How to stop bacterial growth PDF

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H O W T O S T O P B A C T E RIA L G R O W T H Sterilisation: destruction or removal of all microorganisms Disinfection: destruction of pathogenic microorganisms on a surface Disinfectant: antimicrobial agent used on non-living surfaces Antisepsis: destruction or inhibition of microorganisms in living tissues Preservation: prevention of multiplication of microorganisms in natural or formulate products, e.g. in food Antibiotics: selectively toxic agents that kill or inhibit microorganisms; either bacteriostatic or bactericidal Bacteriostatic: inhibition of bacterial growth without killing Bactericidal: killing Why stop bacterial growth?  Biofouling – e.g. fouling through growth of biofilms in industrial reactors  Laboratory culture – need to work with pure microorganisms, free of contaminants  Water treatment  Food spoilage  Medical/surgical equipment – avoid infection  Control growth of bacteria In vivo treatments – antibiotics  Physical (heat, radiation) and chemical (chemical agents) can be used to kill bacteria Pasteurisation:  Pasteur – wine at 60oC (compromise between killing and retaining flavour)  High temperature short time – 72oC for 15 secs (e.g. milk)  Ultra high temperature – 138oC for 2 seconds (not a common as has more of an effect on flavour)  Destruction of pathogens (elimination) – Mycobacterium tuberculosis, Coxiella burnetti, Salmonellae and Escherichia coli  Destruction of spoilage organisms (reduces the numbers) – Lactococcus, Lactobacillus, Pseudomonas, Proteus and Streptococcus  Does not eliminate all spoilage microorganisms – 1 in 100,000 are retained and will proliferate  A reduction of 5 log order of magnitude is acceptable  The substance (e.g. raw milk) is passed through a series of plates that are progressively heated up to an area (no. 5) where the substance is held for the required amount of time – can be automated very easily, widely used  Heat causes protein/membrane/DNA damage  Most mesophilic organisms (grow best in moderate temperatures) are killed at 55 oC  Moist heat is the most effective as condensation onto cooler surfaces releases latent heat onto the surface of what it is trying to disinfect; it is even more effective in a vacuum Endospores:  Some organisms form dehydrated, resilient, vegetative spores – ensure survival of a bacterium through periods of environmental stress  They require more aggressive treatment as they can survive in a dormant state for extended periods  E.g. Bacillus, Clostridium can make spores – cause food borne and other diseases

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Heat: The autoclave:  Used to administer the amount of heat required for complete destruction of bacteria including spores  Moist heat in an evacuated container  Treated for 15 mins at 121oC and 100 kPa  Most reliable and effective method  Heating by the autoclave is cheap, can be very effective, even against spores  Not all items are suitable, e.g. medical plastics

Irradiation:   

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Gamma rays, X-rays, UV rays - high energy UV radiation is lower energy and causes abnormal DNA bonds Two T bases exposed to UV radiation results in covalent bonds between them to generate a thymine dimer which sits in the double helical structure and interferes with DNA replication  arrest of replication/introduction of mutations Ionising radiation (gamma rays) exposure is more directly damaging to nucleic acids than UV because it can introduce double strand breaks It interacts with small molecules in the cell to generate free radicals, e.g. OH  radial, that cause double stranded brakes Double strand breaks mean there is no longer an intact template that can used for repair The Gray is the SI unit of absorbed radiation (1 J/kg) In humans, 10 Grays is lethal; most bacteria require 10/100 times the dose to be killed; to kill viruses, 1000’s times more radiation is needed

Industrial irradiation:  60Co, 137Cs – very reactive and need to be contained  An expensive and dedicated facility is needed for it to be effective, e.g. sealed and remote-controlled chambers  Very effective for high value, fragile products, e.g. drugs  Food, medical products, drugs and tissue grafts can be sterilised by radiation o Actually very rarely used for food, esp in EU/UK – public acceptance  Variable effectiveness – gamma is more penetrative than UV o UV is good for water treatment in lower volume situations, an in labs to sterilise work areas  Suitable for a wide range of materials – plastics, food  Effective for water sterilisation

Chemical agents:  

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Non-specific Chemical agents work in three different ways – disrupt the membrane; act as oxidising agents (producing radical that react with nucleic acids); or modify the protein component of the cell (alkylation, heavy metals – attach to proteins and effect their function) The membrane disrupting agents are the most widely used group of chemical agents Damage the membrane  destroy the integrity  make it permeable  cell contents leach out/extracellular components move in Chemical agents are cheap and easy to use Can be toxic/harmful to the environment/humans

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Affected by the environment – pH, temp, solvent mixtures, organic matter Chemical agents vary in their effectiveness to different types of bacteria – gram negative bacteria have more extensive outer envelope structures than gram positive which can lead to a reduction in the effectiveness of the agents Spores are less affected

Antiseptics:  Can be used on living surfaces, disrupt the membrane  Alcohol – 60-85%  Chlorhexidine – polar head group  Lots of molecules have the quaternary ammonium chloride (benzalkonium chloride) structure; one substituent will be aromatic, one will a long non-polar unsaturated chain o Lots of toothpastes have this  Iodine – more gentle; suitable for use on living tissues  Hydrogen peroxide – generates oxygen radicals at a lower enough concentration to be useful as an antiseptic Disinfectants:  More aggressive, i.e. used when it doesn’t matter if living surfaces are killed; produce higher concentrations of oxidising agents  Chlorine gas (sodium hypochlorite) – oxidising agent  Chlorine dioxide – oxidising agent  Hydrogen peroxide – oxidising agent  Phenolic compounds, e.g. chloroxylenol  Formaldehyde – alkylating agent (more effective  Iodine – iodinating agent  Heavy metals, e.g. Cu (industrial cooling towers), Hg – ore effective Triclosan:  Phenolic  Membrane disruption  Specific inhibitor for FabI protein (which is involved in bacterial fatty acid synthesis)  Species specificity – less useful as a general disinfectant  Resistance Antibiotics:  Specific target, e.g. an essential biological process, component of a ribosome or a component of a biochemical pathway  Safe to use systemically – mechanisms that are targeted by antibiotics are only in prokaryotes

Containment:   

Different approach to controlling bacterial growth Managing the risks in labs and healthcare premises Microorganisms are categorised into hazard groups o Hazard group 1 – unlikely to cause human disease  E.g. Staphylococcus epidermidis – all carry on our skin o Hazard group 2 – can cause human disease  May be hazardous to employees/immunocompromised, but unlikely to spread to the community  Effective prophylaxis (preventative treatment) or treatment is available  E.g. Staphylococcus aureus (MRSA), Pseudomonas aeruginosa o Hazard group 3 – can cause severe human disease  May be a serious hazard to employees and may spread to the community  Usually effective prophylaxis or treatment available  Level 3 labs may have negative pressure and air filters  E.g. Mycobacterium tuberculosis, Bacillus anthracis, Yersinia pestis o Hazard group 4 – causes severe human disease

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Is a serious hazard to employees and is likely to spread to the community Usually no effective prophylaxis or treatment available Negative pressure, filter input/extract air, airlock is needed in a level 4 lab E.g. Variola virus, Ebola virus, Marburg virus

Summary:      

Requirement to control bacterial growth (Moist) heat is simple and effective where feasible Bacterial spores require special consideration Irradiation is effective and convenient Chemical agents are convenient but often less effective Lab work with microorganisms is tightly regulated

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