Physical and Chemical Agents for Microbial Growth

1. Physical and Chemical Agents for Microbial Growth Chapter 11

2. Decontamination • Process that destroys or removes contaminants • Aka undesirable microbes at a given time/place

3. Ancient Egyptians would have people eat moldy bread when they had a sore throat.

4. Doctors during the Civil War would pack wounds with Sphagnum moss.

5. During the 1700’s plague, doctors wore these funny outfits • The beak was filled with strong perfumes to protect against foul odors and inhaling bad air

6. Sir Joseph Lister From Britain used carbolic acid (phenol), C6H5OH to sterilize wounds. (Hospital smell) Antiseptic methods were introduced during surgery both by soaking wound with carbolic acid and heating surgical tools. Found great success!!

7. What do you know? • What methods/things do you currently know of that could decontaminate something or someone? List as many as you can!

8. Decontamination Categories • The two main ones are • Physical agents • Heat/radiation • Chemical agents • Solutions/molecules/etc.

9. Microbial Control • Process or agent that targets microorganisms that cause infection/spoilage and are commonly found on body or in environment • MANY microbes

10. Who are our contaminants? • Bacterial vegetative cells • Bacterial endospores • Fungal hyphae and spores • Yeasts • Protozoans • Worms • Viruses • Prions

11. Ranking of Microbes according to Resistance to Chemical Germicides • Bacterial Endospores • Certain fungal spores • Mycobacteria such as Mycobacterium tuberculosis • Non-lipid containing viruses (picornaviruses) • Vegetative fungi & most fungal spores • Vegetative bacteria • Lipid-containing & medium sized viruses (influenza viruses & adenoviruses

12. Contaminants • Categorized by resistance • P 318 • Endospores are the most resistant microbe entities

13. Contaminants • The goal of sterilization is the destruction of endospores • Because if they’re dead…

14. Synonyms, but not quite • In everyday language, we use words like disinfect, sterile, and antiseptic interchangeably • In microbiology, it’s a whole different game

15. Sterilization • Destroys or removes all microorganisms, including viruses • Not a “kinda sterilized”; black/white term • Reserved for inanimate objects

16. Microbicidal agents • Agent(s) that target a certain type of microbe • Bactericide • Fungicide • Virucide • Sporicide • This would be a sterilant (kill EVERYTHING)

17. Microbistasis agent • Agent(s) that keep microorganisms alive but prevent further growth • Bacteriostatic, fungistatic, etc • ***Typically the category our drugs are in** • Why not microbicidal drugs?

18. Germicide • Type of microbicide that targets pathogens • Can be used on inanimate objects or in living organisms • Not strong enough for spores

19. Disinfection • Use of physical or chemical means to destroy vegetative cells but not endospores • Inanimate objects because they need [high] to be effective • EX: 5% NaHClO3, iodine, boiling

20. Antisepsis • Prevents sepsis (growth of microbes in blood and other tissues • Ex: Iodine, Hydrogen peroxide, soap

21. Sanitation • Decreases number of microbes on inanimate objects • Decreases risk of infection and/or spoilage • EX: Soaps

22. Degermation • Decreases microbial load by mechanical means from tissues • Alcohol wipes • Surgical scrubs

23. How do we know when a microbe is dead? • No “heart” • Don’t look different when dead • Determine: Can they still grow if conditions improve? • “Permanent loss of reproductive capability”

24. Principles Involved in Killing Microbes • During sterilization, only a fraction of the microorganisms or viruses die during a given interval. Time interval tends to be constant (logarithmic death) ie. 1st 3 minutes kills 90%, next 3 minutes kills another 90%… B. The time it takes to achieve sterility depends in part on the # of organisms present at the beginning of sterilization. The > the microbial load, the longer sterilization will take

25. C. Different microorganisms & viruses may vary in their susceptibility to sterilizing and disinfecting. E. coli like other Gram –‘s are killed easier than Acid-fasts Endospores are really tough to kill D. Numerous conditions alter the effectiveness of agents that kill microbes & viruses Best during log growth phase Temperature, pH, viscosity and presence of organic matter

26. How will I know which to use? • Do I have to kill spores or is vegetative only okay? • Is the item reusable? If disposable, go for quick/cheap • Reusableare heat and pressure ok? • Otherwise, chemicals • Does it make sense? • Is it $$? • Is it safe??

27. How do they work • Four main targets: • Cell wall • Cell membrane • Cellular process (DNA, RNA) • Proteins

28. Cell wall as a target

29. Cell wall as a target • Des damage to bacteria and fungi • Why not you or me? • Can… • Block its synthesis • Digest it • Break down its surface • Causes the bacteria to lyse • EX: antimicrobials (G+) detergents (G-)

30. Cell membrane as target

31. Cell membrane as target • Disrupts lipid membrane • Loses regulation; can’t prevent nutrient loss or entry of harmful materials • EX: surfactants—items that decrease the surface tension of membranes

32. DNA/RNA as target • Ribosomes (RNA) • Chloramphenicol binds to ribosomes and blocks protein formation • DNA • Agents bind and prevent transcription/translation • Mutagenic compounds • Radiation

33. Protein as target • Proteins depend on correct shape • Many agents denature • EX: Moist heat • Alcohols, acids • EX: Metallic ions that bind to active site • Stop metabolism

34. Physical Control • Heat • Cold • Desiccation • Radiation • Filtration

35. Heat • Moist heat • EX: hot/boiling water/steam, • 60-135°C • Dry heat • EX: flame/heating coil • 160C-1000+°C

36. Killing Microbes Using Heat Does not introduce toxic substances into material being treated • Heat kills by: Coagulating the proteins of cells & viruses Denatures them Limited to objects that are small enough to fit into heating vesicle and can withstand the heat – can’t be used on plastics or rooms Cooking kills microbes but not always & especially if it isn’t thoroughly cooked through – Endospores!!

37. Using Heat to Control Microorganisms

38. Dry Heat – requires more time than wet heat to kill Direct flame or oven Dry: 200°C for 1 ½ hours Wet: 121°C for 15 minutes

39. Boiling – original sterilization method (Aristotle & water) Normal water: 10 minutes at 100°C will work okay Viruses also but hepatitis virus, virions may survive Clostridium endospores may survive hours of boiling due to temps never reaching high enough to kill them • Not reliable!!

40. D. Autoclaving – (1860’s) Pressure cooker Uses steam and pressure to evacuate air As pressure >, temperature > 15 lbs pressure above atmospheric pressure at 121°C for 15 minutes Flash autoclaving – 135°C for 3 minutes Method used in emergency surgery or OR’s

41. Pasteurization – Controlled by heating at temps below boiling. - Wine, milk, apple juice and brewing industries Animation 1. HTST – High Temperature, Short Time - 72°C (161°F) for 15 seconds & cooled quickly - Modern milk pasteurizing method

43. 2. LTLT – Low temps, long time - 63°C (145°F) for 30 minutes & cooled - Old Pasteurizing of milk method 3. UHT – Ultra High Temps - Heat above 72°C upward to 140°C & cooled within seconds - Beer industry and Parmalat milk

44. Millipore filter Sterilization By Filtration • Used for fluids that are notheat tolerant. • Space technology, unpasteurized beer production and heat sensitive medicines. • Utilized filters made of porcelain, glass, diatomaceous earth and asbestos. • A microscopic sieve that traps by pore size and electrostatic attraction

45. A. Filter Types and Drawbacks 1. They cannot remove viruses reliably from human serum (too small) 2. Too much pressure applied to speed up filtration can overcome filtration action, allowing bacteria to slip though 3. Proteins and other substances in suspension can neutralize the electrostatic charges on filters 4. Filters may absorb desired bacterial enzymes that will fail to appear in the filtrate

46. B. Membrane Filters - Small pore size - Such as cellulose acetate, cellulose nitrate, polycarbonate or Polyvinylidene fluoride  . - Paper thin and extremely small pore size to trap viruses - 0.4 – 0.2 µm are most common for bacteria “Draft” beer is filtered through such filters. No pasteurization

48. Radiation The shorter the wavelength, the greater the energy

50. Gamma Rays • Ionizing radiation –produces hyperreactive ionswhich destroy DNA • Used on Salmonella, Penicillin and disposable plastics • Used immediately after sterilization (unlike ethylene oxide) • Less effective with Vitamin C, ethyl alcohol and glycerol • Bacterial endospores are mostly resistant but Salmonella& Pseudomonas are most sensitive