2. Physical Methods of Microbial Control
• Heat
• Filtration
• Low Temperatures
• High Pressure
• Desiccation
• Osmotic Pressure
• Radiation
3. A. Heat
- It is one of the most common methods of food
preservation.
- It is also usually used to sterilize laboratory media and
glassware and hospital instruments.
- Appears to kill microorganisms by denaturing their
enzymes.
4. Thermal death point (TDP)
- It is the lowest temperature at which all the
microorganisms in a particular liquid suspension will be
killed in 10 minutes.
Thermal death time (TDT)
- It is the minimal length of time for all bacteria in a
particular liquid culture to be killed at a given temperature.
Decimal reduction time (DRT or D value)
- It is the time, in minutes, in which 90% of a population of
bacteria at a given temperature will be killed .
5. Moist Heat
- It kills microorganisms primarily by the coagulation of
proteins, which is caused by breakage of the hydrogen
bonds that hold the proteins in their three-dimensional
structure.
6. Boiling
- It kills vegetative forms of bacterial pathogens, almost all
viruses, and fungi and their spores within about 10
minutes.
- Endospores and viruses are not killed this easily.
- Not always a reliable sterilization procedure.
7. Autoclaving
- It is the preferred
method of
sterilization, unless
the material to be
sterilized can be
damaged by heat or
moisture.
- Retorts are large
autoclaves
The Relationship Between the Pressure and
Temperature of Steam at Sea level
Pressure (psi in excess of
atmospheric pressure
Temperature (ºC)
0 psi 100
5 psi 110
10 psi 116
15 psi 121
20 psi 126
30 psi 135
8. The Effect of Container Size on Autoclave Sterilization Times
for Liquid Solutions
Container Size Liquid Volume Sterilization Time (min)
Test tube:
18 x 150 nm
10 ml 15
Erlenmeyer flask:
125 ml
95 ml 15
Erlenmeyer flask:
2000 ml
1500 ml 30
Fermentation bottle:
9000 ml
6750 ml 70
9. Several methods that can indicate whether sterilization
has been achieved:
• Heat-sensitive autoclave tape- It has white stripes that
turn black when the appropriate temperature is achieved
during a successful autoclave run.
• Diack tube- It is a glass ampule containing a
temperature-sensitive pellet that melts at the proper
sterilization temperature.
• Biological indicator spore test- It uses either a strip of
paper or liquid suspension of the endospore Geobacillus
stearothermophilus to determine whether the endospores
are killed by the process.
10.
11. Pasteurization
- Developed by Louis Pasteur in
the 1860s.
- It is a heat-treatment process
that destroys pathogenic
microorganisms in certain foods
and beverages.
- The dairy industry routinely uses
a test to determine whether
products have been pasteurized:
the phosphatase test.
12. High-temperature short-time (HTST) pasteurization
- It is applied as the milk flows continuously past a heat exchanger.
- It lowers total bacterial counts, so the milk keeps well under
refrigeration.
Ultra-high-temperature (UHT) treatments
- Despite being left out of the refrigerator, UHT milk sometimes
lasts 6 to 9 months.
- To avoid giving the milk a cooked taste, a UHT system is
used.
13.
14. Equivalent treatments
- As the temperature is increased, much less time is
needed to kill the same number of microbes.
15. Dry Heat Sterilization
- It kills by oxidation effects.
- It kills bacteria, viruses, and fungal spores without
leaving any residue behind.
16. Hot-air sterilization
- Items to be sterilized by this procedure are placed in an
oven.
- Generally, a temperature of about 170°C maintained for
nearly 2 hours ensures sterilization.
Direct flaming
- To effectively sterilize the inoculating loop, you heat the
wire to a red glow.
- A similar principle is used in incineration.
17. B. Filtration
- It is the passage of a liquid or
gas through a screen like
material with pores small enough
to retain microorganisms
- It is used to sterilize heat-
sensitive materials, such as
some culture media, enzymes,
vaccines, and antibiotic
solutions.
18. Membrane filters
- Composed of such substances as cellulose esters or
plastic polymers, have become popular for industrial and
laboratory use.
- These filters are only 0.1mm thick.
- Pores are 0.22 μm and 0.45 μm sizes.
High-efficiency particulate air (HEPA) filters
- Remove almost all microorganisms larger than about 0.3
μm in diameter.
- Filterable viruses are unseen pathogens that pass
through the filters.
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Editor's Notes
Heat- frequently used by microorganisms
Filtration- it is the passage of a liquid or gas through a filter with pores small enough to retain microbes.
Low temperatures: refrigeration and freezing
High pressure- also called pascalization; used to kill bacteria, yeast, molds, parasites, and viruses while maintaining food quality.
Dessication- also known as drying; preserve foods such as raisins, prunes, and jerky
Osmotic Pressure- high concentrations of salt and sugar
Radiation- its effect depend on its wavelength, intensity, and duration.
Heating
It is one of the most common and oldest form of microbial control.
It is used in simple techniques like cooking and canning.
Laboratory media, glassware, and hospital instruments are sterilized by heat.
Heat can kill microbes by altering their membranes and denaturing their enzymes;
The resultant changes to the three dimensional shapes of these proteins inactivate them.
Heat resistance varies among different microbes, these differences can be expressed in terms of thermal death point
Thermal Death Point or TDP
Of a microorganism is the lowest temperature at which all microbes are killed in a 10-minute exposure. Different microorganisms will respond differently to high temperatures with some being more heat tolerant.
A similar parameter, which is the length of time required is the
Thermal Death Time (TDT)
It is the minimal length of time needed to kill all organisms in a sample at a given temperature.
Both TDP and TDT are useful guidelines that indicate the severity of treatments required to kill a given population of bacteria.
The third concept related to bacterial heat resistance is the decimal reduction time.
Decimal reduction time (DRT or D value)
(read slide)
Many different protocols can be used for sterilization in the laboratory or clinic.
These protocols can be broken down into 2 main categories: Dry heat sterilization and moist heat sterilization.
This coagulation of proteins is familiar to anyone who has watched an egg white frying. (expand)
The first type of moist heat “sterilization” is boiling
(read 1st)
Free flowing steam is equivalent to temperature in boiling water.
However, endospores and some viruses are not killed this easily.
Example:
Some hepatitis viruses can survive up to 30 mins of boiling
Some bacterial endospores can resist boiling for more than 20hrs.
However, brief boiling even at high altitudes will kill most pathogens.
Ex: baby bottles
Reliable sterilization with moist heat requires tempratures above that of boiling water.
These high temperatures are most commonly achieved by steam under pressure in an autoclave.
These high temperatures are most commonly achieved by steam under pressure in an autoclave.
Autoclaves
Used to raise temperatures above the boiling point of water to sterilize items such as surgical equipment from vegetative cells, viruses, and especially endospores, which are known to survive boiling- as what I’ve said earlier, without damaging the items.
used to sterilize culture media, instruments, dressings, intravenous equipment, applicators, solutions, syringes, transfusion equipment, and numerous other items that can withstand high temperatures and pressures.
In 1879, Charles Chamberland designed the modern autoclave while working in the laboratory of louis pasteur
Outside laboratory and clinical settings, large autoclaves called retorts allow for moist-heat sterilization on a large scale.
(figure)
The length of exposure depends on the volume and nature of material being sterilized, but it is typically 15 mins or more, with larger volumes requiring longer exposure times to ensure sufficient heat transfer to the materials being sterilized. The steam must directly contact the liquids or dry materials being sterilized, so containers are left loosely wrapped in paper or foil. The key to autoclaving is that the temperature must be high enough to kill endospores to achieve complete sterilization.
Heat-sensitive autoclave tape
It is a tape that has white straps and these turn black when the autoclave is successful in sterilizing materials.
However, it does not provide indication of length of exposure so it cant be used as an indicator of sterility.
Diack tube
- Pellet contained within a glass ampule melts when it reaches proper sterilization temp.
Biological indicator spore test
- A widely used test consists of preparations of specified species of bacterial endospores impregnated into paper strips. After the strips are autoclaved, they can then be aseptically inoculated into culture media. Growth in the culture media means survival of the endospores and therefore inadequate process.
Examples of sterilization indicators.
The strips indicate whether the item has been properly sterilized. The word NOT appears if heating has been inadequate. In the illustration, the indicator that was wrapped with aluminum foil was not sterilized because steam couldn’t penetrate the foil.
Roll of tape. If the autoclave has gotten into a high enough temperature and pressure, it turns black. If it hasn’t, and when you come back, the tape is still white then you know that something is wrong with the autoclave, you need to adjust the settings or use a different autoclave because whatever inside it was not sterilized.
Pasteurization:
-Pasteur used mild heating, which was sufficient to kill the organisms that caused the particular spoilage problem without seriously damaging the taste of the product. The same principle was later applied to milk to produce what we now call pasteurized milk.
It reduce spoilage organisms and pathogens by heating materials for a short time.
It does not sterilize things, this is considered as a method of disinfection because it is targeted at food spoilage organisms and pathogens.
- phosphatase test is routinely used by the dairy industry to determine whether products have been pasteurized.
If the product has been pasteurized, phosphatase will have been inactivated.
Pateurization is similar to an autoclave, there’s heating for a short period of time. However, it’s less time, less intense heat, and does not have the intense pressure also
Figure:
The liquid you want to disinfect, in this case it is milk, it runs trhough pipes that go next to hot water pipes so that heats it up for a certain amount of time and then it is run to cold water pipes to cool it down. Runs in cycle.
The methods used for milk pasteurization balance the temperature and the length of time of treatments.
One method,
HTST pasteurization
Exposes milk to a temperature of 72 C for 15secs, which lowers bacterial numbers while preserving the quality of milk
Milk can also be sterilized by UHT
UHT Treatments
-read
UHT sterilized milk can be stored for a long time in sealed containers without being refrigerated. However, very high temperatures can change its smell and taste, so to avoid this, a UHT system is used.
It is used in which the liquid milk never touches a surface hotter than the milk itself while being heated by steam. The milk falls in a thin film through a chamber of superheated steam and reaches 140°C in less than a second. It is held for 3 secs in a holding tube and then cooled in a vacuum chamber, where steam flashes off. With this process, in less than 5 seconds, the milk temperature rises from 74C to 140C and drop backs to 74C
The heat treatments discussed illustrate the concept of Equivalent treatments.
For example, suppose that the destruction of highly resistant endospores might take 70 minutes at 115°C, whereas, in this hypothetical example, only 7 minutes might be needed at 125°C. Both treatments yield the same result.
A simple analogy is the slow burning of paper in a heated oven, even when the temperature remains below the ignition point of paper.
One of the simplest methods is direct flaming.
Direct flaming
To effectively sterilize the inoculating loop, one must heat the wire to a red glow. The flame of the bunsen burner is employed for a few seconds to sterilize the bacteriology loop before removing a sample for a culture tube and then preparing a smear.
Incineration
It is an effective way to sterilize and dispose contaminated paper cups, bags, and dressings.
- It is the process of burning hazardous materials at temperatures high enough to destroy contaminants.
Another form of dry heat is hot air sterilization.
Another form of dry heat is hot air sterilization.
hot air sterilization
- The longer period and higher temperature (relative to moist heat) are required because the heat in water is more readily transferred to a cool body than is the heat in air. For example, imagine the different effects of immersing your hand in boiling water at 100°C (212°F) and of holding it in a hot-air oven at the same temperature for the same amount of time.
Filtration
- Passage of substances through a screenlike material
A vacuum is created in the receiving flask; air pressure then forces the liquid through the filter
read
Figure:
4 Filter sterilization with a disposable, presterilized plastic unit. The sample is placed into the upper chamber and forced through the membrane filter by a vacuum in the lower chamber. Pores in the membrane filter are smaller than the bacteria, so bacteria are retained on the filter. The sterilized sample can then be decanted from the lower chamber. Similar equipment with removable filter disks is used to count bacteria in samples
High-efficiency particulate air (HEPA) filters
- It is where air is commonly filtered.
- It may be found in vacuum cleaners, heating and air-conditioning systems, and air purifiers.
- HEPA Filters have effective pore sizes of 0.3 micrometer, small enough to capture bacterial cells, endospores, and many viruses, as air passes through these filters, nearly sterilizing the air on the other side of the filter.
- Not small enough for most microbes
In the early days of microbiology, hollow candle-shaped filters of unglazed porcelain were used to filter liquids.
Unseen pathogens that passed through the filters are called Filterable viruses.
Membrane filters
- The pores of membrane filters, for example, 0.22-mm and 0.45-mm sizes, are intended for bacteria.
- Some very flexible bacteria, such as spirochetes, or the wall-less mycoplasma will sometimes pass through such filters, however.
- Filters are available with pores as small as 0.01 mm, a size that will retain viruses and even some large protein molecules
