CONTROL OF MICROBIAL GROWTH Flashcards
Sterilisation
Sterilization: The killing or removal of ALL living organisms and viruses from on/in a material.
from surfaces,
from gasses
from liquids (e.g. growth media).
Criterion of death is the irreversible loss of the ability to multiply under ANY circumstances
Disinfection
Disinfection: The elimination of most pathogenic microorganisms from inanimate objects or surfaces (inanimate = not alive).
Disinfectants are usually chemicals but disinfection can also be done by using steam or boiling water etc.
There are different levels of disinfectant (high, intermediate and low).
STERILISATION vs DISINFECTION
Sterilizationdescribes a process that destroys or eliminates ALL forms of microbial life (including bacteria, viruses, fungi, and spores) and is carried out by physical or chemical methods.
Steam under pressure,
Dry heat,
EtO gas,
Hydrogen peroxide gas
plasma,
Liquid chemicals
Disinfectiondescribes a process that eliminates most pathogenic microorganisms, except bacterial spores, on inanimate objects. Objects are usually disinfected by
Liquid chemicals,
Wet pasteurization.
Decontamination
What is contamination?
The action or state of making or being made impure by polluting or poisoning.
“the risk of contamination by dangerous bacteria”
Decontamination: Removal of dangerous substances to make an object or inanimate surface safe to handle.
Sanitisation
Sanitization: The use of agents that reduce, but may not eliminate, microbial numbers to a safe level.
Sanitizing is typically recommended for food contact surfaces as the chemicals are in concentrations that are considered safe.
Sanitisation is essential to avoid food poisoning!
Antiseptics
Antiseptics: Chemical agents that kill or inhibit the growth of microorganisms but are sufficiently non-toxic to be applied to living tissues.
They are (in theory) similar to disinfectants but used at lower concentrations.
Do not confuse antiseptic with aseptic.
Aseptic means the absence of microbial contamination.
Aseptic Technique
Aseptic means the absence of microbial contamination as in
‘aseptic techniques’.
- Handwashing
- Autoclaving
SEPSIS
Sepsis is a life-threatening reaction to a bacterial infection.
It happens when your immune system overreacts to an infection and starts to damage your body’s own tissues and organs.
It is sometimes called septicaemia or blood poisoning
BACTERIA KILLERS - MODE OF ACTION
Agents that destroy or kill bacteria are referred to as bacteriocides.
-cide/cidal suffix:
Fungicide kills fungi.
Virucide deactivates or
destroys viruses.
Algicide kills algae.
Pesticide kills pests (insects).
Agents that inhibit bacterial growth are said to be bacteriostatic.
(Prevent growth but cells may not be killed.)
MICROBIAL DEATH
Bacterial death occurs at a constant, usually, exponential rate.
Killing curves usually plotted as log10 number of survivors vs time.
Factors affecting death rate
Microbial characteristics, growth phase, culture conditions, presence of resting cells.
Environmental factors.
Concentration or intensity of treatment.
Time of exposure.
Number of organisms does not affect rate of death but will affect time of treatment.
Methods to Kill Pathogens
Killing methods
Physical methods
e.g. heat, irradiation, filtration.
Chemical methods
e.g. gases or liquids
MECHANISMS OF KILLING:
Alteration of membrane permeability.
Damage to proteins.
Damage to nucleic acids.
Methods to Kill Pathogens
Thermal sterilisation = Heat
Non-thermal sterilisation
Irradiation
Filtration
Chemical (Gas – liquid)
Metals
1- HEAT STERILISATION
One of the most widespread method used for controlling microbial growth is the application of heat.
For all microorganisms there is a maximum temperature for growth, beyond which viability decreases.
At very high temperatures virtually all macromolecules lose their structure and their ability to function.
In other words: macromolecules are denaturated. This process is called denaturation
DECIMAL REDUCTION TIME – D VALUE
D-value (decimal reduction time) isthe time (in minutes) required to reduce viable bacteria by 90% at a defined temperature.
It is used to calculate the time that is required to achieve sterilisation. For example in the canning industry.
This is the most precise way to characterise heat sterilisation.
DRY HEAT vs MOIST HEAT
Heat can be in the form of ‘dry heat’ or ‘moist heat’ and the nature of the heat is important.
Moist heat has better penetrating power than dry heat and produces a faster reduction in the number of living organisms at a given temperature.
For comparison:
sterilisation with dry heat at 160-170°C takes 1-2 hrs
sterilisation with moist heat at 121°C takes 15 minutes.
MOIST HEAT
Can be done with water at liquid state or vapour state (steam).
A temperature of > 63°C is sufficient to kill the vegetative cells of most pathogens BUT not spores or thermoduric or thermophilic organisms.
Boiling for 10 min will kill most vegetative cells, fungi and viruses.
Functions by denaturation of proteins by breakage of hydrogen bonds.
Probably kills by denaturing membrane proteins.
Disadvantage: Increased rate of chemical reactions (e.g. in nutrients).
Thermophilic: Bacteria those thrive at 50-110 C growing conditions and this needs for their optimum growth.
Thermoduric: those bacteria which withstand higher temperatures that actually not suitable for their optimal growth
MOIST HEAT + PRESSURE = AUTOCLAVING
Autoclave is a pressure vessel.
Pressure increases the boiling point of water – higher temperatures can be reached!
121°C 15-20 min 15 psi or 103.5
kPa (most common).
132°C 2-3 min 30 psi or 207 kPa.
Kills all organisms including endospores
A special autoclave cycle called ‘vacuum cycle’
used for dry objects such as dressings. The vacuum inhibits condensation of water so that the objects do not get wet
How to monitor efficiency of autoclave?
Physical indicators: pressure and temperature recording devices,
Chemical indicators: indicators that change colour after being exposed to specific temperatures, such as temperature sensitive tape.
Biological indicators: Bacillus stearothermophilus spores are used, due to their resistance to heat.
ENDOSPORES AND HEAT STERILISATION
Under typical autoclave conditions (121°C, 15-20 min., 15 psi):
Endospores require about 4-5 min. for a decimal reduction.
Vegetative cells require only 0.1-0.5 min. at 65°C.
Water content and the concentration of SASPs (small acid soluble proteins) of the endospore determine heat resistance of endospores.
Low water content + high SASP concentration = high heat resistance.
pH is also an important factor. Acid foods such as tomatoes fruits and pickles are much easier to sterilise than neutral pH foods such as corn and beans.
TYNDALLISATION
Tyndallisation is a method for killing endospores without need for very high temperatures. (Not considered totally reliable and not often used today.)
Step 1: Kill vegetative state cells at 80oC (10 mins)
Step 2: Cool and incubate at 30-37oC – trigger germination.
Step 3: Heat at 80oC (10 mins) – kills all germinated cells (now at vegetative state!)
Step 4: Repeat the cycle 2-3 times.
Used for liquids that support endospore germination.
Used for heat sensitive materials (e.g. vitamins, plant seeds).
PASTEURISATION
Pasteurisation is NOT sterilisation.
Pasteurization isthe process of heating, and then rapidly cooling, liquids or food in order to kill microbes that may expedite their spoilage or cause disease.
Objective is to kill pathogens and increase shelf life
Low temperature pasteurisation: 63°C for 30 min (Milk, ham, fermented sausages e.g. salami).
High temperature Short time (HTST) pasteurisation: 72°C for 15 sec. Refrigerated shelf life of approximately two weeks.
Ultra High temperature (UHT) 135°C for 1-2 sec. provides same level of protection, but along with packaging, increases shelf life to 3 months under refrigeration.
DRY HEAT
Dry heat kills microorganism by oxidizing molecules.
Several types of dry heat sterilisation are:
1- Hot air oven
2- Incineration
3- Open flame
Ovens: 180°C for 1 hr or 160°C for 2 hr or 121°C for 16 hr
Used for glassware, surgical instruments, oils and heat resistant powders.
IRRADIATION
Ionising radiation e.g. gamma radiation or high energy beta particles (electron beams) are used for sterilisation of medical devices and treat wastewater.
Often cobalt 60 high-energy photons are used for gamma radiation.
Used on disposable glass and plastic-ware, pharmaceuticals, sutures, syringes and needles etc.
Lead shielding is needed to protect process workers.
Water can be sterilised by ionising radiation. Ionisation of water forms highly active free radicals such as hydroxyl radicals, ·OH and hydride radicals ·H. . These radicals react with proteins, nucleic acids and lipids – and impair their structure/function – and lead to death of pathogens.
RADAPPERTISATION
Radappertisation means exposing food in sealed containers to ionizing radiation.
The dose of radiation is high enough to reduce the number and activity of viable microorganisms to such an extent that very few, if any, are detectable in the treated food.
The shelf life of radappertised foods – if correctly packaged - mainly depends on the service life of the packaging material and its barrier properties.
NON-IONISING RADIATION – UV-LIGHT
Ultraviolet irradiation (UV-A, UV-B and UV-C).
UV-C is the short wavelength UV-radiation and is most efficient to kill microorganisms (UVC, 254 nm, close to the absorption maximum of nucleic acids)
Causes the formation of thymine dimers and hydroxylation of pyrimidines, producing mutations and inactivating DNA.
Damage skin (sunburn) and especially cornea of eye. Excessive exposure can cause skin cancer.
Thymidine dimer formation by UV light
Cyclobutene thymine dimers distort the double helix and block replication and transcription. Can be repaired.
6-4 photoproduct is formed less frequently but tends to be much more mutagenic.
OTHER WAVES AND PRESSURE
1- Microwaves work by heating but care must be taken as most items are unevenly heated.
2- Strong visible light has a bacteriocidal effect by oxidising light sensitive molecules such as riboflavin.
3- Ultrasound (>20,000 hertz or cycles sec-1) works by disrupting the cells.
4- High pressure (>300 atmospheres) Pascalisation of food. Disrupts cells without affecting flavour or consistency. Some controversy regarding effectiveness.
FILTER STERILISATION
Heat-sensitive liquids may be sterilised by filtration.
Filter is a device with pores too small for the passage of microorganisms but large enough to allow the passage of liquids and gases.
Historically selective filtration methods used to define and isolate viruses: 20 nm to 250 nm size range.
There are three main types of filters:
Depth filters
Membrane filter
Nucleopore filter
DEPTH FILTERS
Simplest type of filter.
Composed of fibrous sheets or mats made from random layers of paper, asbestos or borosilicate (glass) fibres.
Very porous, often used as prefilters.
Often used in biosafety applications.
Example: High-efficiency particulate air (HEPA) filters:
HEPA filters remove 0.3 µm particles with >99.97% efficiency.
Used in construction of clean rooms.
MEMBRANE FILTERS
Most common type for liquid sterilisation.
Composed of polymers with high tensile strength such as cellulose acetate, cellulose nitrate and polysulfone.
Can adjust polymerisation conditions to control the size of pores in the filter.
About 80-85% of the surface consists of open pores.
Acts like a high flow-rate sieve.
NUCLEOPORE FILTERS
Nucleopore filters are made from polycarbonate (plastic) films.
These filters contain uniform holes of any desired diameter to allow even a virus to be filtered.
They are used to trapcellswhile removing all other fluids and smaller particles.
Because the filters have a flat surface, the cells are trapped on top of the filter and remain visible, unlike other types of filters where the cells may be trapped inside the filter.
HOW TO GRADE CHEMICAL DISINFECTANTS
High Level Disinfectants destroy all viruses and vegetative microorganisms but not endospores. Some are sterilants used at lower concentrations or for a short time.
Intermediate Level Disinfectants destroy all vegetative cells including mycobacteria and most, but not all, viruses.
Low Level Disinfectants have no activity on mycobacteria, non-enveloped viruses or endospores
CHEMICAL STERILANTS/DISINFECTANTS
1- Gaseous chemical sterilants/disinfectants
Ethylene oxide
2- Liquid chemical sterilants/disinfectants
Hydrogen peroxide (oxidising
agent)
Peroxy and peroxo acids
Chlorine
Iodine
Alcohols
Aldehydes
Phenol & Phenolics
ETHYLENE OXIDE
Ethylene oxide is an alkylating agent for proteins and nucleic acids
Used for heat sensitive materials,
It is highly penetrating.
Formerly widely used (plastics, sutures, textiles, baby talc etc) but now banned in EU due to toxicity (carcinogen).
Explosive in air so used in sealed rooms with freon or nitrogen, controlled humidity, batch mode several hours
HYDROGEN PEROXIDE
Forms highly active superoxide radicals.
Works best against obligate anaerobes (Aerobes have catalase enzyme which can inactivate hydrogen peroxide).
Hydrogen peroxide is increasingly used – its vapourised form is used for room disinfection.
Used in hospitals to disinfect surfaces and it is used in solution alone or in combination with other chemicals as a high-level disinfectant.
PEROXY AND PEROXO ACIDS
Peroxycarboxylic acids and inorganic peroxo acids are strong oxidants and extremely effective disinfectants.
Peroxyformic acid
Peracetic acid
Peroxypropionic acid etc
They break down in water to form hydrogen peroxide (H2O2 )and acetic acid.
They work by oxidation of membrane components.
CHLORINE (HALOGEN)
Usually used as hypochlorite, NaOCl (bleach).
Domestos 10–25% solution of sodium hypochlorite used as 1:10 dilution in water.
Strong oxidizing agent.
Other slow-release forms e.g. chloramine-T breaks down to hypochlorite in water; chlorine dioxide; dichloroisocyanuric acid (used in swimming pools).
IODINE (HALOGEN)
Iodine combines with tyrosine and also oxidises SH groups on cysteines. It oxidises amino/fatty acids, nucleotides, lipids in the cell membrane, and enzymes in the cytosol, ultimately promoting their denaturation and deactivation
Found in emergency survival kits, used both to disinfect wounds and to sanitize surface water for drinking.
Povidone-iodine (PVP-I), also known as iodopovidone, is an antiseptic used for skin disinfection before and after surgery.
ALCOHOLS
High level disinfectants.
Alcohols are approved for use as hospital grade disinfectants.
100% alcohol typically denatures only external membrane proteins.
Most effective when combined with distilled water to facilitate diffusion through the cell membrane.
70% ethanol or isopropanol diluted in water is effective against a wide spectrum of bacteria.
ALDEHYDES
Formaldehyde, glutaraldehyde.
Form crosslinks with functional groups on proteins.
Wide microbiocidal activity as well as being sporicidal and fungicidal.
Used for hospital instruments and also as a preservative.
PHENOL AND PHENOLICS
Generally low-level disinfectants.
Phenolics are active ingredients in some household disinfectants.
Also found in some mouthwashes and in disinfectant soap and handwashes.
Phenol, formerly known as carbolic acid, is probably the oldest known disinfectant. It was first used by Lister (Listerine).
METALS
The oligodynamic effect is a biocidal effect of metals, especially heavy metals, that occurs even in low concentrations.
The metals react with thiol (-SH) or amine (-NH(1,2,3)) groups of proteins
METALS CONTINUED
Silver: metabolism of bacteria is adversely affected by silver ions at concentrations of 0.01–0.1 mg/L.
Silver salts e.g. AgNO3 can be used.
Slow release form silver sulfadiazine used on burn wounds.
Can be incorporated into medical implants and devices such as catheters.
Mercury salts e.g. thiomersal, mercurichrome were used but are very toxic.
Copper, primarily copper or alloys e.g. brass surfaces. Antibacterial properties of copper recognised by ancient civilisations (predating microbe concept).
QUATERNARY AMMONIUM COMPOUNDS
Quaternary ammonium compounds (“quats”) are a large group of related compounds.
Effective low-level disinfectants. (Newer low-alcohol formulations are highly effective broad-spectrum disinfectants)
Quats are biocides that also kill algae and are used as an additive in large-scale industrial water systems to minimize undesired biological growth.
Quats can be found in shampoos, toilet cleaners, hand soap, shaving cream, baby wipes, body wash, sunscreens, moisturizers, disinfectant sprays, liquid fabric softeners, anti-cling dryer sheets, disinfectants.
SURFACTANTS
Surfactants are compounds that lower the surface tension between two liquids, between a gas and a liquid, or between a liquid and a solid. Example: Soaps and detergents.
They mainly work through mechanical removal of microbes through scrubbing.
Soap breaks oily film on skin – emulsification.
Little antimicrobial effect.
However, washing, especially hands, is essential to remove bacteria picked up from the environment e.g. when handling food, infected patients.
PRESERVATIVE
A number of chemicals are added to food to limit microbial growth:
Nitrates and nitrites.
Benzoate.
Proprionate.
Sulfite.
Formaldehyde and phenolics (from food smoking processes).
Sorbic acid.
