Name: Calabano, Mark Jasper C.

Section: II- BS Medical Technology

Date: 02/02/2021
 
ACTIVITY SHEET NO. 1
 
1. What other sterilization techniques are used in the laboratory/hospitals? State the
principle involved; indications for used; temperature and time

A mixture of heat, chemicals, irradiation, high pressure and filtration will achieve sterilization,
such as steam under pressure, dry heat, ultraviolet radiation, gas vapor sterilants, chlorine dioxide
gas, etc. For working in a laboratory/ hospitals, successful sterilization techniques are important and
failure of this could lead to serious consequences, it could even cost a life.

As based from sources, various methods of medical sterilization is being utilized in the laboratory/
hospitals. And these are  Plasma Gas Sterilizers, Heat Method, Filtration, Radiation sterilization, and
Gas sterilization.

Plasma Gas Sterilizers

In order to destroy all living microorganisms in medical and dental devices, including
bacteria, spores, viruses and fungi, plasma sterilization uses low-temperature hydrogen peroxide
gas plasma inside a chamber. When vaporized hydrogen peroxide is added to the chamber and the
equipment within the enclosure become sterile. Once the vapor is removed from the chamber, a low
temperature plasma is produced, ensuring complete sterilization for all tools. In this method of
sterilization, the sterilized materials can be handled safely, either for immediate use or storage. The
process operates in the range of 37-44°C and has a cycle time of 75 minutes.  The remnants of this
process are water and oxygen, making operation safe for both medical staff and the environment.
Although plasma sterilization is a more expensive method, its highly effective and ideal for moisture
sensitive medical tools. The biological indicator used with this system is Bacillus atrophaeus spores.

Heat Method

This is the most prevalent sterilization process. The heat is used to destroy the microbes that
are in the material. The heat temperature and heating period influence the degree of sterilization. On
the basis of type of heat used, heat methods are categorized into: Wet Heat/ Steam Sterilization and
Dry heat sterilization.

 Wet Heat/ Steam Sterilization

This is a commonly used process in most laboratories, which is performed in

autoclaves. Steam heated to 121–134 °C under pressure is used by autoclaves. This is a
very successful approach that destroys all pathogens, bacterial spores and
viruses/deactivates them. Autoclaving destroys bacteria by hydrolysis and cellular protein
coagulation, which, in the presence of water, is effectively accomplished by extreme heat.
Out of the steam comes the extreme heat. Pressurized vapor has a high latent heat and it
retains 7 times more heat at the same temperature at 100 ° C than water. In general, except
for the fact that almost all the air is removed from the autoclave before the heating process
begins, autoclaves can be compared to a conventional pressure cooker used for cooking. As
for example in autoclave, to be effective, the autoclave must reach and maintain a
temperature of 121° C to 124 °C for at least 30 minutes by using saturated steam under at
 least 15 psi of pressure. The temperature should be used to control and monitor the process;
the pressure is mainly used to obtain the required steam temperature. Alternative conditions,
with different combinations of time and temperature:

Temperature (°C) kPa Minimum sterilization time

(min.)
126-129 250 (~2.5 atm) 10
134-138 300 (~3.0 atm) 5

Aqueous solutions in glass containers usually reach thermal equilibrium within 10 minutes for
volumes up to 100 mL and 20 minutes for volumes up to 1000 mL.

Porous loads, such as surgical dressings and related products, should be processed in an
apparatus that ensures steam penetration. Most dressings are adequately sterilized by
maintaining them at a temperature of 134 - 138 °C for 5 minutes.

In certain cases, glass, porcelain, or metal articles are sterilized at 121 - 124 °C for 20
minutes.

Fats and oils may be sterilized at 121 °C for 2 hours but, whenever possible, should be
sterilized by dry heat. In certain cases (e.g. thermolabile substances), sterilization may be
carried out at temperatures below 121 °C, provided that the chosen combination of time and
temperature has been validated. Lower temperatures offer a different level of sterilization; if
this is evaluated in combination with the known microbial burden of the material before
sterilization, the lower temperatures may be satisfactory. Specific conditions of temperature
and time for certain preparations are stated in individual monographs. The bioindicator strain
proposed for validation of this sterilization process is: spores of Bacillus stearothermophilus
(e.g. ATCC 7953 or CIP 52.81) for which the D-value (i.e. 90% reduction of the microbial
population) is 1.5-2 minutes at 121 °C, using about 106 spores per indicator.

 Dry Heat Sterilization

In dry-heat processes, the primary lethal process is considered to be oxidation of cell

constituents. Dry-heat sterilization requires a higher temperature than moist heat and a
longer exposure time. The method is, therefore, more convenient for heat-stable, non-
aqueous materials that cannot be sterilized by steam because of its deleterious effects or
failure to penetrate. Such materials include glassware, powders, oils, and some oil-based
injectables. Preparations to be sterilized by dry heat are filled in units that are either sealed
or temporarily closed for sterilization. The entire content of each container is maintained in
the oven for the time and at the temperature given in the table below. Other conditions may
be necessary for different preparations to ensure the effective elimination of all undesirable
microorganisms.

Temperature (°C) Minimum sterilization time (min.)

160 180
170 60
180 30

Specific conditions of temperature and time for certain preparations are should be followed.
The oven should normally be equipped with a forced air system to ensure even distribution
of heat throughout all the materials processed. This should be controlled by monitoring the
 temperature. Containers that have been temporarily closed during the sterilization procedure
are sealed after sterilization using aseptic techniques to prevent microbial recontamination.
The bioindicator strain proposed for validation of the sterilization process is: spores of
Bacillus subtilis (e.g. var. niger ATCC 9372 or CIP 77.18) for which the D-value is 5-10
minutes at 160 °C using about 106 spores per indicator.

Filtration

It is the quickest way to sterilize solutions without heating. This method involves filtering with
a pore size that is too small for microbes to pass through. Generally filters with a pore diameter of
0.2 um are used for the removal of bacteria. Membrane filters are more commonly used filters over
sintered or seitz or candle filters. It may be noted that viruses and phage are much smaller than
bacteria, so the filtration method is not applicable if these are the prime concern. The effectiveness
of the filtration method must be validated if larger pore sizes are employed. To confirm the integrity
of filters, both before and after filtration, a bubble point or similar test should be used, in accordance
with the filter manufacturer's instructions. This test employs a prescribed pressure to force air
bubbles through the intact membrane previously wetted with the product, with water, or with a
hydrocarbon liquid.

Radiation sterilization

This method involves exposing the packed materials to radiation (UV, X-rays, gamma rays)
for sterilization. The main difference between different radiation types is their penetration and hence
their effectiveness. UV rays have low penetration and thus are less effective, but it is relatively safe
and can be used for small area sterilization. X-rays and gamma rays have far more penetrating
power and thus are more effective for sterilization on a large scale. It is, however,  more dangerous
and thus needs special attention. UV irradiation is routinely used to sterilize the interiors of biological
safety cabinets between uses. Using 100-200 nm UV light and 30 minute exposure time is
recommended for UV sterilization.  X-rays are used for sterilizing large packages and pallet loads of
medical devices. Gamma radiation is commonly used for sterilization of disposable medical
equipment, such as syringes, needles, cannulas and IV sets, and food. Gamma sterilization is a
“cold” sterilization technique, where temperature is not a key parameter. Temperature may increase
slightly in the product due to ionization, but gamma sterilization may be effective at ambient,
refrigerated, or even frozen conditions. The key parameter is the dose received by the product. The
dose is dependent on the presentation to the source and the time exposed to the gamma ray source.

Gas sterilization

The active agent of the gas sterilization process can be ethylene oxide or another highly
volatile substance. The highly flammable and potentially explosive nature of such agents is a
disadvantage unless they are mixed with suitable inert gases to reduce their highly toxic properties
and the possibility of toxic residues remaining in treated materials. The whole process is difficult to
control and should only be considered if no other sterilization procedure can be used. It must only be
carried out under the supervision of highly skilled staff. In order for the sterilization to be effective,
four significant parameters should be meet: gas concentration (450 to 1200 mg/l); temperature (37 to
63°C); relative humidity (40 to 80%)(water molecules carry ETO to reactive sites); and exposure time
(1 to 6 hours).
Efficiency must be monitored each time using the proposed bioindicator strains: spores of
Bacillus subtilis (e.g. var. niger ATCC 9372 or CIP 77.18) or of Bacillus stearothermophilus, (e.g.
ATCC 7953 or CIP 52.81). The same quantity of spores should be used as for "Heating in an
autoclave" and "Dry-heat sterilization".
 
2. Discuss the mechanisms by which autoclaving can kill bacteria and other infectious
agents.
 

Under the steam sterilization, in autoclave, the high pressure increases the boiling point of
water and thus helps achieve a higher temperature for sterilization. Water usually boils at 100°C
under normal atmospheric pressure (760 mm of Hg); however, the boiling point of water increases if
the pressure is to be increased. Similarly, the high pressure also facilitates the rapid penetration of
heat into deeper parts of the material, and moisture present in the steam. This principle is employed
in an autoclave where the water boils at 121°C at the pressure of 15 psi or 775 mm of Hg. When this
steam comes in contact on the surface, it kills the microbes by giving off latent heat. The condensed
liquid ensures the moist killing of the microbes; the heat that an autoclave delivers via highly-
pressurized steam kills bacteria and other microorganisms by causing the microbes' structural
proteins and enzymes to lose their shape in an irreversible way, denaturing and coagulating them
and making them nonfunctional. Once the sterilization phase is completed (which depends on the
level of contamination of material inside), the pressure is released from the inside of the chamber
through the whistle. The pressure inside the chamber is then restored back to the ambient pressure
while the components inside remain hot for some time.

3. Why is infection control important in clinical microbiology laboratories?

 
We all know that clinical microbiology laboratories play an important role in patient care by
providing the cause of infection and antimicrobial susceptibility data to physicians; since that the
rapid diagnosis of pathogens is important for initiating effective antibiotic administration and
improving the outcomes of treatment. With this matter, in the side of laboratory workers it is crucial
that infection control is always observed. Exposure of laboratory workers to infectious agents in the
clinical microbiology laboratory is considered as an occupational risk for them. So in this regard,
infection control is important in clinical microbiology laboratories in order to maintain a safe
environment for everyone--reducing the risk of the potential spread of disease and to prevent the risk
of nosocomial infections for the laboratory workers/ health workers. Through infection control, the
danger of transmission of microorganism from one individual to another, from laboratory worker to
another hospital personnel, and hospital personnel to the patient or to the community will be
eliminated. Hence, through infection control, the spread of infections or a potential outbreak of
infections in whole microbiology laboratories/ healthcare settings to the community could be
prevented, ensuring the safety of everyone, whether among patients, from patients to staff, from staff
to patients, or among staff. 

4. Discuss the difference between disinfection and sterilization. Enumerate other ways by
which disinfection is done in the clinical laboratory?
 
Sterilization is a process mainly used to completely kill all forms of microorganisms and its
spores. This process is carried out to maintain a sterile environment. It is usually done through
combinations of heat, irradiation, filtration, high pressure etc.. While disinfection is to destroy or
irreversibly inactivate bacteria, viruses and fungi but not necessarily the spores on inanimate object.
In sterilization, the medium is made completely free from all microbes. On the other hand, in
disinfection, the number of harmful microbes is minimized to a negligible level. In such cases as
wounds, wounds cannot be sterilized – as it may kill surrounding healthy cells, but wounds could be
disinfected – with agents such as hydrogen peroxide or rubbing alcohol. Sterilization completely rids
microbes from the surface whereas disinfection only reduces the effect of microbes. In sterilization,
high temperatures, steam, radiation, filtration are the techniques being utilized. While in disinfection,
phenol, alcohol, chlorine, iodine (disinfecting agents) are used. Sterilization could result into an
extreme cleanliness. On the other hand, only adequate cleanliness is done through disinfection.
Sterilization is primarily used during surgery by autoclaving (steam sterilization). While disinfection is
used in daily life. In a clinical laboratory setting, chemical germicides are used for the surface
disinfection. These germicides are classified as high-level, intermediate, or low-level disinfectants
based on their activity and strength.  The most commonly recommended disinfectant for lab surfaces
is ethanol (70%, vol/vol), and a 10-percent solution of sodium hypochlorite (or bleach), which is
considered an intermediate-strength chemical germicide. Intermediate-level disinfection will
eliminate most bacteria (including Mycobacterium tuberculosis) and all fungi, and it inactivates
viruses. Traditionally, labs made a 1:10 solution, as most bleach products were sold in the past at a
5.25 percent concentration. Since many available commercial bleach concentrations now are at 8.25
percent, labs should make a 1:16 solution for disinfection purposes if they are using that bleach.
Laboratory workers should be sure to check the label of the bleach product purchased before
making dilutions. Sodium hypochlorite is a corrosive chemical, and even in its diluted form it can
cause damage to some surfaces over time. In order to prevent this, the disinfected surface should
be rinsed off with sterile water or 70 percent ethanol (another intermediate chemical germicide). In
the Anatomical Pathology section of the laboratory, formaldehyde is sometimes used as a
disinfectant. Depending on its concentration, formaldehyde can be considered a high-level
disinfectant. The chemical, a known carcinogen, is placed inside a cryostat, and fumes generated
buy a heat source are used to decontaminate the inside of the apparatus. However, the
effectiveness of the disinfection is unclear, and the dangers of using a carcinogen mark this method
as less than satisfactory. Check with the cryostat manufacturer for cleaning guidelines, but many
instruments can be disinfected using 70 percent alcohol.
 

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