How to control bacterial growth?
We usually control bacterial growth by sterilization and disinfection.
Sterilization: the process by which all viable organisms including spores are removed or killed.
Disinfection: the process by which most and not all viable organisms are removed or killed.
SterilizationA. Physical methods :• Dry heat.• Moist heat.• Filtration.• Radiation.
B. Chemical methods:• Chemicals.• Gases.
The efficacy of these methods depends on • Processing time.• Material being treated.• Presence of organic material.
A. Physical methodsI. Dry heat:Mechanism:
Mainly by oxidation; Dry heat coagulates the proteins in any organism, causes oxidative free radical damage, which causes drying of cells and can even burn them to ashes, as in incineration.
A- Flaming to red hot: Used for metal instruments such as loops, dental
mirrors and scalpels.First put the metal in alcohol and then burn it off.
B. Hot air oven:
oThey are electrical devices used in sterilization.
o There is a digitally controlled thermostat
controlling the temperature.
o Their double walled insulation keeps the heat in .
o There is also an air filled space in between to aid
insulation.
o An air circulating fan helps in uniform distribution
of the heat.
oThey are fitted with the adjustable wire mesh
plated trays or aluminum trays.
oTemperature sensitive tapes or other devices like
those utilizing bacterial spores can be used to
work as controls, to test for the efficacy of the
device in every cycle.
disadvantagesAs they use dry heat instead of moist heat, some
organisms may not be killed by them every time.
Advantages
They do not require water and there is not much
pressure build up within the oven, unlike an
autoclave, making them safer to work with.
Uses:These are widely used to sterilize articles that can
withstand high temperatures and not get burnt, like
glassware.
The standard settings for a hot air oven are:
1.5 to 2 hours at 160 °C
....plus the time required to preheat the chamber before
beginning the sterilization cycle.
C. Incineration:
complete burning of material in an incinerator.
This is used for the safe disposal of items such as
contaminated dressings and lab cultures.
II. Moist heat:Moist heat, as the name indicates, utilizes hot air that is
heavily loaded with water vapour and where this
moisture plays the most important role in the process of
sterilization. It is more rapid and efficient than dry heat.
Mechanism:
Moist heat coagulates the proteins in any organism and
this is aided by the water vapour that has a very high
penetrating property, leading to their death.
I. Autoclave
Autoclaves commonly use steam heated to 121 °C or 134
°C. To achieve sterility, a holding time of at least 15
minutes at 121 °C or 3 minutes at 134 °C is required. The
pressure reached is 15 pounds /square inch.
Additional sterilizing time is usually required for liquids
and instruments packed in layers of cloth, as they may
take longer to reach the required temperature.
Proper autoclave treatment will inactivate all fungi,
bacteria, viruses and also bacterial spores, which can
be quite resistant.
To ensure the autoclaving process was able to cause
sterilization, most autoclaves have meters and charts
that record or display information such as
temperature and pressure as a function of time.
Biological indicators can also be used to confirm
autoclave performance. Most contain spores of the
heat resistant microbe Bacillus stearothermophilus,
among the toughest organisms for an autoclave to
destroy.
There also chemical indicator strips that contains a
compound that changes color upon an efficient
sterilization cycle.
For effective sterilization, steam needs to penetrate
the autoclave load uniformly, so an autoclave must
not be overcrowded, and the lids of bottles and
containers must be covered.
During the initial heating of the chamber, residual air
must be removed.
II. Boiling water: For 10 to 30 min
It is still used in some clinical situations but they don’t
destroy spores.
III. Pasteurization:Used to destroy vegetative cells in milk
(Mycobacteria,Salmonella, Brucella), not the spores. The
main principal involves heating followed by sudden
cooling.
It is done by one of three methods:
Holder method - peaks at 63°C for 30 minutes .
Flash method - peaks at 72°C for 20 seconds.
Ultra high temperature (UHT) method - peaks at
125°C for a few seconds
III. Filtration:• Clear liquids that would be damaged by heat,
irradiation or chemical sterilization can be sterilized
by mechanical filtration.
• This method is commonly used for sensitive
pharmaceuticals and protein solutions in biological
research.
• A filter with pore size 0.2 µm will effectively remove
bacteria.
• If viruses must also be removed, a much smaller
pore size is needed.
• Vacuum pump is usually used since solutions
filter slowly through membranes with smaller
pore diameters.
• To ensure the best results, pharmaceutical sterile
filtration is performed in a room with highly filtered
air or in a laminar flow cabinet.
IV. Radiation sterilizationMany bacteria are readily killed by radiation.
a.Gamma rays
- They are very penetrating and are commonly used
for sterilization of disposable medical equipment, such
as syringes, needles, cannulas and IV sets.
-Gamma radiation requires storage of a
radioisotope (usually Cobalt-60), which continuously
emits gamma rays.
- This process does not result in rise in temp; but the
time required for sterilization is long (48hrs).
- Only used within hospitals due to its hazardous
effects.
b. X-rays
They are less penetrating than gamma rays and tend
to require longer exposure times, but require less
shielding.
C. Ultraviolet light irradiation
- It is useful only for sterilization of surfaces and
some transparent objects.
- Many objects that are transparent to visible light
absorb UV.
- UV irradiation is routinely used to sterilize the
interiors of biological safety cabinets
B. CHEMICAL METHODS
Advantages:
1. Chemicals are also used for sterilization of heat-
sensitive materials such as biological materials, fiber
optics, electronics, and many plastics where heat
sterilization can’t be used.
2. The chemicals used as sterilants are designed to
destroy a wide range of pathogens.
Disadvantages: We must be sure that article to be sterilized is
chemically compatible with the sterilant being used.
I. GASES
a. Ethylene Oxide (EO) Ethylene oxide gas is commonly used to sterilize objects sensitive to temperatures
greater than 60 °C such as plastics, optics and electrics.
Advantages:
1. penetrates well, moving through paper, cloth, and some plastic films
2. highly effective
3. Ethylene oxide sterilizers are used to process sensitive instruments which cannot be
adequately sterilized by other methods
4. It can kill all known viruses, bacteria and fungi, including bacterial spores and is
satisfactory for most medical materials, even with repeated use.
5.
Disadvantages:
1. highly flammable
2. requires a longer time to sterilize than any heat treatment
3. The process also requires a period of post-sterilization
aeration to remove toxic residues.
Spore testing
Bacillus atrophaeus a very resistant organism, is used as a
rapid biological indicator for EO sterilizers. If sterilization
fails, incubation at 37 °C causes a fluorescent change within
four hours. Fluorescence is emitted if a particular (EO
resistant) enzyme is present, which means that spores are
still active.
b. OzoneOzone is used in industrial settings to sterilize water and
air, as well as a disinfectant for surfaces.
Advantages:
1. ozone is a very efficient sterilant because of its strong
oxidizing properties and it is capable of destroying a
wide range of pathogens.
2. Able to oxidize most organic matter.
II.LIQUIDS
a.Glutaraldehyde and Formaldehyde
They are accepted liquid sterilizing agents, provided
that the immersion time is sufficiently long.
To kill all spores in a clear liquid can take up to 12
hours with glutaraldehyde and even longer with
formaldehyde.
The presence of solid particles may lengthen the
required period or render the treatment
ineffective.
Glutaraldehyde and formaldehyde are volatile,
and toxic by both skin contact and inhalation.
Many vaccines, such as the original
Salk polio vaccine, are sterilized with
formaldehyde.
b. Hydrogen PeroxideHydrogen peroxide is another chemical sterilizing agent. It is
relatively non-toxic when diluted to low concentrations
(3 % ).
Advantages:
1. Hydrogen peroxide is strong oxidant and these oxidizing
properties allow it to destroy a wide range of pathogens.
2.It is used to sterilize heat or temperature sensitive articles
such as rigid endoscopes.
3.Short cycle time compared to ethylene oxide but its
penetrating ability is not as good as ethylene oxide.
Disadvantages:
1. Hydrogen peroxide is primary irritant
2. the contact of the liquid solution with skin will cause
bleaching or ulceration depending on the concentration
and contact time.
3. The vapor is also hazardous with the target organs
being the eyes and respiratory system.
Disinfection:It is usually by chemical agents. Disinfectants are generally not
intended to achieve sterilization. Most reduce the microbial
populations to safe levels or remove pathogens from objects.
An ideal disinfectant or antiseptic kills microorganisms in the
shortest possible time without damaging the material treated.
Disinfectants are antimicrobial agents that are applied to non-
living objects to destroy microorganisms while antiseptics
destroy microorganisms on living tissue.
The choice of disinfectant for different hospital applications
is made on the basis of their antimicrobial activity,
inactivation by organic materials and their toxicity.
Examples of commonly used disinfectants:
1.Ethanol:• used at concentrations between 70 – 95%
• Denature proteins; disrupt membranes
• Kills vegetative cells of bacteria & fungi but not spores.
• Used in disinfecting surfaces; thermometers; “ethanol-
flaming” technique used to sterilize glass plate spreaders or
dissecting instruments at the lab bench.
2. phenols:
• Aromatic organic compounds with attached -OH
• Denature protein & disrupt membranes
• Commonly used as disinfectant and is effective in presence
of organic matter.
• It has disagreeable odor & is skin irritant.
Evaluation methods of disinfectants:
To evaluate an antiseptic or disinfectant, the phenol coefficient
test is used.
Phenol coefficient test (Rideal Walker method)
It is a measure of the bactericidal activity of a chemical
compound in relation to phenol.
Phenol Coefficient Test is done by measuring the concentration at
which a chemical is equal in effectiveness to phenol.
a. If a chemical is equal in effectiveness to phenol at the same
concentration, its phenol coefficient is 1.
b. If the concentration of the chemical to be tested must be
twice that of phenol, its phenol coefficient is 1/2.
c. If the solution is less concentrated than the phenol standard,
its phenol coefficient is greater than 1.
Phenol Coefficient = Conc. of chemical Conc. of phenol
Phenol Coefficient Test A series of dilutions of phenol and the experimental
disinfectant are inoculated with Salmonella typhi
Samples are removed at 2.5 min intervals and
inoculated into fresh broth.
The cultures are incubated at 37°C for 2 days
The highest dilution that kills the bacteria after a
7.5min exposure, but not after 5 min, is used to
calculate the phenol coefficient
The reciprocal of the maximum effective dilution for the
test disinfectant is divided by the reciprocal of the
maximum effective dilution for phenol to get the phenol
coefficient
For example:
Suppose that, on the test with Salmonella typhi
The maximum effective dilution for phenol is 1/90
The maximum effective dilution for “Disinfectant X” is
1/450
The phenol coefficient for “Disinfectant X” with
S. typhi = 450/90 = 5
EVALUATION OF THE EFFICACY OF AN ANTISEPTIC BY RIDEAL WALKER TEST
Materials: Salmonella typhi suspension 5ml of phenol (1:95) 5 ml of different dilutions of the
antiseptic under test.(1:50,1:100,1:150……………….
4 sterile broth tubes for each dilution.
Loop 1sterile pipette 1 ml
Methods: Add 0.2 ml of S.typhi suspension to each of the dilutions of the
phenol and antiseptic leaving 30 seconds intervals between each
tube.
At 2.5 minutes intervals inoculate one loopful of each of the
previous dilution tubes into a sterile broth tubes.
Shake all the tubes and incubate at 37oC for 2 days.
Record the results as – if there is no growth and + if growth occurs.
Calculate the phenol coefficient of the given antiseptic.
Results:
Antiseptic1:50 1:100 1:150 1:200
phenol1:95
Time of Sampling(min)
+ + + _ + 2.5
+ + _ _ + 5
+ _ _ _ _ 7.5
_ _ _ _ _ 10
Disadvantages:1. Absence of organic matter. (Chick
Martin Test: yeast extract)2. Test time is so short: 10 min
(Chick Martin: 30min)3. It is only applicable for phenolic
compounds4. It uses Salmonella typhi as the
test organism only.