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Controlling Microbial Growth in the Environment

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1 Controlling Microbial Growth in the Environment

2 Terminology of Microbial Control
Sterilization (a) Sterilization is the killing of all microorganisms in a material or on the surface of an object (b) A surface or an object is either sterile or it is not sterile, there are no gradations in sterility (c) Typically the last things to die when one attempts sterilization is the highly heat- (and chemical-, etc.) resistant endospores Aseptic Degerming

3 Terminology of Microbial Control (cont.)
Disinfection/disinfectants (a) Disinfection means reducing the number of viable microorganisms present in a sample (b) A disinfectant is a chemical or physical agent that is applied to inanimate objects to kill microbes (c) Not all disinfectants are capable of sterilizing, but, of course, all disinfectants are employed with the hope of disinfecting Antisepsis/antiseptic Antiseptic (a) Typically an antiseptic is a chemical agent that is applied to living tissue to kill microbes (b) Note that not all disinfectants are antiseptics because an antiseptic additionally must not be so harsh that it damages living tissue (c) With this constraint imposed on antiseptics, in general antiseptics are either not as cheap or not as effective at killing microbes as disinfectants

4 Terminology of Microbial Control (cont.)
Sanitization Sanitization is the cleaning of pathogenic microorganisms from public eating utensils and objects such as that done by the kitchen of a restaurant Pasteurization Suffix –stasis/-static  Bacteriostatic means that the antimicrobial inhibits bacterial growth but does not kill the bacteria; consequently, removal or dilution of the antimicrobial can result in a resurgence of bacterial growth Suffix-cidal An antimicrobial that kills a microorganism (or, more specifically, a bacterium) is said to be bactericidal

5 Microbial Death Rates

6 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

7 Alteration of Cell Walls and Membranes
Cell wall maintains integrity of cell When disrupted, cannot prevent cell from bursting due to osmotic effects Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell When damaged, cellular contents leak out Viral envelope responsible for attachment of virus to target cell Damage to envelope interrupts viral replication Nonenveloped viruses have greater tolerance of harsh conditions

8 Damage to Proteins and Nucleic Acids
Protein function depends on 3-D shape Extreme heat or certain chemicals denature proteins Chemicals, radiation, and heat can alter or destroy nucleic acids Can produce fatal mutants Can halt protein synthesis through action on RNA

9 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

10 Factors Affecting the Efficacy of Antimicrobial methods
Nature of site to be treated Degree of susceptibility of microbes involved Environmental conditions that pertain

11 Site to Be Treated Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects Method and level of microbial control based on site of medical procedure

12 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

13 Relative Susceptibility of Microorganisms

14 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

15 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.

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

17 Environmental Conditions

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

19 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

20 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

21 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

22 Disk-Diffusion Method

23 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

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

25 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

26 Decimal reduction time

27 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

28 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

29 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


31 Autoclave

32 Autoclave

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

34 Sterile Services Department (cont.)
Workflow of the SSD - in the ward, dirty re-usable instruments are collected and put into clearly labelled containers and delivered to the SSD. The dirty instruments are then received in the dirty area of the SSD. All equipment is first washed in hot water and detergent. The equipment is then inspected for cleanliness and damage. Instruments are then packed into individual trays for ward use and autoclaved and/or disinfected as required. The packaged trays are then inspected to ensure that they are dry and then sorted for ward collection. The sterile packs should be stored in well-ventilated rooms ready for dispatch to the wards. Collections should be regular and there should be a written record of receipt and delivery.


36 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

37 Pasteurization Milk Batch method – 30 minutes at 63ºC
Flash pasteurization – 72ºC for 15 seconds Ultrahigh-temperature pasteurization – 134ºC for 1 second

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

39 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 means of sterilization Loops (1500°C), carcasses, dressings

40 Refrigeration and Freezing
Refrigeration is a great short term solution, 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

41 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

42 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)


44 Filtration Used to sterilize ophthalmic 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)

45 Filtration

46 Filtration


48 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

49 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


51 Ionizing Radiation Wavelengths shorter than 1 nm – electron beams, gamma rays, and X rays Ionizing radiation is radiation that ionizes water; this temporarily turns water into an oxidizing agent Create ions by ejecting electrons from atoms they strike Ions disrupt hydrogen bonding, oxidize double covalent bonds, and create hydroxide ions; hydroxide ions denature other molecules (DNA) One potential application of ionizing radiation is as an antimicrobial is in food preservation

52 Ionizing Radiation Electron beams – effective at killing but do not penetrate well Used to sterilize some microbiological plastic ware, and medical and dental supplies Gamma rays – penetrate well but require hours to kill microbes Used to sterilize some medical instruments X-rays require too much time to be practical for growth control


54 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

55 Chemical Methods of Microbial Control
Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA Effect varies with temperature, length of exposure, and amount of organic matter Also varies with pH, concentration, and age of chemical Tend to be more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa

56 General Rules Certain rules should be followed when using disinfectants. The manufacturer’s instructions should be followed. The expiry date should be checked and the optimum dilution used. Articles to be disinfected should be washed and cleaned before disinfection. The disinfectant container should not be refilled without sterilizing between each use and not be topped up. Disinfectants should be supplied ready for use from the pharmacy and empty containers should be returned to the pharmacy. Empty containers should not be discarded or used to store any other solutions as this is potentially dangerous. Open containers of disinfectant should not be tolerated as there is a serious risk of contamination with multiple antibiotic-resistant bacteria, such as Pseudomonas species and spores.

57 General Rules Disinfectants should be used according to instructions at the optimum dilution. Some disinfectants may be rapidly inactivated by organic matter, therefore any object that is to be disinfected should be cleaned thoroughly with warm water and detergent prior to disinfection. When disinfectants are indicated for use on surfaces, they should be applied by wiping rather than bathing as bathing wastes disinfectants. Sustained-action disinfectant should be used for hand hygiene by staff and for cleaning the skin and mucous membranes of patients. Alcohol preparations over 40% are not recommended because of the risk of fire when use in conjunction with diathermy. In general, hard surfaces do not require disinfectants - warm water with detergent is usually sufficient to remove all organic contamination. The exceptions are where persistence of potentially dangerous pathogens, such as HIV or HBV is suspected, when the surface should be wiped by a disinfectant afterwards.

58 Chemical Methods of Microbial Control
Major Categories Phenols Alcohols Halogens Oxidizing agents Surfactants Heavy Metals Aldehydes Gaseous Agents Antimicrobics

59 Phenol and Phenolics Intermediate- to low-level disinfectants
Denature proteins and disrupt cell membranes Effective in presence of organic matter and remain active for prolonged time Commonly used in health care settings, labs, and homes (Lysol, triclosan) Have disagreeable odor and possible side effects


61 Alcohols Intermediate-level disinfectants
Denature proteins and disrupt cytoplasmic membranes Evaporate rapidly – both advantageous and disadvantageous Swabbing of skin with 70% ethanol prior to injection Alcohols should not be applied to wounds since they can cause tissue damage Tinctures

62 Halogens Intermediate-level antimicrobial chemicals
Believed that they damage enzymes via oxidation or by denaturing them Iodine tablets, iodophores (Betadine®), chlorine treatment of drinking water, bleach, chloramines in wound dressings, and bromine disinfection of hot tubs


64 Oxidizing Agents Peroxides, ozone, and peracetic acid kill by oxidation of microbial enzymes High-level disinfectants and antiseptics Hydrogen peroxide can disinfect and sterilize surfaces of objects Catalase neutralizes; not useful for treating open wounds Ozone treatment of drinking water Peracetic acid – effective sporocide used to sterilize equipment

65 Surfactants Surfactants are substances that are soluble in water but are able to dissolve lipids ie. “Surface active” chemicals that reduce surface tension of solvents to make them more effective at dissolving solutes Soaps and detergents Soaps have hydrophilic and hydrophobic ends; good degerming agents but not antimicrobial Detergents are positively charged organic surfactants Quats – colorless, tasteless, harmless to humans, and antimicrobial; ideal for many medical and industrial application (benzalkonium chloride) Low-level disinfectants Pseudomonas


67 Heavy Metals Ions are antimicrobial because they alter the 3-D shape of proteins, inhibiting or eliminating their function (cysteine) Low-level bacteriostatic and fungistatic agents 1% silver nitrate to prevent blindness caused by N. gonorrhoeae Thimerosal used to preserve vaccines Copper controls algal growth in reservoirs, fish tanks, swimming pools, and water storage tanks; interferes with chlorophyll


69 Aldehydes Compounds containing terminal –CHO groups
Cross-link with amino, hydroxyl, sulfhydryl, and carboxyl groups to denature proteins and inactivate nucleic acids Only one liquid chemical, if properly used, is capable of rendering an item sterile. That chemical is glutaraldehyde. The item to be sterilized must be totally submerged in the glutaraldehyde solution for 10 hours. Before immersion, the item must be thoroughly cleansed and rinsed with sterile water or sterile normal saline. It should be noted that this chemical is extremely caustic to skin, mucous membranes, and other tissues i.e.Glutaraldehyde both disinfects (short exposure) and sterilizes (long exposure) Formalin used in embalming and disinfection of rooms and instruments

70 Gaseous Agents Ethylene oxide, propylene oxide, and beta-propiolactone used in closed chambers to sterilize items Denature proteins and DNA by cross-linking functional groups Used in hospitals and dental offices Can be hazardous to people, often highly explosive, extremely poisonous, and are potentially carcinogenic The most effective method of gas chemical sterilization presently available is the use of ethylene oxide (ETO) gas. ETO gas sterilization should be used only for material and supplies that will not withstand sterilization by steam under pressure.

71 Development of Resistant Microbes
Little evidence that extensive use of products containing antiseptic and disinfecting chemicals adds to human or animal health The use of such products promotes the development of resistant microbes

72 Waste disposal All hospital waste should be disposed of so that it presents no risk of injury or contamination. Clinical waste is generated during routine patent care and surgery. It is potentially dangerous and should be clearly labelled as high risk. Examples of clinical waste include dressings, body fluids, pathology waste, IV needles and syringes, drainage bags etc. Laboratory waste should also come under the high risk category and should be autoclaved before leaving the department and labelled biohazard. Other hospital waste include non-clinical waste and kitchen waste. The latter should be disposed of properly because it is a potential source of pests and vermin and thus may pose an indirect threat to the hospital. Provision must also be made for the safe disposal of radioactive waste. A simple colour-coding system should be used to separate waste so that the different components can be treated safely. In general, non-clinical waste is taken to a compactor and clinical waste to an incinerator or if not possible, to a lime-pit. Under no circumstances should clinical waste, needles or syringes be sent to the municipal dump. All staff handling clinical waste should be adequately trained especially with regards for the protocol of action in case of accidents. All staff must be provided with adequate protective clothing and replacement garments. Hepatitis B immunization should be offered to all staff and proper records kept for such immunization.

73 Waste disposal It is totally unjustified to recycle needles and syringes. Broken glass should be included in the sharps category. To dispose safely of broken glass, thick gloves should be worn and the pieces collected with a newspaper. The glass is then wrapped securely in the paper and either disposed of into the sharps container or if not practicable, into a cardbox box which is then marked and sealed. Sharps containers must be leak and puncture-proof. Have a handle that allows lifting, a non-reopenable lid, and carry a biohazard sign. They should be sealed and replaced when it is no more than two-thirds full. Sharps containers are treated as clinical waste and should be put into clinical waste bags before being incinerated. In the event of an inoculation or contamination accident, it must be documented by the senior manager and reported to the Infection Control Team so that appropriate action can be taken. There must be clearly defined policy on the action to be taken after the injury and all staff should be aware of the policy.

74 General Use Articles Bedpans and Urinals - gloves should be worn to empty the bedpan and its contents directly into the bedpan disinfector. Alternatively, the contents may be flushed down the sluice or toilet and then washed thoroughly with warm water and detergent to remove all signs of organic contamination and dried. The bedpan disinfector is the preferred method and it functions at no less than 80oC for 1 minute. Washing bowls - these must be washed thoroughly between each patient and stored and inverted to dry. Fresh water and towels should be used for each patient. Towels, soaps, hairbrushes, shaving brushes etc. - all these items should be for individual use only and should never be shared. Crockery and cutlery - each patient should have an individual set. the crockery and cutlery should be washed in very hot water (>60oC) and detergent. Disposable crockery is only necessary in cases of strict isolation such as rabies. Mattresses and pillows - they are a major source of cross-infection. Wet mattresses must be changed. Contaminated mattresses should be washed with warm water and detergent. Mattresses should be covered with an impervious layer so that they can be cleaned thoroughly between patients. Damaged mattresses should be discarded as they may easily trap microorganisms. Thermometers - they should be washed in warm water and dried before being wiped over with a swab soaked in 70% isopropyl alcohol. They should never be soaked in disinfectants. Trolley tops - they should be washed with warm water and detergent  

75 General Use Articles Endoscopy Unit
Fibre-optic endoscopes are usually heat-labile and therefore require chemical disinfection. 2% glutaraldehyde should be used under strict controlled conditions as required by the COSHH regulations. Protoscopes and sigmoidoscopes may be disposable or reusable. The latter must be cleaned, sterilized, or disinfected by heat or 2% glutaraldehyde. Disposable Equipment Syringes and needles - disposable syringes and needles should be used. Under no circumstances should fine-bore needles be recycled because they cannot be cleaned. If syringes are to be recycled, it must be well controlled. After thorough cleaning, the syringes must be autoclaved or processed in ethylene oxide. Administration sets - administration sets for IV fluids must be disposable Urinary catheters and drainage bags - these should be single-use and disposable

76 ICUs, Operating Theatres
Ventilatory circuits - multiple-use circuits must be heat-disinfected for at least 80oC for 3 minutes or sterilized by autoclave or ethylene oxide between each patient. Filters may be used between circuits and if properly maintained, a ventilated patient may use the same circuit for 4-5 days before disinfection. Humidifiers - humidifiers are a common source of viruses and Gram-negative bacilli and should be emptied daily and refilled with distilled water. They should be disinfected when contaminated or when the respiratory circuit is changed. Routine heat disinfection is essential after each patient use and if humidification is required for a prolonged period, the humidifier should be cleaned thoroughly, dried, and filled daily with distilled water. Endotracheal suction catheters - they are usually disposable but may be used up to 24 hours on the same patient. Endotracheal tubes - these may be recycled after thorough cleaning and autoclaving. Disposable endotracheal tubes are available but are more expensive. Ambu-bags - they are extremely difficult to disinfect and become contaminated very quickly. Heat is the most reliable method and 2% glutaraldehyde is a less acceptable alternative. Oxygen-delivery face masks - these can be disposable or reusable. If reused, they should be washed thoroughly and wiped over with 70% isopropyl alcohol. Suction and drainage bottles - these are usually disposable, with a self-dealing inner container held in a clear outer plastic container. The outer container should be heat-disinfected or autoclaved after each use. Non-disposable bottles must be changed every 24 hours or sooner if full. They must then be washed and autoclaved. Recyclable connector tubing should be cleaned thoroughly and sterilized. The system must be closed and risk to staff from body fluids should be minimal.

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