Microbial Control

1. Microbial Control

2. Terminology for Microbial Control • Sterilization- removal or destruction of all forms of microbial life • Commercial sterilization- subjects canned food to only enough heat to destroy the endospores of Clostridium botulinum • Disinfection- is the destruction of vegetative pathogens on a surface, usually with chemicals • Spores and viruses are not necessarily destroyed • Antisepsis- is the chemical disinfection of living tissue, such as skin or mucous membranes

3. Terminology for Microbial Control • Asepsis- is the absence of pathogens on an object or area, as in antiseptic surgery • Degerming (degermation)- is the removal of transient microbes from the skin by mechanical cleansing or by antiseptic • Sanitation- is the reduction of microbial populations on objects to safe public health levels • A biocide or germicide- kills microorganisms • Fungicides kill fungi, virucides kill viruses • Suffix – cidemeans the killer of a specified microorganism • Suffix- statused in this way indicates only that the substance inhibits – for example bacteriostasis

4. Rate of Microbial Death • Bacterial populations killed by heat or chemicals tend to die at constant rates—for example, 90% every 10 minutes. Plotted logarithmically, these figures form straight descending lines.

5. Factors that influence effectiveness of anantimicrobial treatment: • 1. Number of microbes • -more cells, more time needed to kill all • 2. Environmental influences • -organics often inhibit chemical agents (blood, feces, vomit) • -temperature (disinfectants work better in warm temperatures) • -pH (heat is more effective in an acid pH)

6. Factors that influence effectiveness of anantimicrobial treatment: • 3. Time of exposure • -Chemical antimicrobials require a certain amount of exposure; same agent may need longer on resistant organisms or spores • -with heat, lower temps require longer to kill • 4. Microbial characteristics • -resistance genes, protective structures • (e.g.capsules) etc. caninhibit action • -bioflims prevent penetration

7. Type and Age of Microbe • Bacteria- susceptible to protein denaturing BUT mycobacteria is not because of its hydrophobic coat • Growth cycles- physiologically young bacteria(early in growth cycle) susceptible to heat • Endospores more resistant to heat the older they get

8. Properties of Materials: • Plastic and rubber- not heat tolerant • Cutting edges of surgical instruments- no moisture or corrosive chemicals • Edges can become pitted due to rust and corrosion, microbes can hide there • Fabrics- no chemical disinfection; some too fragile

9. Actions of Microbial Control Agents • 1. Alteration of membrane permeability • The plasma membrane controls the passage of nutrients and wastes into and out of the cell. • Damage to the plasma membrane causes: • leakage of cellular contents • interferes with cell growth. • = Leak lysis, death

10. Actions of Microbial Control Agents • 2. Damage to proteins and Nucleic Acids • Enzymes and other proteins are essential for cell function • Denatures proteins • Enzymes (no reactions) • Proteins necessary for bacteria metabolism • Hydrogen bonds are broken • Covalent bonds are also broken • 3. Damage to nucleic acids • Prevent replication, transcription, or translation

11. Physical Methods of Microbial Control • -to disinfect objects, food, and solutions common methods: • -Temperature: kill or inhibit growth • Heat • Low Temps. • -Filtration: physical removal • -Desiccation: inhibit growth • -Osmotic pressure • -Radiation: kill

12. Methods involving Heat: • Heat: Common food preservation • Denatures protein (changes shape) • Thermal Death Point (TDP) = lowest temp at which all microbes in liquid suspension will be killed in 10 min • Thermal Death Time (TDT)= minimal length of time for all microbes in liquid suspension to be killed at given temp • *Both are different for different species due to microbial variation in heat tolerance • Decimal Reduction Time (DRT) = Time in minutes in which 90% of bacteria at a given temperature will be killed

13. Methods involving Heat: • 1. Moist heat will always kill faster than dry heat at the same temperature • Kills microbes by coagulation of cell proteins • A. Boiling(100°C) kills vegetative forms of bacterial pathogens, many viruses, and fungi within 10 minutes • Some mo’s and viruses are resistant • Endospores (up to 20 hrs.) and some viruses (30 minutes) survive boiling for longer times.

14. Methods involving Heat: • B. Autoclaves • Moist Heat (steam) and pressure for sterilization • Achieves higher temps. than boiling • Preferred method (sealed chamber, air is exhausted, and steam under pressure is injected) for all materials that can withstand it • Kills all organisms and their endospores in about 15 - 20 minutes • An autoclave is shown in Figure 1.1and 1.2

15. Autoclave Figure 1.1 Figure 1.2 Autoclave tape

16. Methods involving Heat: • C. Pasteurization • Louis Pasteur • Mild Heating • Kills most pathogens • Kills bacteria that cause spoilage • Preserves taste in product • Lowers bacterial numbers

17. Methods involving Heat: • 2. Dry Heat • Kills by burning to ashes or by oxidation • Flaming- we use this on loops in labs • Incineration- burning of contaminated paper • Hot air sterilization • Hot ovens • Mainly used for items not suitable for autoclaving • Oily substances, powders • Large amounts of glassware

18. Low Temperature • Lower temp inhibits growth, rapid freezing limits moisture (bacteriostatic) • Refrigerator temperatures (0° to 7°C) slow the metabolic rate of microbes; however • Psychrotrophic species still grow slowly. • Some organisms grow at temperatures slightly below freezing, but microbes at the usual temperatures of freezer compartments are completely dormant.

19. Filtration • Liquids • Heat- sensitive materials • Small pores prevent passage of bacteria • High-efficiency particulate air filters (HEPA) • Operating rooms • Special clean rooms • Masks • Used with solutions for: renal dialysis, heart bypass machines, I.V.’s • Catch particles autoclaves miss

20. Desiccation • Microbes require water for growth, and adequately dried (desiccated) foods will not support their growth, therefore inhibiting growth • Absence of Water • Lyophilization- rapid freeze drying • Used for blood products , serum products, enzymes, cultures • Avoids ice crystal formation; cells burst when water expands

21. Osmotic Pressure • High salt or sugar concentrations cause water to leave the cell; this is an example of osmosis. • Generally, molds and yeasts resist osmotic pressures better than bacteria.

22. Radiation • Ionization Radiation • includes X rays, gamma rays, and high-energy electron beams • very short wavelengths and high levels of energy • Penetrate deeply • Ionizes water to form hydroxyl radicals • These destroy cell components, especially DNA • Kills: vegetative cells, viruses, most endospores with adequate exposure • Applications: food preservation, sterilization of pharmaceuticals, medical supplies, mail

23. Radiation • Nonionizing radiation • Have a longer wavelength and less energy • Ultraviolet (UV) light is the common example • Causes the formation of thymine dimers, which interferes with DNA replication and formation of mRNA. • UV lamps are used in hospitals and in food service • This method does not sterilize, but it does reduce bacterial growth • Penetrating power is very low, so any type of covering protects microbes. • Sunlight has some weak antimicrobial effects (biocidal), but the wavelengths of sunlight are too long to work well.

24. Types of Chemical Agents • 1. Phenols and Phenolics • A. Phenol (carbolic acid) • Irritating to skin and mucous membranes • Bad odor • Rarely used today • B. Phenolics • Chemicals derived from phenol • Chemically altered to make it less irritating and more effective • Damages plamambs., inactivates enzymes, denature proteins • Often used as disinfectants as they remain active in the presence of organic matter

25. Types of Chemical Agents • C. Bisphenols • contain 2 phenolic groups connected by a bridge • Hexachlorophene • pHisoHex is an example • Prescription antibacterial lotion • Used in nurseries to control gram + bacteria • Skin bacteria: Staphylococcus and Streptococcus • Triclosan • Found in antibacterial soap • Effective against G+ and G- bacteria

26. Types of Chemical Agents • 2. Biguanides • Chlorhexidine is an example • Hibiclenssoap • Used on skin and mucous membranes • Similar to phenolics but less toxic • Disrupt plasma mb. • Broad spectrum • Effective against most vegetative bacteria and fungi, but not against endospores and many viruses. • Damaging to eyes

27. Types of Chemical Agents • 3. Halogens- Effective alone or in compounds • A. Iodine (I2) • One of the oldest and most effective • Very effective on: all bacteria, many endospores, fungi and some viruses • Combines with amino acids in proteins and denatures proteins • Alters plasma mb. • Negative Aspects • Staining • Sometimes irritating to the skin • May trigger allergies • Applications • Skin disinfection, wound treatment, water treatment

28. Types of Chemical Agents • B. Chlorine (Cl2) • Action: forms hypochlorus acid with water → oxidizing agent, denatures proteins • Broad spectrum: bacteria, fungi, some endospores, some viruses • Positive aspects: -effective against all vegetative cells including Mycobacterium • -cost effective • Negative aspects: -action inhibited by organics • -can form carcinogenic compounds • Applications: water and sewage treatment, surface and instrument disinfection • Sodium hypochlorite (NaClO): Is active ingredient of bleach

29. Types of Chemical Agents • C. Alcohols- frequently used for skin degerming • Kill bacteria, fungi, but not endospores or naked viruses. • Act by denaturing proteins and disrupting cell membranes. • Used to mechanically wipe microbes off skin before injections or blood drawing, instrument disinfection • Not good for open wounds, because cause proteins to coagulate and leave bacteria unharmed • Ethanol (ethyl alcohol): Optimum concentration is 70%. • Isopropanol: Rubbing alcohol • Better disinfectant than ethanol • Usual concentration is 90% • Also cheaper and less volatile.

30. Types of Chemical Agents • 5. Heavy metals and their compounds • Oligodynamic action- Very tiny amounts are effective • Includes- silver, mercury, copper, zinc • Bind sulfur groups causing inactivation or denaturing of proteins • A. Silver: • 1% silver nitrate used to protect infants against gonorrheal eye infections until recently. • B. Mercury • Organic mercury compounds like merthiolate and • mercurochrome are used to disinfect skin wounds. • C. Copper • Copper sulfate is used to kill algae in pools and fish tanks (fungicidal, algicidal) • D. Zinc • Used in mouthwashes • Superficial fungal and bacterial infections

31. Types of Chemical Agents • 6. Surface-Acting Agents • Disrupt plasma membrane & denature proteins • Decrease surface tension • Include soaps and detergents • Washing with soap breaks up the oily film that covers skin and allows microbes and dirt to be washed away • 7. Quaternary Ammonium Compounds • Widely used surface active agents • Denature proteins & disrupt cell membranes • Cationic (positively charge) detergents • Effective against gram positive bacteria, less effective against gram-negative bacteria. • Also destroy fungi, amoebas, and enveloped viruses.

32. Types of Chemical Agents • 8. Aldehydes • These can act very effectively against microbes • Inactivate proteins • Action: cross-link (thus inactivate) nucleic acids and proteins • High activity (sterilization) • biocidal including endospores • Positive aspects • achieves sterilization • Negative aspects • unstable • -toxic • -volatile with noxious fumes • Applications: specimen preservation (embalming), vaccine sterilization

33. Types of Chemical Agents • 9. Gaseous Sterilizers • Chemicals that sterilize in a chamber similar to an autoclave. • Denature proteins, by replacing functional groups with alkyl groups • A. Ethylene Oxide • Kills all microbes and endospores , but requires exposure of 4 to 18 hours. • Toxic and explosive in pure form. • Highly penetrating. • Most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment.

34. Types of Chemical Agents • 10. Peroxygens • Oxidize cellular components • A. Hydrogen Peroxide • Common household antiseptic • Not good for open wounds because quickly broken down by catalase present in human cells • used in deep wounds because it releases oxygen as it breaks down, which makes conditions unfavorable for anaerobic bacteria • Effective in disinfection of inanimate objects (kills endospores) • Sporicidalat higher temperatures • Used by food industry and to disinfect contact lenses

35. Types of Chemical Agents • B. Benzoyl Peroxide • Main ingredient in many acne treatments • May be used in treating wound infections caused by anaerobes • C. Peracetic Acid • One of the most effective liquid sporicidesavailable • Sterilant : • Kills bacteria and fungi in less than 5 minutes • Kills endosporesand viruses within 30 minutes • Used widely in disinfection of food and medical instrument because it does not leave toxic residues