Physical methods of Microbial Growth Control

Microbial control by Physical Methods & Agents

  A variety of physical agents and methods can be used for microbial control. Of these, high temperature, radiation and filtration are the agents of choice for achieving sterilization of the objects and environment in laboratories, pharmaceuticals, fermentation industries etc.

Microbial control by use of high temperature

Use of high temperature is one of the most commonly used method for control of microorganisms. It is used as a sterilizing agent obtain total destruction of micro-organisms from the materials. 

  High temperature causes inhibition of microbtal growth by its ability to denature cellular proteins and enzymes as well as other thermo labile substances, Their inactivation kills the organisms.

  The killing effect of heat and sensitivity of organisms to high temperature can be expressed by determination of thermal death point. thermal death time and decimal reduction rate.

Thermal death point -

  The temperature at which all organisms from the substance are killed within 10 minutes is called thermal death point. Resistant organisms possess higher TDP.

Thermal death time and decimal reduction rate -

  The shortest amount of time required to kill all organisms from a given substance under prescribed set of conditions at a given temperature is called thermal death time.

  TDT also indicates degree of temperature resistance of organisms. The death of organisms at high temperature is logarithmic and reduction in microbial number occurs by a specific factor. This is referred to a decimal reduction rate.

   Theoretically 100 % death of organisms is never possible at a given temperature at any time. There Is always a probability of survival of organisms. This rate of reduction in number of microorganisms is also referred to as D value or (del) factor. The time required for reduction of microbial number by one log value is referred to as D value.

              ∇ = K ln N2/N1
∇ = del factor
K = Constant indicating set of conditions
N1 = Initial number of micro-organisms
N2 = Final number of micro-organisms

The graphical presentation of decimal reduction rate is shown in figure

The graph indicates concept of decimal reduction rate and D value.

Types of heat used

  Heat can be applied for the control of micro-organisms in three manners.
1. Direct heat
2. Dry heat
3. Moist heat

1. Control by direct heat

  Application of direct heat by exposing materials to flames causes incineration of the microorganisms present on the surface of materials. It can be achieved by ......
a). Flaming the material by passing it through burner flame.
b). Exposing the materials to infrared radiation. Infrared radiations Increase the temperature of the surface of materials and cause the effect similar to direct heat.

Limitation of direct heat sterilization :
1. The method causes destruction of microorganisms only from the surface and not those, which occur beneath the surface.

2. Only the materials, which can withstand direct flaming. can be sterilized by this method.

Application of direct heat :
Materials like inoculation loops. slides, scalpel, spatula etc. can be sterilized by this method.

2. Control by dry heat

  Another common mode of application of high temperature for control of microorganisms is use of dry heat or hot air. Dry heat kills microorganisms by causing Irreversible denaturation of cellular proteins by oxidation of proteins.

   Various devices are used for control of micro-organisms from materials based on this principle. Hot air ovens are the most common amongst them. The temperature required for obtaining sterilization of materials by this method is 150°C - 200°C for about two hours.

Limitations of dry heat sterilization :
   Dry heat has low penetrating power as well as less effectivity. Therefore, high temperature is to be used for long time achieve sterilization. Therefore, only those materials, capable of withstanding these conditions can be sterilized by this mode.

Application of dry heat sterilization :
This method of steriltzation applied for sterilization of laboratory glass wares, dry powders or similar such materials.

3. Control by moist heat

  Application of moist heat as sterilizing agent is one of the most common approaches to achieve sterilization. It is achieved by exposing materials to steam.

  Moist heat causes death of microorganisms by denaturing cellular proteins. It causes coagulation of cellular proteins. Compared to dry heat, moist heat has more penetration power and effectivity. Therefore, to achieve sterilization, relatively steam at lower temperatures and application for shorter durations is required.

  Amongst all forms of microorganisms, spores of bacteria are most resistant. They are killed by steam at 121.6°C within 10 to 15 minutes, whereas similar results are obtained by dry heat within 2 hours.

Modes of application of moist heat :

Moist heat sterilization can be achieved in three different modes of treatments.
a). At temperature below 100°C. 
b). At 100 C.
c). At temperature above 100°C.

a). Sterilization at temperature below 100° C -

  This method is used when the materials to be sterilized are thermo labile and cannot withstand higher temperature of treatment. Sterilization by this method involves application of the principle of fractional sterilization or tyndallization.

• Tyndallization -

This method of control was initially evolved by Tyndall. Therefore, It is called tyndallization. It is also known as fractional sterilization or intermittent sterilization due to its mode of application. The method does not involve destruction of all the organisms at a time, but they are killed intermittently or fractionally in different phases.

• Method of tyndallization -

  Tyndallization involves treatment of the material at 70°C - 80°C for about 15 minutes in water bath for three successive days, to achieve total destruction of microorganisms. This treatment achieves sterilization as under.

1). When the material is exposed to 70°C - 80°C for 15 minutes in water bath on the first day, It will kill all vegetative cells from the material but not spores. This is because, spores are heat resistant.

2). Now when the material is incubated for 24 hours after first exposure to heat, the spores will germinate and get converted into vegetative forms. These spores are then killed by exposure of material to 70°C - 80°C again for 15 minutes in water bath.

3). Still, there is a possibility that some spores might not have germinated. They may survive during this second heat treatment. To ensure the destruction of these surviving spores, the material is again incubated at 37°C for 24 hours. 

  During this period, the surviving spores will also germinate to form vegetative cells. They are then killed by again heating the material at 70°C to 80°C for 15 minutes in water bath on the third day. Thus, the method Involves killing of all organisms from the objects by use of relatively low temperature.

• Use of tyndallization -

  Inspissator is the most commonly used instrument for sterilization of materials by tyndallization. The method is used to achieve sterilization of various thermo labile materials, such as egg media, serum and serum containing media etc.

b). Sterilization at of temperature 100°C -

Sterilization at 100°C temperature is achieved by use of boiling water bath or Arnold sterilizer. The method is used for sterilization of materials and objects which resist boiling. Since boiling cannot destroy bacterial spores, principle of tyndallization is employed to achieve total destruction of all forms of micro- organisms.

c). Sterilization at temperature above 100°C -

  Steam under pressure is used to achieve sterilization by moist heat at temperature above 100°C. Temperature of steam is directly proportional to pressure as shown in the table.

Table : Relationship between steam pressure and temperature
Steam pressure
Temperature °C
    0     100.0
5 109.0 
10 115.0 
15  121.6 
 20 126.5 

Steam under pressure, has greater penetration power and effectivity. Hence the death of organisms is rapid at higher steam pressure and temperature. Usually, steam at 15 Ibs/sq in pressure is recommended for sterilization of objects. At this treatment, even spores of bacteria are killed, Autoclave or steam sterilizer is the most commonly used instrument, used in laboratory, working on this principle.

Application of moist heat sterilization :

   Materials, which can withstand high stem pressure and temperature, can only be sterilized by use of this method. The method is suitable for sterilization of routine bacteriological media, glassware, surgical instruments, gauges, clothes ete.

Control by use of low temperature

  Decrease in temperature of incubation results in the decrease in the overall rate in the cellular metabolism and hence the rate of growth. Therefore, low temperature is widely used for the control of microblal activities in the materials, especially for preservation of foods, food products and beverages. 

 Control of organisms, using low temperature can be achieved at
a). refrigeration temperature, i.e. at 4°C to 7°C
b). freezing temperature i.e. at temperature below 0°C.

Control at refrigeration temperature -

  The method is most commonly used for preservation of foods and food products in households and culture preservation. Low temperature causes retardation in microbial activities and exerts bacteriostatic effect.

Control at freezing temperature -

  Exposure of organisms at subzero temperature results in to freezing and formation of ice crystals within the cell. Hence when organisms are expressed to subzero temperature cellular water content gets frozen. This result in -
a). Mechanical damage to cells and some of the organisms are killed.
b). Arrest of cellular metabolism and hence growth of organisms.

   However organisms can survive for long time even the low temperature. Hence, use of freezing temperature achieves microbial control by inhibiting growth rather than killing of organisms.

Microbial Growth Control by Desiccation

  Removal of water is called desiccation. Desiccation of microblal cells and their environment stops microbial growth and activities. This is because Water required for activity of cellular enzymes as well as growth is not available.

   This is one of the most widely used methods for microblal control of food spoilage as well as the preservation of cultures during freeze drying method.

   A number of factors decide the efficiency of microbial control by desiccation. They include:
a). The type of micro-organisms.  
b). The type of substances on which organisms are dried.
c). Physical conditions to which organisms are exposed to after desiccation such as light. temperature, humidity etc.
  Usually, gram-negative bacteria are most musceptible to desiccation.

  They include gram-negative cocci, spirochetes etc. Gram- positive cocci and rods are more resistant to desiccation.

  Spore formers as well as capsulated organisms can withstand conditions of desiccation for long duration. Mycobacteria are one of the classical examples of the organisms which can survive after desiccation for exceptionally long durations. This is because of the layer of myclic acid in their cell wall, which protects cytoplasm from desiccation. Fungal spores, conidia are also resistant to desiccation.

Control by use of osmotic pressure

  The difference in the concentration of solutions across semi permeable membrane causes passage of solvent from lower solute concentration to higher solute concentration tiIl the equilibrium is achieved. This phenomenon is called osmosis. The pressure resulting due to the passage of solvent on the membrane is called osmotic pressure.

  When the cells are exposed to high osmotic conditions (high concentrations), water from cells come out resulting into plasmolysis of cells. This results into desiccation of cells that inhibit metabolic activities, and cause control of microbial growth.

  Similarly, when cells are exposed to low osmotic conditions, water from outside enter the cell causing plasmoptyses. The cells get ruptured as a result of this phenomenon. Thus also helps in achieving microbial control.

   The practical application of this method is in the control of food spoilage where foods can be preserved using high brine solution syrups etc., in which development of high osmotic conditions exert control.

Microbial Control by Sonication

Bacteria can be disrupted by using sonic and ultrasonic waves. Sound waves with a frequency of 720000 cycles/sec. are more effective. They can be achieved by use of electrically vibrating needle or disc.

Mode of action :

   When the bacteria, suspended in liquid, are exposed to ultrasonication, the sound waves cause cellular damage by cavitation. The passage of sound through liquid causes formation of cavities. The cavities grow in size, until they collapse violently. 

  This results in to disintegration of cells. Thus, sonic waves kill the organisms. Gram-negative bacteria are most susceptible to sonication as compared to cocci and gram-positive rods.

Microbial Control by Radiations

  Radiations, which have shorter wavelength, are called energy radiations. They possess a higher quantum of energy. These radiations are lethal to the organisms.

  Therefore, these radiations are used for the control of microorganisms. These high energy radiations are of different types:
- Ultraviolet rays,
- X-rays,
- Gamma rays and
- Cathode rays.
  Sterilization by use of radiation Is also known as cold sterilization.

Ultraviolet rays

The light radiations with wavelength between 2000 A° to 4000 A° are called ultraviolet rays. They are able to kill organisms due to their ability to -
a). denatures cellular macromolecules.
b). cause ionization of the cellular molecules.

Denaturation of cellular macromolecules by UV rays :

  Of the different macromolecules of cells, nucleic acids and proteins are the most sensitive molecule which gets denatured by UV rays. This is mainly due to the absorption of UV light by nucleic acids and proteins.

- Effect of UV on DNA

   DNA can absorb maximum UV light at 2654 A° λ. This results into formation of pyrimidine dimers in to DNA. Formation of the pyrimidine dimers in the DNA affects the DNA structure.

  This, in turn affect the normal functioning of DNA and interfere with DNA transcription and its replication. This ultimately influences viability of the cells, resulting into death of cells

- Effect of UV on RNA and proteins

  RNA and protein molecules can also absorb maximum UV light at 260 nm. This results into alteration in the structure and conformation of cellular RNA and proteins, which in turn, contribute to the killing of cells by UV rays.

Ionizing activity of UV rays :

   UV rays. being energy radiations, also possess some ionizing effect in the cell. They cause lonization of water molecules present in cell, which generate toxic effect.

Use of UV in microbial control :

UV rays are widely used for obtaining sterilization in closed room areas and inoculation chambers. It is also widely used in operation theaters, aseptic areas of pharmaceutical industries etc. to achieve sterilization.

Ionizing radiations

   Electromagnetie radiations with extremely short wavelength possess extremely high energy. Such radiations are therefore able to cause lonization of the target molecules. Therefore, these radiations are called ionizing radiations. These radiations are lethal to the organisms. lonizing radiations include X-rays, gamma rays and cathode rays.

- X-rays

X-rays possess wavelength, smaller than UV rays. They were first discovered by Rontgen. Hence, they are also called Rontgen rays.

Mode of action :
  Since these radiations possess very short wavelength. they possess a high penetration power as well as lonizing property. They act by -
1. Damaging DNA and RNA due to their ability to break phosphodiester bond in the nucleie acids.
2. Ionizing various molecules of cytoplasm. especially water, by forming OH- ions, which are toxic.

  Though, X-rays possess antimicroblal activity, their practical application is rare. This is because -
1. They are expensive.
2. Their controlled use is difficult because of their ability to disperse in all direction.

- Gamma rays
These are also high energy radiations, emitted by radioactive isotopes. Usually radio isotope Co⁶⁰ is used for this purpose. These radiations also act by caustng lontzation of almost all cellular molecules, especially water, by forming hydroxyl ions, having toxie effect.

Application :
   Gamma rays are widely used commercially for sterilizing various materials in packed conditions. These include plastic injection syringes, needles, Petri dishes and other surgical materials. food and food products etc. The commercial application of gamma rays for obtaining the packed materials has become possible due to their high killing as well as penetration power.

- Cathode rays
  Cathode rays have the smallest wavelength (0.05 A°). Therefore, they possess maximum energy component and maximum lonizing ability. Cathode rays can be generated by creating a high voltage difference between cathode and anode potentials. A very high voltage of electric current is gven to a cathode, which emits electrons. These electrons are called cathode rays.

Mode of action :
   The cathode rays act in the same manner, as other ionizing radiations act. They cause ionization of call cellular molecules leading to the death of cells.
   Though cathode rays possess a high killing power than all other ionizing radiations, their use in microblal control is limited because of their high cost. However, they can be applied to obtain sterilization of surgical materials. drugs etc.

Microbial control by filtration

  Physicall removal of organisms from materials / system is one of the most commonly employed methods to achieve microbial control. Use of bacteriological filters for the purpose is a widely used technic.

Principle of microbial control by filtration-

  Passage of liquid or gas through a filter having a specific pore size has a capability to retain particles of larger size. Hence employing the use of a filter having pore retain small enough to micro-organisms of certain size causes removal of these organisms from the liquid or gas treated with.

   In addition, certain filters cause removal of organisms by adsorption also. Charged surface of these filters ald in removal of negatively charged bacteria from the system by causing their adsorption on the filter surface.

Types of filters used for microbial control

A variety of filters made of different materials are employed for the removal of micro-organisms from liquids to be sterilized. These filters are made of materials like clay, paper, asbestos, glass, diatomaceous earth, cellulose acetate etc.

   These filters a designed to have pores with diameters small enough to retain bacteria. The earliest filters of these types are Chamberland filter, developed by Chamberland at Pasture's laboratory. Following are the different types of bacteriological filters.

• Porcelain filters

  These are made of unglazed porcelain. They are available in different grades. These were the earliest filters to be developed by Chamberland. Therefore, these filters are also known as Chamberland filters.

• Berkfeld filters

  These filters are made from a mixture of diatomaceous earth, asbestos, plaster of Paris and water. They are made in form of a hollow candle with graded pore size. The fluid to be filtered is forced by pressure or suction from outside to inside or vice versa, allowing microorganisms to be retained on the fiter surface. These filters are also commonly referred to as candle filters.

• Sintered glass filters

  These filters are made from finely ground glass beads. The glass beads are allowed to fuse to adhere sufficiently to allow the beads to adhere each other leaving pores between them. These are available in form of disc, fused with glass funnel.

• Asbestos filters

   A sheet of asbestos can also provide a fltering surface and hence it can be used as filter. Seitz filter is one of these types of filters. In this filter, asbestos sheet is clamped between two perforated metal dises with a metal funnel assembly.

• Membrane filters

   These filters are also known as molecular sieve filters. They are manufactured from a variety of polymeric materials such as cellulose diacetate, cellulose nitrate, polycarbonate, polyester etc. These niters were first introduced by Millipore inc. as milipore filters.

  The membrane filters are available with destred grade of pore size ranging from 0.015 nm to 12 nm. Specially designed assembly is used to hold membrane filters for sterilization of liquids.

Preparation of bacteriological filters for the use -

  A special care has to be taken before a bacteriological filter is used for the sterilization of liquids.
1). The filter should be properly cleaned before use. If asbestos filter of membrane filters are used, new filter sheet is required for the purpose.
2). The filter assembly is properly sterilized by autoclaving before its use.
3). Usually vacuum flask, connected to vacuum pump is used to collect filtered liquid. Vacuum creates a negative pressure in the flask and helps in rapid filtration.

Applications of bacteriological filters -

  Bacteriological filters are employed to steriltze liquids like water, broth media, antibiotics, vitamin and amino acid solutions, hyper immune sera etc. In general. the liquids, which are thermo labile and unable to get sterilized by heat, are sterilized by use of bacteriological filters.

High Efficiency Particulate Air (HEPA) filters :

  HEPA filters are made from cellulose acetate, pleated around aluminum foil. They are available in various sizes for different applications. These filters have ability to remove 99.97% of the particles having size 0.3 nm or more from air. These filters are commonly employed In obtaining clean air environment in pharmaceuticals, laboratories, Industries, hospitals etc.

Laminar air flow system

   Laminar air flow system is one of the commercial application of HEPA filters to obtain sterile environment in a given area. Here, the air is allowed to flow in one direction with uniform velocity in a confined are after its passage to HEPA filters. It removes air bore particles and maintains a clean environment in the area through which air is allowed to flow.

Application :

   Laminar air flow has application not only in obtaining sterile area in inoculation chambers in microbiology laboratories, but has also got wide applications in creating clean room environment. especially in pharmaceuticals. operation theatres, electronic industries, biotechnology laboratories etc.