants, antiseptics, sanitizing agents, tx of H2O supplies </li></ul></ul></ul></ul>
  30. 30. Types of Disinfectants <ul><li>Iodine – more germicidal than chlorine , found in marine seaweed </li></ul><ul><ul><li>Tincture of iodine (iodine dissolved in ROH) – used as antiseptic for wounds </li></ul></ul><ul><ul><li>Iodophors = iodine-detergent complexes that release iodine over a long period of time ex. Betadine – for local wounds </li></ul></ul>
  31. 31. Iodine & Iodophores
  32. 32. Types of Disinfectants <ul><li>Alcohols – denature proteins and disrupt membranes </li></ul><ul><li>ETOH and isopropyl ROH are effective in killing vegetative bacteria and fungi but not endospores or nonenveloped viruses </li></ul><ul><ul><li>Used to disinfect thermometer, medical instruments, mechanically remove bacteria from skin before drawing blood or giving a shot </li></ul></ul>
  33. 33. Heavy Metals <ul><li>Hg, Ag, Cu – denatures proteins </li></ul><ul><li>Small amts of heavy metal (Ag, Cu) have antimicrobial effects = oligodynamic action </li></ul><ul><ul><li>Demonstrated when place a coin on petri dish w/ bacteria and growth is inhibited from diffusion of metals into media </li></ul></ul><ul><ul><li>1% silver nitrate solution used to be placed in eyes of newborns to guard against Neisseria gonorrhoeae </li></ul></ul><ul><ul><ul><li>Can cause blindness if contracted while going thru birth canal </li></ul></ul></ul><ul><ul><ul><li>Can cause eye irritation so now use antibiotics </li></ul></ul></ul>
  34. 34. Heavy Metals <ul><li>Hg used in the form of mercuric chloride used for skin disease but toxic to skin </li></ul><ul><ul><li>Mercury compound – thimerosal – vaccines , 1999 CDC advised removal </li></ul></ul><ul><li>Copper as Cu sulfate is used as an algicide to control algal and cyanobacteria growth in swimming pools, fish tanks, and H2O supplies </li></ul><ul><li>Zinc chloride used in mouth washes , Zn oxide is used in paints as an anti-fungal </li></ul>
  35. 35. Types of Disinfectants <ul><li>Soaps and detergents are surfactants that emulsify particles and reduce surface tension, good degerming agents </li></ul><ul><li>Aldehydes – denature proteins </li></ul><ul><ul><li>Formaldehyde </li></ul></ul><ul><ul><ul><li>Gas at high temps used to sterilize hospital gowns, medical instruments </li></ul></ul></ul><ul><ul><ul><li>MC as formalin a 37% aqueous solution used to preserve biological specimens, embalming fluid, inactivate bacteria and viruses in vaccines </li></ul></ul></ul>
  36. 36. Aldehydes <ul><li>Glutaraldehyde – disinfectant that can be considered as a sterilizing agent </li></ul><ul><ul><li>Kills bacteria and viruses in 10 min. and spores in 3 -10 hrs </li></ul></ul><ul><ul><li>Used to sterilize endoscopes, respiratory therapy equipment </li></ul></ul><ul><ul><li>Also used for embalming </li></ul></ul>