lecture 7 Flashcards
what is the difference between sterilisation and growth inhibitors?
rilisation is killing/removal/inactivation of all viable organisms
Control methods discussed so far may inhibit growth by creating unfavourable conditions
spores are the most difficult part of an organism to kill. sterilisation is a term reserved for them
Examples of these control methods include manipulation of:
temperature, pH, water levels (aw), oxygen levels
These growth control methods still used in food industry, for microbial selection in the lab, etc
Q. Listeria can cause death in utero and meningitis in the immunocompromised. It grows at 4oC. What are the implications?
listeria causes salmon food outbreak. unless ur pregnant u wont know if u have it, to the very old or young or immunocompromise - they are affected.
what is pastuerisation?
kill organisms who dont have spores. we use this on objects that dont pass through the skin because spores cant penetrate through the skin unless through a needle because they are steralised.
Pasteurisation (71°C for 15 seconds)
Boiling (100°C) - not a form of sterilisation because it doesnt kill spores
Hot air oven (160-180°C)
Incineration (>800°C)
Autoclaving (steam at >100°C) - we do this by putting it in a pressure cooker. the container is sealed making molecules more agitated, the temp goes above 100*c
Foods can be boiled at 100°C
doesn’t kill endospores
may change taste and physical properties of heat-sensitive foods
Heat sensitive foods can be pasteurised:
Named for Louis Pasteur, first used to prevent spoilage of wine
Not a process of sterilisation, reduces microbial numbers and kills pathogenic bacteria including Listeria and Campylobacter
Pasteurisation routinely used for juice and milk
Pasteurisation retards growth of spoilage organisms, increases shelf life
Emphasize that heating and cold storage inhibits growth but may not kill microorganisms
Sterilisation:
Killing/removal of all viable organisms, including endospores
Disinfection:
Killing/removal of all vegetative forms, not including endospores
where do we find endospores and give an example
Allow microorganisms to survive in extreme conditions
Endospore-forming bacteria most commonly found in soil
Heat resistant, can withstand temperatures of 150°C
Also resistant to UV radiation and chemical disinfectants
example tetanus
in shepparton - anthrax
explain sterilisation by heat and give an example
Heat kills cells by denaturing proteins
Autoclaving is commonly used for sterilisation, kills endospores
Uses moist heat (steam) at pressure to achieve high temperatures
Moist heat penetrates better than dry heat - kills more organisms
Typically used at a temperature of 121°C
At 121°C, sterilisation can be achieved in 10-15 minutes
Pressurizing the steam allows temperatures of 121 degrees to be reached. Sterilisation essential when preparing microbiological media and for treating surgical instruments.
how do you use an autoclave machine?
Generally heat till 1210C and then hold for 15 minutes
Flash sterilisers (tend to be smaller bench-style) 1340C for 3 minutes
Important to allow flush out of air as air trapped in load poor transfer of heat
Condensation on items allows transfer of heat rather than conduction
Drying cycle important as bugs can pass through wet packs
definition of decimal reduction time test?
Decimal reduction time (D) the most precise way to measure heat sterilisation
Time required for a 10-fold reduction in viable cells (90% cells killed)
Exponential, straight line obtained when the logarithm of D is plotted against time
Can calculate time required for sterilisation
Time required varies for different organisms, e.g. a mesophile or a thermophile
So how long do we need to sterilise before all the organisms are dead ? EXAM
ONE FROM PRAC ONE (WORKING OUT THE NUMBER OF ORGANISMS IN AN ORIGINAL SAMPLE) AND THIS ONE AND LOG GRAPH, C1V1=C2V2, 1 CHI SQUARE EXAMPLE
From the previous slide we saw that the number of organisms reduced by a power of 10 each minute.
At 3 minutes, 10 organisms were left
At 4 minutes, 1 organism was left
At 5 minutes, there is a 1/10 chance of an organism being alive
At 6 minutes, there is a 1/100 chance of an organism being alive
how are autoclaves checked?
Print out of cycle (Physical)
Heat sensitive papers or tape (Chemical)
Spore-test (commercially available) and should be performed weekly(Biological)
what are the factors that influence autoclaving?
Dry items take longer to sterilise than moist ones, high amounts of sugars, proteins or fats in the medium decreases heat penetration
must be autoclaved at higher temperatures for longer times
Size/volume of item to be autoclaved influences sterilisation time, time must be increased so that all of the item reaches 121°C for 10-15 minutes
Endospores of Bacillus stearothermophilus used in hospitals to test sterilisation effectiveness
Vials of endospores included in each run, incubated after autoclaving, a colour change in vial means organisms have survived and grown
how do we treat heat sensitive items or equipment or air?
Ethylene oxide but toxic by contact and explosive! Confined to industry and commercial plant available to send packs for sterilisation
Gamma-irradiation, again used in industry and useful for large boxes of plastic goods (check coloured indicator on outside of box to ensure processed!)
how do we sterilise by radiation?
Ionising radiation used for sterilisation of lab/hospital materials
Eg petri dishes, wound dressings, etc
High-energy radiation particles which produce electrons and free radicals from molecules they collide with
These radicals disrupt DNA and proteins in the cells, leading to death
Some foods are also irradiated with low doses of gamma (y) radiation
Low doses used, lowers microbial numbers but will also produce free radicals
can we use UV light as a sterilising agent?
Generated by a lamp that also produces blue light so you can see when on
Effective as it denatures proteins
Does not penetrate glass or get around objects
Only suitable for flat surfaces, air or clear water
other methods of sterilisation?
Not all objects that need sterilising can be autoclaved
Heat sensitive materials, eg antibiotics and growth factors such as amino acids, are often added to microbiological media for growth/selection
These must be sterile to avoid contaminating the media, so microorganisms are removed by physical filtration
Membrane filters are most commonly used, made of polymers, polymerisation conditions can be altered to change pore size
Membrane filters with a 0.2 µm (1/20,000th of 1 mm) pore commonly used
Filters can also be used to concentrate microorganisms from large volumes, eg. for water testing. The filter will trap but not kill the bacteria, filter can then be placed on nutrient medium
A pore size of 0.2 micrometres allows liquid to pass through but traps bacteria at the membrane surface
Filtration can be used for liquids, removes down to 0.3 microns (micrometres) as minimum pore size 0.2. Not suitable if liquid is cloudy – why?because the cloudy will block the filter and it will break the filter
HEPA (High efficiency particulate air) filters can be used to filter air for special rooms like operating theatres (99.97% efficient and remove particles as small as 0.3 microns)
what is the most logical size of this filter if it filters out bacteria?
0.2 micrometers
how do you use UV radiation?
surface sterilisation.
UV radiation: between 220-300 nm wavelength light, causes modifications and double stranded breaks in cell DNA, leading to death
“Biohazard” cabinets use UV light to sterilise surfaces before/after use
Also have HEPA (high efficiency particulate air) filter to sterilise air within the cabinet during use – can remove 0.3 µm particles
Some bacteria (eg Serratia) have pigments which absorb UV radiation, increasing survival time
Endospores more resistant to UV radiation than vegetative cells
