Sterilisation & Contamination Flashcards
what is the definition of sterile
free from all viable forms of life
population never reaches zero
what does SAL stand for
sterility level assurance
what does PNSU stand for
probability of a non-sterile unit
what is the bioburden
how many bacteria are in a product before sterilisation and what level of contamination is acceptable
what level of contamination is acceptable
1 in 10^6
what are industry disadvantages of long sterilisation times
drug may degrade
process is time consuming and expensive
what is the process of sterilisation
- destruction- flaming/chemical oxidation (not practical for pharmaceutical products)
- killing/inactivation - used in most methods, may not remove endotoxins
- removing- filtration or centrifugation *not reliable for pharmaceutics)
steam sterilisation is most effective and reliable
what sterilisation processes are in the European Pharmacopeia
steam sterilisation, dry heat, ionising radiation, gaseous sterilisation, filtration
what is moist heat sterilisation
involves steam at 121-134 degrees C
very effective with widespread application
what is moist heat sterilisation used for
dressings, sheets, equipment, containers, aqueous injections, ophthalmic preparations, contaminated waste materials
what is dry heat sterilisation
usually in the 160-180 range
less effective than moist heat sterilisation
what is dry heat sterilisation used for
glasswear, metal surgical instruments, non-aqueous thermostable liquids, thermostable powders
how is radiation used in sterilisation
gamma rays, accelerated electrons, x-rays and uv are used
alternate method for heat sensitive products
mainly for articles in dried state
what articles is radiation used to sterilise
surgical instruments, sutures, plastic syringes, dry pharmaceutical products
what is gaseous sterilisation
uses ethylene oxide or fomaldehyde
only used for heat sensitive items
what products are gaseous sterilisation used for
reusable surgical instruments, medical/diagnostic equipment, surface sterilisation of powders
what is filtration
for sterilisation (0.2 to 0.22 micron) filters removes particulates from gases and liquids
what is the only process that removes microorganisms
filtration
what articles is filtration used for
heat sensitive injections, ophthalmic preparations, biological products, air/gases to supply asceptic areas
what are the limiting factors of sterilisation processes
cost, nature of product and nature of microbial contamination
what is terminal sterilisation
product is sterilised in its final container
what is asceptic processing
using pre-sterilised components to assemble the product, required a clean room
what does the initial shoulder of a survival curve indicate
clumping - so each cell needs to be ‘hit’ before CFU goes from 1 to 0
repair mechanisms in cell - some need to be ‘hit’ twice to result in death
what does a tailing curve graph show
initial exponential phase then flatter
mixed population - different bacteria present, each with different resistance to sterilisation process
protective effects - lysis of cells protects surviving cells
what does an activated survival curve look like
initial hump but then exponential
germination of spores upon heat stimulation
what is a D value
decimal reduction time - time needed to reduce population factor by 10
can be used for heat and radiation, refers to specific temperature or radiation dose
what is a z value
the increase in temperature needed to reduce the D value ten fold
only used in heat sterilisation
what is the equation used to find a D value
D= t2-t1
———–
logN1-logN2
what is the equation used to find a Z value
Z= t2-t1
———–
logD1-logD2
what is the inactivation rate constant
k= 2.303/D
what does Q10 mean
change in k from a 10degree change in temp
what does IF stand for
inactivation factor
measure of total microbial inactivation
what does F0 mean
equivalent time of moist sterilisation at 121degrees with a Z value of 10degrees
what is autoclaving
moist heat with temp >100degreesC only achieved under pressure
organisms killed by temp, time, hydration
what is superheated steam
water in the vapour phase and behaves like a gas
pressure decreases if temp decreases
what is supersaturated steam
in liquid phase and is made up of small droplets of water held in suspension by convection currents
what is dry saturated steam
exists only on the phase boundary
will condense if temp is lowered
what form of steam is suitable for sterilisation
only dry saturated steam
due to release of heat, hydration and penetration
what happens in the release of heat stage of moist heat sterilisation
sensible heat when exchanged, results in change in temperature
latent heat; when exchanged, results in change of physical state- no temp change
what type of heat does supersaturated steam release
sensible heat
what type of heat does superheated steam release
sensible and latent, but only releases sensible heat in absence of condensation
what type of heat does dry saturated steam release
both sensible and latent heat
what is the second step of moist heat sterilisation
hydration
what happens in the hydration stage of moist heat sterilisation
dry saturated steam = moist heat reaction (rapid heat transfer)
superheated steam= dry heat reaction (oxidation, slower heat transfer)
what is the third stage of moist heat sterilisation
penetration
what happens in the penetration stage of moist heat sterilisation
dry saturated steam and condensation causes a local vacuum and rapid penetration of spores
superheated steam and no condensation means no vacuum so no penetration
what are the advantages of a large scale autoclave
they prevent wet steam formation
they have a jacket to assist with heat retention or cooling
they have a boiler and heating coils
they have a separate boiler and autoclave
how do large scale autoclaves work
gravity displacement autoclave
gravity displacement- steam added from top and air removed from bottom
used for lab media, water, pharmaceutical products, waste, non-porous items
not suitable for porous loads as air may become entrapped in packaging or material
how do large scale autoclaves work
porous load autoclave
vacuum assisted
fitted with vacuum pump to remove air before adding steam
used for dressings, wrapped or hollow instruments
what are the stages of autoclaving
air removal and steam admission, heating up/exposure, drying and coolung (porous loads may get damp; drying by vacuum and heat from outer jacket) cooling necessary to shorten the cycle (pumping water through jacket or spray cooling)
what is air ballasting
sterile fluids can be packaged in flexible plastic containers
pressure in airspace above fluid may lead to bursting
to prevent this, some air may be retained to create overpressure in specifically designed autoclaves
to prevent layering of air, such autoclaves may require fan or spraying mechanism to mix air and steam
what are some problems with sterilising
superheated steam- too dry/overheating/excessive pressure reduction/hydration of over-dried fabrics
air in steriliser
what is the effect of air in an autoclave
with steam/air mixtures, total pressure is equal to sum of partial pressures, which are proportional to relative amount of steam and aire present
1. unjacketed bench autoclave, 2. jacketed autoclave, 3. layering
what are the effects of an unjacketed bench autoclave
usually only controlled by pressure
air leads to lower temp
what are the effects of a jacketed autoclave
both pressure and temperature controlled
air can lead to superheating
what is the effect of layering
density of air must be twice as dense as steam
air forms layer in any container in an upright position
air will be cooler
container must be at an angle so air can flow out
what is dry heat sterilisation
inactivation is principally by oxidation
oxidation is less effective than hydrolysis in inactivating bacterial spores - required higher temps for longer
smaller margin for error
what are the requirements for dry heat sterilisation
temp 150-180
30mins-2hrs
higher temps needed for pyrogen destruction
what are the requirements for instruments used in dry heat sterilisation
raise temp quickly
even distribution of heat
maintains temp within 5degrees
what are dry heat steriliser tunnels
heated by infrared irradiation or forced convection in laminal flow tunnels
items sterilised on conveyor belt
shorter processing times
used mainly for glasswear
what is dry heat sterilisation used for
substances that cannot be subject to moisture, materials that are not penetrated by steam, glasswear, metal surgical items
what are some disadvantages of dry heat sterilisation
may take longer, temp must be higher, some objects oxidise at higher temperatures
when is sterilisation by gases used
for temperature sensitive materials; plastics, devices with electronics
what gases are used in gas sterilisation
ethylene oxide, formaldehyde
what are some advantages of gas sterilisation
good alternative for materials sensitive to heat/radiation
some processes work at RT
what are some disadvantages of gas sterilisation
slow, toxic, the gas may be absorbed so require more input
what is ethylene oxide
used for gas sterilisation, colourless liquid, contact toxicity, readily absorbed by fabrics and plastics, highly flammable
what are some advantages of using ethylene oxide
broad spectrum, non-selective, bactericidal, can act at room temp
what are some disadvantages of ethylene oxide
expensive, difficult, potentially toxic, need to be stored safely while gas leaches out and disperses
what is fomaldehyde
used in gas sterilisation, low bp, aqueous solution used as disinfectant, gas obtained by heating with steam to low-temp-steam fomaldehyde, not flammable
how is fomaldehyde used
low temp steam-fomaldehyde
operates with sub-atmospheric pressure steam, air removed and steam admitted to heat load, formaldehyde released with steam flushing after sterilisation
what are some advantages of using fomaldehyde
broad spectrum, non-selective, bactericidal, non-flammable or explosive
what are some disadvantages to using fomaldehyde
toxic, low penetration power, slow acting, may leve residue of polymers, need to store products after sterilisation while gas leaches out
what is the most common source of radiation for sterilisation
gamma rays
how does radiation sterilisation work
mainly targets DNA, direct damage through ionisation or indirect through radiolysis of water
resistance decreases with presence of moisture or dissolved oxygen; most vegetative cells are sensitive to radiation; bacterial spores and viruses are most resistant
what do gamma ray radiation sterilisers look like
held as pellets packed into metal rods
required reinforced concrete building and stored underwater when not in use
what is filtration
sterilisation, removal of bacteria/fungi, includes clarification
only for liquids or gases/air
what is clarification
removal of particulates that would be hazardous if injected
what happens during filtration
cold process so good for heat sensitive products, fast, aseptic, not absolute so some growth may occur
what mechanisms are involved in filtration
sieving, adsorption, trapping in filter matrix #9depth filter), low holding capacity(screen filter)
what is dead end filtration
fast, but filter ‘cake’ builds up and requires cleaning or replacement
what is crossflow filtration
slower but self-cleaning
what materials are used in filtration
polyether sulfone, cellulose acetate, nylon, PTFE
what is the advantage of using polyether sulfone for filtration
wide pH range, low protein absorption
what is the advantage of using cellulose acetate for filtration
thermal stability, low adsorption
what is the advantage of using nylon in filtration
resistant to solvents and alkaline solutions
high, non-specific binding
what is the advantage of using PTFE in filtration
hydrophobic so used for gas/air
useful at high temps
what is breakthrough in filtration
some microbes or particles pass through depth filters
raw materials with low bioburden or multiple filters can be used to overcome this problem
what is growthrough in filtration
bacteria and grow and divide through a filter if left there for too long
limiting time of filtration process can prevent this
what is high intensity light
new method of sterilisation
pulse with 10^5 times intensity of sunlight, UV, used for water or injectables
liquid and container must be UV transarent
what is low temperature plasma
new technology for sterilisation
gas subjected to electrical field, generates ionised molecules
mainly for medical devices as not suitable for liquids or powders
what are some non-viable contaminants
particulates, microbial products
what are some viable contaminants
bacteria, fungi, viruses
what causes spoilage in aspirin mixture
hydrolysis by acinetobacter
what causes spoilage in atropine eye drops
atropine loss by pseudonomas
what causes spoilage in prednisolone tablets
steroid transformed by cladosporium
what are some sources of contamination
air, raw materials, packaging, equipment, premesis, people
what are some common microbes in air
spore formers (bacillus, clostridium), non-spore formers (stapholycoccus, streptococcus), moulds (penicillium), yeasts
what are some common contaminants in water
gram negatives: pseudonomas, e.coli
gram positives: micrococcus, bacillus
what are water storage and distribution systems used for
maintain water at 80degreesC, circulate water at positive pressure, decrease pipeline, include sterilisation system
how is water treated
chemicals, filtration, UV light
what is the presence of microbes in raw materials
high levels of microflora, ensures microbiological quality, consider sterilisation
what is the microbe contents of synthetic raw materials
low levels of microflora
stored at low moisture content; liquids stored at constant temp
how are sterilisation processes validated
ca;ibration of equipment, testing quantity of steam, leak tests, software testing, physical/chemical/biological indicators, documentation
how is the environment controlled in a manufacturing area
air; settle plates and air samplers
surfaces; contact plates, swabbing
what are physical indicators of contamination
heat sterilisation- digital record of temp, taken at coolest part of loaded steriliser
gaseous sterilisation- leak tests, pressure testing
radiation- plastic dosimeter that darkens in proportion to radiation
filtration- bubble point pressure test
what are chemical indicators for sterilisation
undergo melting or changing colour
does not correspond to microbiological sterility
what are Browne’s tubes
acid is produced upon heating, indicator changes colour, speed of reaction determined by colour, different types for dry/moist heat sterilisation
what is an Attest biological indicator
outer tube, sealed glass ampoule with broth and pH indicator, spore strip inside
after sterilisation, inner ampoule is crushed and tube incubated at 55degreesC
what is a sterility test
only recognises organisms that grow under conditions of test, sample size is restricted, sampling itself may introduce contamination, all samples in batch must be treated similarly
what does a sterility test indicate
passing sterility test does not mean batch is sterile, sterility test indicates gross failure, used as additional check, not standalone
what is parametric release
system of release that gives the assurance that product is of the intended quality, based on process data collected during manufacturing instead of sterility testing
all parameters must be accurately controlled and measured
parametric release is only applied to products that are technically sterilised in their final container
what validation is used in parametric release
heat distribution and penetration, bioburden, container, cycle lethality studies
reduces pre-sterilisation bioburden testing of each batch
each cycle includes chemical or biological indicators
what conditions are required for pyrogen removal
250degreesC, distillation, ultrafiltration, ion-exchange chromatography, alkali or oxidising agent treatment
what pyrogen testing occurs
rabbit pyrogen testing, limulus amoebocyte lysate test, monocyte activation tests
what happens during rabbit pyrogen testing
three animals are injected and their temperature is recorded over three hours
if individual temp increases of one rabbit then the product fails
what are the disadvantages of rabbit pyrogen testing
not very sensitive, depends on gender/age of rabbit, repeated animal testing can lead to endotoxin tolerance, low reactivity to endotoxin from certain bacteria, some drugs may influence body temp, not quantative
what is limulus amoebocyte lysate
blood cells isolated from horseshoe crabs and cells lysed in water, lysate purified (enzymes inactivated by endotoxins)
if mixed with endotoxins, coagulation is observed
more sensitive than RPT
what are the disadvantages of limulus amoebocyte lysate testing
detects only LPS, some compounds may interfere with clotting system, can only be used if product has p6-8
can now use protein from horseshoe crabs produced by recombinant DNA technology
what is a monocyte activation test
predicts human response to pyrogens, monocytes (from whole blood or cell line) mixed with sample, measures formation of cytokine IL-1beta
