1. Controlling Microbial Growth in the Environment

2. Action of Antimicrobial Agents
Many types of chemical and physical microbial controls
Modes of action fall into two basic categories
Alteration of cell walls or cytoplasmic membranes
Interference with protein and nucleic acid structure

3. Selection of Microbial Control Methods
Ideally, agents should be:
Inexpensive
Fast-acting
Stable during storage
Control all microbial growth while being harmless to humans, animals, and objects

4. Factors Affecting the Efficacy of Antimicrobial methods
Nature of site to be treated
Degree of susceptibility of microbes involved
Environmental conditions that pertainالمتعلقة التابعة

5. Organismal differences
Microorganisms differ in their susceptibility to antimicrobial agents."
Often what fails to be killed by a disinfectant are endospores though certain viruses and some vegetative bacteria are also highly resistant to disinfection
Also, the same organism may differ in susceptibility depending on growth phase with actively growing organisms typically more susceptible to disinfection than not-growing cultures

6. Relative Susceptibility of Microorganisms

7. Relative Susceptibility of Microorganisms
Effectiveness of germicides classified as high, intermediate, or low
High-level kill all pathogens, including endospores
Intermediate-level kill fungal spores, protozoan cysts, viruses and pathogenic bacteria
Low-level germicides kill vegetative bacteria, fungi, protozoa, and some viruses

8. Environmental conditions that pertain
Increasing temperatures increases the efficacy of most chemical antimicrobials
The converse of this statement is that relatively cold temperatures result in relatively poor disinfection
The fewer organisms present, the shorter the time needed to achieve sterility. Thoroughly cleaning objects before attempting to sterilize them is a practical application of this principle. Clearing objects of tissue debris and blood is also important because such organic matter impairs the effectiveness of many chemical agents.

9. Environmental conditions that pertain (cont.)
Concentration effects: Generally, the use of more disinfectant provides better killing than the use of less disinfectant

10. Environmental Conditions

11. Methods for Evaluating Disinfectants and Antiseptics
Phenol Coefficient
Use-Dilution Test
Disk-Diffusion Method
In-Use Test

12. Phenol Coefficient
Evaluating the efficacy of disinfectants and antiseptics by determining the ratio of agent’s ability to control microbes to that of phenol
Greater than 1.0 indicates that agent is more effective than phenol
Has been replaced by newer methods

13. Use-Dilution Test
Especially useful for determining the ability of disinfectants to kill microorganisms dried onto a typical clinical surface (stainless steel)
Metal cylinders dipped into broth cultures of bacteria and dried
Contaminated cylinder immersed into dilution of disinfectant for 10 minutes
Cylinders removed, washed, and placed into tube of medium for 48 h
Most effective agent entirely prevents growth at highest dilution

14. Disk-Diffusion Method
A method that requires less manipulation to judge the efficacy of disinfectants
Here filter paper is soaked with disinfectant and then simply placed on the agar surface of a petri dish that has been inoculated with a lawn of test organism
The clear area around the disk following incubation is used as an indication of disinfectant efficacy

15. Disk-Diffusion Method

16. In-Use Test
Swabs taken from objects before and after application of disinfectant or antiseptic
Swabs inoculated into growth medium and incubated
Medium monitored for growth
Accurate determination of proper strength and application procedure for each specific situation

17. Physical Methods of Microbial Control
Exposure to extremes of heat
Exposure to extremes of cold
Desiccation
Filtration
Osmotic pressure
Radiation

18. Heat-Related Methods Effects of high temperatures
Denaturation of proteins
Interference with integrity of cytoplasmic membrane and cell walls
Disruption of structure and function of nucleic acids
Thermal death point – lowest temperature that kills all cells in broth in 10 minutes
Thermal death time – time to sterilize volume of liquid at set temperature

19. Moist Heat Used to disinfect, sanitize, and sterilize
Kills by denaturing proteins and destroying cytoplasmic membranes
More effective than dry heat; water better conductor of heat than air. Moist heat is also more penetrating than dry heat
Methods of microbial control using moist heat
Boiling
Autoclaving
Pasteurization
Ultrahigh-Temperature Sterilization

20. Boiling
Kills vegetative cells of bacteria and fungi, protozoan trophozoites, and most viruses within 10 minutes at sea level
Temperature cannot exceed 100ºC at sea level; steam carries some heat away
Boiling time is critical
Water boils at lower temperatures at higher elevations; requires longer boiling time
Endospores, protozoan cysts, and some viruses can survive boiling

21. Autoclaving
Pressure applied to boiling water prevents steam from escaping
Boiling temperature increases as pressure increases
Autoclave conditions – 121ºC, 15 psi, 15 minutes
Volume
Contact
Wrapping
Testing

22. Autoclave

23. Sterile Services Department
A sterile services department (SSD) is vital for an effective infection program. Dirty, recyclable equipment should be collected from the wards عنابرand transferred to the SSD, where it is washed, inspected, sterilized, packed, and dispatched back to the wards.

25. Pasteurization Pasteur’s method
Pasteurization is the application of moist heat of less-than boiling temperatures to foods to prevent the growth of food-spoiling organisms as well as various heat-labile pathogens
Pasteur’s method
Today, also used for milk, ice cream, yogurt, and fruit juices
Not sterilization; heat-tolerant and heat-loving microbes survive
These do not cause spoilage prior to consumption
These are generally not pathogenic

26. Ultrahigh-Temperature Sterilization
140ºC for 1 second, then rapid cooling
Treated liquids can be stored at room temperature
Chemical degradation

27. Dry Heat
Used for materials that cannot be sterilized with or are damaged by moist heat
Denatures proteins and oxidizes metabolic and structural chemicals
Requires higher temperatures or longer time than moist heat
Nevertheless, application of dry heat is cheap and easy
Typically one bakes materials in an oven at
(i)   171ºC for at least one hour
(ii)  160ºC for at least two hours
(iii) 121ºC for at least 16 hours
Incineration – ultimate في نهاية المطافmeans of sterilization
Loops (1500°C), carcasses, dressings

28. Refrigeration and Freezing
Refrigeration is a great short term solutionحل عظيم على المدى القصير , it decreases microbial metabolism, growth, and reproduction i.e. it merely slows the growth of organisms rather than preventing it
Chemical reactions occur slower at low temperatures
Liquid water not available
Psychrophilic microbes can multiply in refrigerated foods
Refrigeration halts growth of most pathogens (Listeria, Yersinia)
Slow freezing more effective than quick freezing
Organisms vary in susceptibility to freezing

29. Refrigeration and Freezing
For organisms that survive the freezing process, freezing constitutes a reasonably good long-term preservation method with prevention of deterioration increasing as temperatures are lowered
Lower temperatures result in greater long-term storage:
(i)  -20ºC is the temperature of a standard freezer
(ii) -80ºC is a good temperature for long-term storage of microorganisms
(iii)-180ºC is the temperature of liquid nitrogen, a common medium for long-term storage of microorganisms

30. Desiccation and Lyophilization (Freeze-drying)
Drying inhibits growth due to removal of water; only microbiostatic. Desiccation prevents organism metabolism, and is a good means of preventing organism growth
Desiccation is an effective means of organism storage for those organisms capable of remaining viable upon desiccation
Lyophilization used for long term preservation of microbial cultures
Prevents formation of damaging ice crystals
Freeze-drying involves freezing something and then evacuating air so that boiling occurs at low temperatures; this desiccates material thereby preventing deterioration and spoilage
Liquid nitrogen or frozen carbon dioxide (dry ice)

32. Filtration
Used to sterilize ophthalmic للعيون solutions, antibiotics, vaccines, liquid vitamins, enzymes, culture media eg, tissue culture
Membrane filters (nitrocellulose) with pore sizes 25µm to less than 0.01µm
High-efficiency particulate air (HEPA) filters are used to filter the air flowing into aseptic environments ( e.g. operating rooms) and out of potentially contaminated ones (e.g., containment facilities)

33. Filtration

34. Filtration

36. Osmotic Pressure
High concentrations of salt or sugar in foods to inhibit growth (jam, jerky, pickled food, salted fish)
Water in cell is drawn out by osmosis; cell desiccates
Fungi have greater ability than bacteria to survive hypertonic environments

37. Radiation
Electromagnetic radiation (enrgy without mass travelling in waves at the speed of light, 3x105km/sec)
Shorter wavelength equals more energy and greater penetration
Radiation described as ionizing or nonionizing according to effects on cellular chemicals

40. Nonionizing Radiation
Wavelengths greater than 1 nm
Excites electrons and causes them to make new covalent bonds
Affects 3-D structure of proteins and nucleic acids
UV light causes thymine dimers in DNA
UV light does not penetrate well
Suitable for disinfecting air, transparent fluids, and surfaces of objects