1. Physical and Chemical Control of Microbes
Chapter 11
Physical and Chemical Control of Microbes

2. Controlling Microorganisms
General Considerations in Microbial Control
The methods of microbial control belong to the general category of decontamination procedures, in that they destroy or remove contaminants
Most decontamination methods employ either physical agents, such as heat or radiation, or chemical agents such as disinfectants and antiseptics

3. Microbial Control Methods

4. Relative Resistance of Microbial Forms
Highest resistance
Bacterial endospores
Moderate resistance
Protozoan cysts
Fungal sexual spores (zygospores)
Naked viruses
Mycobacterium tuberculosis, Staphylococcus aureus, and Pseudomonas species
Least resistance
Most bacterial vegetative cells
Ordinary fungal spores and hyphae
Enveloped viruses
Yeasts
Trophozoites

5. Terminology and Methods of Microbial Growth
Sterilization is a process that destroys or removes all viable microorganisms, including viruses
Bactericide is a chemical that destroys bacteria except for those in the endospore stage
Fungicide is a chemical that can kill fungal spores, hyphae, and yeasts
Virucide is any chemical known to inactivate viruses, especially on living tissues
A sporicide is an agent capable of destroying bacterial endospores

6. Terminology and Methods of Microbial Growth
Bacteriostatic agents prevent the growth of bacteria on tissues or on objects in the environment
Fungistatic chemicals inhibit fungal growth
Germicde, also called a microbicide, is any chemical agent that kills pathogenic microorganisms
Disinfection refers to the use of a physical process or a chemical agent to destroy vegetative pathogens but not bacterial endospores
Sepsis is defined as the growth of microorganisms in the body or the presence of microbial toxins in blood and other tissues

7. Terminology and Methods of Microbial Growth
Asepsis refers to any practice that prevents the entry of infectious agents into sterile tissues and thus prevents infection
Antiseptics are applied directly to exposed body surface, wounds, and surgical incisions to destroy or inhibit vegetative pathogens
Sanitization is any cleansing technique that mechanically removes microorganisms to reduce the level of contaminants
Degermation is a process usually involves scrubbing the skin or immersing it in chemicals, or both

8. Factors that affect Death Rate
The number of microorganisms
The nature of the microorganism (vegetative vs. endospore)
The temperature and pH of the environment
The concentration of the agent

9. How Antimicrobial Agents Work
The mode of action of the agent (destructive or inhibitory)
Damage to cell wall
Damage to cell membrane (surfactants)
Inhibit protein and nucleic acid synthesis
Alteration of protein function
The presence of solvents, interfering organic matter, and inhibitors

10. Mode of Action of Surfactants on the Cell Membrane
Surfactants inserting in the lipid bilayer disrupt it and create abnormal channels that alter permeability and cause leakage both into and out of the cell

11. Mode of action affecting protein function
Some agents denature the protein by breaking all or some secondary and tertiary bonds, resulting in complete unfolding or random bonding and incorrect folding
Some agents react with functional groups on the active site and interfere with bonding

12. Physical Methods for Controlling Microorganisms
Moist Heat: Use of hot water or steam in disinfection to kill vegetative cells or in sterilization to kill spores. Mode of action: denaturation of proteins, destruction of membranes, and DNA
For Sterilization
Autoclave uses steam under pressure (15 psi/121C/10-40 minutes)
Applied on heat-resistant material that steam can penetrate
Not recommended for oils, powders, heat-sensitive material
Intermittent sterilization uses free-flowing unpressurized steam at 100C applied for minutes on 3 successive days
Used for substances that cannot be autoclaved, especially media and certain canned foods

13. Sterilization using Steam under Pressure

14. Physical Methods for Controlling Microorganisms
For Disinfection
Pasteurization is application of heat less than 100C to liquids; employs the flash method to heat liquid to 71C for 15 seconds
Targets non-spore forming milk-borne pathogens, such as Salmonella and Listeria, and reduces spoilage by lowering the overall microbial count; also applied to beer and wine
Only reduces microbial content; many thermourics survive
Boiling water at 100C used to destroy pathogens (not spores) on household supplies; materials may become recontaminated

15. Physical Methods for Controlling Microorganisms
Dry Heat: Use of hot air with low moisture content to sterilize
Mode of action: depending on the temperature, combusts, dehydrates, or coagulates proteins
Incineration
Open flame: Materials are placed for a few seconds in an 800C to 1800C flame of a Bunsen burner
Furnace: Incinerator chamber equipped with 600C C flame that burns materials to ashes
Used for decontamination of hospital and industrial wastes; may add pollutants to atmosphere
Dry Oven: Closed chamber heated to 150C - 180C for 2 – 4 hours
Used for sterilizing metals and glass, but too vigorous for liquids, plastics, paper, cloth

16. Dry Heat Incineration
Infrared incinerator with shield to prevent spattering of microbial samples during flaming

17. Physical Methods for Controlling Microorganisms
Cold Temperatures
Refrigeration (0C - 15C) or freezing (below 0C) is not a reliable mode of disinfection
It is only microstatic, meaning it slows the growth rate of most microbes
Refrigeration is used for preserving foods, media, and cultures
Drying/Dessication is the gradual withdrawal of water from cells by exposure to room air, leading to metabolic inhibition
Effective in preservation, but not reliable for pathogens, and not really an effective method for infection control

18. Radiation/Irradiation
Use of energy in the form of waves (electromagnetic) and particles that can be transmitted through space
A method of cold sterilization

19. Radiation/Irradiation
Ionizing radiation uses high-energy, short waves and particles that can dislodge electrons from atoms
This causes direct damage to DNA by the formation of breaks and mutations
Types are gamma rays, X rays, cathode rays (high speed electrons) used in irradiation and used to sterilize heat sensitive medical materials in packages; also to sterilize and increase storage time of fresh products; very penetrating
Somewhat more expensive and dangerous than other methods

20. Radiation/Irradiation
Nonionizing radiation uses moderate energy, medium-length ultraviolet waves that excite, but do not ionize, atoms
Rays act on DNA molecule, forming dimers between adjacent pyrimidines, and produce toxic products
UV lamps disinfect air in medicine and industry; treat water, sera, vaccines, drugs; disinfect solid surfaces
Does not penetrate most solids and can damage human tissues (skin, eyes)

21. Formation of pyrimidine dimers by the action of ultraviolet (UV) radiation

22. Sterilization by Filtration
Filtration involves the physical removal of microbes from liquids and air by trapping them in fine filters
The fluid passes through tiny pores into a container
Can be used to remove viruses
Filters can sterilize heat-sensitive liquids (vaccines, serum, drugs, media, water)
Can remove microbes from air in hospital rooms, isolation units, and “clean rooms”

23. Chemical Control of Microorganisms
General uses: Disinfectants, antiseptics, sterilants, preservatives, sanitizers, degermers
Physical states: Solutions come in aqueous form (chemicals dissolved in water) and tincture form (chemicals dissolved in alcohol); solutions expressed as dilutions, percents, and parts per million
Halogens
Chlorine: Forms are Cl2, hypochlorites, chloramines
Denaturation of proteins by disrupting disulfide bonds; can be sporicidal with adequate time
Elemental chlorine (1-2 ppm) disinfects water by destroying pathogenic vegetative pathogens
Hypochlorites (chlorine bleach) are used extensively for disinfection and sanitization
Chloramines are clinical disinfectants and antiseptics and alternative water disinfection agents
Chemical action may be retarded by high levels of organic matter

24. Water is passed through a chlorination tank

25. Chemical Control of Microorganisms
Iodine: Forms are free iodine (I2) and iodophors (iodine complexed to organic polymers)
Interfere with protein interchain bonds, causing denaturation; can be sporicidal
Weak solutions are topical antiseptics; iodine tincture is for a high degree of surgical asepsis and disinfection
Iodine solutions stain and corrode; stronger solutions are too irritating and toxic to use on tissue
Iodophors are aqueous solutions with 2-10% iodine (Betadine, Povidone); they are milder medical and dental degerming agents, disinfectants, and ointments

26. Use of providone-iodine sponge

27. Chemical Control of Microorganisms
Phenolics
Phenols act on microbes by disrupting cell membranes and precipitating proteins
They are bactericidal, fungicidal, and virucidal, but not sporicidal
Phenol is an older toxic disinfectant; 1-3% cresol in soap is a common housekeeping disinfectant and cleaner
Orthophenyl phenol (Lysol) is a milder phenolic used in air sprays and hospital disinfectants
Triclosan is an antibacterial additive to soaps, detergents, and numerous household products
Most phenolics are to toxic for use as antiseptics and are relatively insoluble

28. Chemical Control of Microorganisms
Chlorhexidine is available as Hibiclens and Hibitane
It is a surfactant and protein denaturant, with bactericidal, some antiviral and antifungal effects, but not sporicidal
An aqueous or alcohol solution is a skin degerming agent for preoperative scrubs, skin cleaning, and burns
Product is relatively mild, nontoxic, and fast-acting

29. Chemical Control of Microorganisms
Alcohols include ethyl and isopropyl, usually in solutions of 50-95%
Act as a surfactant to dissolve membrane lipids and coagulate proteins; work on bacterial vegetative cells and fungi, but are not sporicidal
Ethyl alcohol (70-95%) is a germicide in the clinic, laboratory; used at home for degerming, low-level disinfection, and sanitization
Isopropyl alcohol has similar uses, though it is more toxic and less safe

30. Chemical Control of Microorganisms
Hydrogen peroxide is available in weak (3%) to strong (25%) solutions that produce highly active hydroxyl-free radicals and damage proteins and DNA molecules; also decomposes to water and O2 gas, which can be toxic to anaerobes; strong solutions are sporicidal
3% hydrogen peroxide is used for skin and wound antisepsis, care of mucous membrane infections; and disinfection of utensils
6-25% can be used to sterilize equipment
Decomposes in presence of light and catalase

31. Chemical Control of Microorganisms
Detergents and Soaps
Cationic detergents are known as quaternary ammonium compounds or quats
They are surfactants that alter cell permeability
Their action is limited to some bacteria and fungi; they are not sporicidal
Benzalkonium and cetylpyridinium chlorides are quats used in environmental disinfection, as cleaners in clinics and the food industry, and as presevatives
Soaps are not very microbicidal but function in the mechanical removal of grease and soil on skin, utensils, and environmental surfaces
Soaps do not destroy the tubercle bacillus or hepatitis B virus and are inactivated by large quantities of organic matter

32. Detergents

33. Chemical Control of Microorganisms
Heavy metal compounds include mercury and silver solutions and tinctures; work in high dilutions (oligodynamic action) and precipitate proteins; are germicidal but not sporicidal
Organic mercurial tinctures (thimerosal, nitromersol) are skin antiseptics and preservatives
Silver nitrate solutions are used as a topical antiseptic; silver sulfadiazine ointment can prevent burn infections; colloidal silver preparations are used for mouth and eye rinses
Metal solutions are highly toxic, cause allergies, and are neutralized by organic substances

34. Chemical Control of Microorganisms
Aldehydes include glutaraldehyde and formaldehyde or formalin solutions
They kill cells by alkylation of amino and nucleic acids, with wide microbial action
Glutaraldehyde in 2% solutions (Cidex) is used as a sterilant for heat-sensitive instruments in the medical and dental office, and for some types of environmental disinfection
Formalin in 1-8% is limited to disinfection of some instruments, rooms, and as a preservative
Glutaraldehyde is somewhat unstable; formaldehyde is toxic and irritating

36. Chemical Control of Microorganisms
Gases and aerosols include ethylene oxide (ETO), propylene oxide, and betapropiolactone, which inactivate nucleic acids and proteins
All are sporicidal, but only ETO is an approved sterilant
Ethylene oxide is added to a special chamber for sterilizing plastic supplies and to treat spices and dried foods
Propylene oxide can disinfect food (nuts, starch)
Betapropiolactone is used to disinfect rooms and some biological materials
Toxic and has poor penetration
ETO is somewhat dangerous because of its explosiveness and toxicity, and it is slow-acting