PHYSICAL METHODS OF STERILIZATION

I. Heat methods

Heat methods are used for sterilization of the instruments or the products which can withstand higher temperatures. Heat can be used to sterilize thick materials, as it can penetrate thick materials easily where chemicals cannot reach. Advantage of heat method is due to its efficiency and faster sterilization, while it cannot be used for sterilization of heat sensitive and heat labile articles and preparations.

There are various terms related to the heat method of sterilization:

• Thermal death point: The minimum temperature at which all the bacteria present in 24 hrs old broth are killed in 10 mins., when kept at a neutral pH.
• Thermal death time: It is the time required to kill the microbes present in any broth at a specific temperature.
• Decimal reduction time (DRT or D value): It is the time required to kill 90% of the population of bacteria at a given temperature. The temperature in this is indicated by the subscript (D₅₀^o_C)

a) Dry heat

Dry heat is used for the sterilization of metal objects. However, oils and powders can only be sterilized by the dry heat method.

Time required for the sterilization of the objects under different temperatures according to BP 1988  can be summarized in the following table:

*or longer, depending on the volume.

Applications:

• Sterilization of inoculating loop
• Sterilization of mouth of culture tubes
• Metal instruments
• Some glassware

b) Moist heat

Moist heat or steam has more penetrating power than the dry heat. Most of the microbes can be killed at the boiling temperature of water, but vegetative cells and endospores requires a higher temperature to be killed. For this purpose, 2% sodium carbonate is added to elevate the boiling temperature of water or boiling under pressure technique is used. Autoclaves are used most widely for the latter technique.

In the autoclave, according to BP, the pressure of 15 lb/in² above the atmospheric pressure should be maintained for minimum of 15 mins. in order to each the temperature up to 121^oC. At this temperature, vegetative cells, endospores can be easily killed and nucleic acid of the viruses can also be disrupted. The packing of articles in a crowded manner in the autoclave can increase the duration of autoclave up to 1 hour.

Two rules should be applied during autoclaving:

i. Articles in the autoclave should be placed in such a manner that steam can easily penetrate the articles.

ii. Evacuation of the chamber in the autoclave, so that steam can be filled in it effectively.

c) Pasteurization

This technique was invented by Pasteur. It can kill the pathogens that are present in the milk like, Mycobacterium and Salmonella. Two methods are used for pasteurization:

i. Flash method: Pasteurization of milk by heating it to 71.6^oC for 15 seconds.

ii. Holding method: Pasteurization of milk by heating it to 62.9^oC for 30 minutes.

Ultrahigh temperature processing: in this process, the temperature of the milk is elevated from 74^oC to 140^OC and then dropping back to 74^oC in less than 5 seconds. This helps in the prevention of developing a cooked flavor in the milk.

II. Cold

Cold does not kills many microorganisms but it instead retards the growth of the microorganisms by limiting the important enzyme-reactions. Cold methods are commonly used for storage and preservation purposes. However, on keeping the articles or preparations in low temperatures for long can kill parasites present in them.

Advantage of cold treatment is it can be used for the preservation of preparations and food for longer durations, while it cannot have power to kill the microbes and spores present in the preparations.

a) Refrigeration

This technique includes preserving fresh foods from spoiling by keeping them at 5^oC. it can only preserve food for a few days as some microbes like Clostridium botulinum can still grow at this temperature and can spoil food after few days.

b) Freezing

Freezing the food at -20^oC prevents the food from spoilage by retarding the growth of microorganisms. It is not a method for sterilization. Repeated freezing and thawing makes the food susceptible to microorganism contamination. Freezing is also used for the preservation of the microorganism at temperature of -78^oC or in liquid nitrogen at -180 ^oC

c) Freeze drying or lyophilization

It is the drying of materials directly from the frozen states. The technique found useful in producing various coffee and in the preservation of cultures of microorganisms. Microorganisms can be kept alive for years through this technique.

III. Radiation

Radiations are very effective methods for sterilization. It can penetrate the objects deeply and can sterilize it. Disadvantages of radiations includes its complexity and need of specialized people for using these techniques. It can be fatal if not used properly.

a) Ultraviolet radiations or UV radiations:

It consists of light of a wavelength from 40 nm to 390 nm. The most effective wavelength for killing microbes is 200 nm. UV rays can kill microbes by altering the purine and pyrimidine molecules of the DNA and RNA. It is very effective for killing viruses, however, endospores are resistant to UV rays.

UV rays found useful in the sterilization of sewage water, empty rooms and laundry cloths.

b) Ionizing radiations:

Radiation such as X-rays and Gamma rays can dislodge the electrons form the atoms and thus, create mutation by altering the DNA and RNA of microbes. It damages the DNA and produces peroxides which further leads to death of microorganism.  Humans can withstand up to 50 rads of radiations. This technique found useful in sterilization of sea foods, poultry meats, fruits, milk, etc. Gamma rays can be used for sterilization of tissue allocrafts and medical devices.

c) Microwave radiations:

This is effective in sterilizing the materials which are containing at least some amount of water. Metals, glasses and paper-like things cannot be sterilized from this. Endospores are also resistant to microwaves.

d) Visible light:

Visible light produces bactericidal effects by oxidizing the light-sensitive materials such as riboflavins and porphyrins. Visible light s used with a combination of dyes such as eosin and methylene blue to get rid of viruses and bacteria

e) Sonic and ultrasonic waves:

These waves kill bacteria through the process of cavitation. Disruption of cells with the help of sound waves is called sonication. They are not practical means for sterilization.

IV. Filtration

Filtration is used for sterilizing the materials which are sensitive to heat. It uses filters with very small pores which can allow the media to pass through it but blocks the bacteria. Membrane filters made up of nitrocellulose with a specific pore size of 25 microns to less than 0.025 microns is widely used. This method is effective and inexpensive, however, it can allow various viruses and mycoplasma to enter into media. HEPA filters are also used for sterilizing the air entering to the room where microbial contamination control is needed, such as in laboratories and food processing industries. Syringe filters of 0.45 microns are used for general filtration and particle removal application. 0.2-0.22 microns can be used as solution sterilization.

V. Osmotic pressure

Adding high concentration of salt or sugar to a solution creates a hyperosmotic solution that can draws out the water from the microorganism cells which kills the microbes through plasmolysis. This technique is used for preserving jellies, jams, syrups, pickles, etc.

VI. Desiccation

Microbes required some amount of water in order to grow properly. Desiccation in the removal of water from the media to make it completely dry. This limits the growth of microorganisms but as soon as the water is made available again, they started to multiply. Microbes can sustain viability for years under desiccative conditions. It can be achieved by cryogenic condensation, absorption into glycols and absortion onto silica gel.

MCQs

1. Heat methods can be used for sterilization of?

a. Equipments made of plastic

b. Equipments that can withstand high temperature

c. Heat methods can be used for sterilizing every pharmaceutical preparation

d. Heat method is not for the purpose of sterilization

2. Oils and powders can be sterilized by using which method?

a. Dry heat

b. Moist heat

c. Filtration

d. Desiccation

3. According to BP, the pressure inside autoclave to reach a temperature of 121^oC must be?

a. 5 lb/in² above atmospheric pressure

b. 10 lb/in² above atmospheric pressure

c. 15 lb/in² above atmospheric pressure

d. 0 lb/in² above atmospheric pressure

4. Radiations which can be used for sterilization includes?

a. Ioninzing radiations

b. X-ray

c. Gamma rays

d. All of the above

5. The correct sequence for true/false from the following statements can be?

• The time requires to kill all the microbes present in any broth at a specific temperature is called thermal death point.
• The time required to kill 90% of the population of the bacteria at given temperature is called as D value.
• Oils and powders can only be sterilized using dry heat method
• Clostridium botulinum can grow in the refrigerated food

a. FTTT

b. TFFT

c. TFFF

d. TTTF

6. Which of the following statements are INCORRECT related with the time required for complete sterilization at given temperature through dry heat method?

I. 171^oC :  30 minutes

II. 160^oC :   2 hours

III. 121^oC :   16 hours

a. II , III

b. I, III

c. I

d. III

7. Match the following methods of pasteurization with correct temperature and time-

a. A-III-I ; B-IV-ii

b. A-IV-iv ; B-I-i

c. A-IV-iii ; B-I-ii

d. A-I-iv ; B-II-iv

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ANSWERS

1-b

2-a

3-c

4-d

5-a

6-c

7-c

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