We all are aware that microbes are present everywhere. The microorganisms can be useful and harmful for us. Their harmful part is experienced by all of us. They spoil our food, water and make us sick. And hence, we need methods for controlling Microbial Growth. The practices of sterilization and disinfection have been followed from ages. In this chapter, we will see what different measures are taken to control the growth of unwanted microorganisms.
Let’s understand the important keywords of control of microorganism-
Sterilization: It is the process by which the living cells are completely eliminated by either killing or removing from biological or non biological material. The agents that are used for sterilization are called as sterilant. The sterilants may be physical (UV, temperature, radiation) or chemical agents (phenol, alcohol, halogen).
Disinfection: It is the method for eliminating microbes from inanimate surface. It is the killing of microorganism, inhibiting or removal microbes which can cause disease. The main motive of disinfectant is to destroy potential pathogens, but disinfection also substantially reduces the total microbial population.
Antisepsis: It is chemical agent used for killing or controlling the microorganism on living tissues. You might have seen antiseptic is applied on the surface of wounds because the open lesion attracts the microbe and that causes the infection so in order to avoid this antiseptics are applied.
Sanitizer: It is a chemical agent that removes the microorganism from surface to safe level.
Static: The agent that inhibit the growth of microorganisms.
Cidal: The agents that kills the microorganisms.
If the name of the agents that are used for controlling the microbes ends Static or cidal then you should be able to understand whether the agent will inhibit or kill the microorganisms.
The Microbial Death –
The microbes are not killed as soon as they come in contact with the sterilizing agent. The microbial death also show exponential graph similar to log phase of bacterial growth curve. When the microbes are exposed to sterilizing agent, the same number of fraction of population is killed at regular and constant interval. If the number of bacteria killed data is plotted against time then we will obtain straight graph.
This rate of microbial death is measured and calculated to test the efficiency of the sterilizing agent. It also estimate the time required by the sterilizing agent to kill the given microbe.
Factors Affecting the Efficiency of Antimicrobial Agents –
The effectiveness of antimicrobial agent depends on the following factors: –
- The size of the microbial population, more the populated longer time it takes to kill the microorganisms.
- The composition of the population, whether they are vegetative or resistant. The sterilizing agent kills the vegetative cells much faster than resistant ones.
- The amount of concentration of the antimicrobial agent used. Higher the concentration of sterilizing agent, higher will be rate of killing.
- The time of exposure, the longer the time duration the microbes are exposed the more the organism are killed.
- With the rise in the temperature the chemical often enhance its activity.
- The local environment factors if the factors are not in the favour of the microbes it will lead to lead to death.
Physical Methods Used for controlling Microbial growth
1) Heat –
Use of heat and fire for killing microbes is one the most ancient method. It is one of the most reliable methods. In tropical countries, sunlight is still use for killing microorganisms. The presence of UV and heat makes it more efficient for killing microorganisms. The spores are resistant to sunlight and hence the sunlight does not kill spores. The mechanism of killing microorganisms is denaturation of proteins and enzymes and it also cause oxidative stress of cells. The heat may also melt the lipid content of cell membrane.
The heat could be used in either dry form or moist form. It has been found that moist heat is more effective than dry heat. In case of heat sterilization, temperature is inversely proportional to time, it means higher the temperature lower the time is required. Material that is sensitive to high temperature can be exposed to low heat for longer duration.
To measure the efficiency of heat, time is considered as an important parameter and hence it is expressed as Thermal Death Point (TDP). It is the minimum time required to kill the given microorganism.
Types of Dry heat–
- Red heat/direct flame: In this method the material that need to sterilised is directly sterilised in the burning flame. Materialised like scalpel, forceps and inoculating look are sterilised in direct flame. They are hold in flame until they become red hot and then before using them, they are cooled in sterile environment.
- Flaming: In this method, the material that need to be sterilised are passed few times through the flame without making it red hot. Flaming method is employed for glass slide, coverslip, test tube and flask neck. This method is not at all efficient to kill the spores. In practise, this method is used for sterilised material and employed just before using them.
- Incineration: In common language incineration means burning and hence this method is used only for disposable material. Its motive is to destroy the contaminated material example contaminated microbial culture, dressings, pathological material etc which cannot be reused.
- Hot Air Oven: It is an insulated electrical instrument in which heat is produced by electricity; this artificial heat is used to kill the organisms. The temperature range for such hot air oven is in the range of 100 to 450˚C. It is usually operated at 160 or 180˚C. The hot air oven is equipped with fan which ensures uniform distribution of heat. The Hot oven has display and regulators for temperature.
Types of Moist Heat-
As compared to dry heat, moist heat penetrates the microbial cell and hence it is more efficient to kill the microbial cell.
- Boiling water: It is one the most ancient and common method employed. This method is efficient to kill only that temperature which cannot resist 100˚C because boiling water temperature cannot exceeds than that. So what do you think, will this method will kill spores and extremophiles?
- Autoclave: Autoclave is one of the most important and efficient sterilizing instrument used in Microbiology. It works on steam under pressure principle. The steam is a type of moist heat. The autoclave is efficient than boiling because water boils at 100˚C but the extremophiles and spores can survive at this temperature. The autoclave makes use of moist heat and pressure, the pressure allows raising the water temperature above its boiling point. The studies have found that the microbes (vegetative/spores/extremophiles) die in autoclave at 121˚C at 15 pounds of pressure when it is run for 15 min and hence this has become standard settings or conditions for running autoclave.
- Pasteurization: This method was invented by Louis Pasteur and hence it is called as Pasteurization. This method is usually used to kill or inhibit the microbes in consumable products. The most common known example is milk. The Pasteurisation is done at either 63° C for not less than 30 min. or 72° C for not less than 16 sec. This temperature does not kill all the microbes and hence packed and refrigerated milk may also get spoiled.
2) Refrigeration –
It does not kill the microbes but inhibit its growth. The low temperature slows down the biochemical and molecular process. This method is not used for sterilization but it is used for preserving or storing the culture.
3) Desiccation –
It is the process by which the water content or moisture is evaporated. It is also called as state of extreme dryness. As we know, water is one of the most important constituent of cell when the cell is dried the water gets evaporated and hence cell fail to conduct its important life processes. In dehydrated state, the microbes cannot divide but the some microbes undergo spore formation and get resisted and survive in such dehydrated state. When they again come in contact with water, the microbes start multiplying and hence desiccation does sterilize completely.
4) Osmotic Pressure –
As you know, that molecules always tend to move from higher concentration to lower concentration across the semi permeable membrane. This movement creates a pressure on both sides of the semi permeable membrane called as osmotic pressure. In microbial cell, this pressure is maintained by the cell wall.
The hypertonic solution causes movement of water molecules from inside to outside making microbial cell dehydrated and inhibiting their growth.
5) Filtration –
As the name suggest, the sample is filtered through membrane filter. The pore size of the membrane filter is in the range of 0.2 to 0.45 µm. When sample is passed through such membrane the microbes larger than the mentioned size are hold or the filter while the sterile sample or solution get pass through the membrane. This method is employed for heat labile liquid materials such proteins or enzyme solution, vitamins, antibiotics etc. Such biomaterial is heat sensitive because the heat denatures its structures and affecting its functions.
6) Radiation –
When energy is emitted or transmitted in the form either particles or waves, it is called as radiation. The electromagnetic radiation includes UV, visible, radio, X-rays and gamma rays. X, gamma rays and high energy UV (very low wavelength) are called as ionising radiation because of their ability to detach or remove the electron from atoms or molecules making them ionised atom or molecule. To control microbial growth, ionising radiation is employed. The ionising radiation may break the backbone of DNA and cause chemical and structural change.
UV spectrum range from 10 nm to 400 nm and it has high energy and low wavelength. The Nucleic acid molecule absorbs UV (non-ionising) in the range of 260 to 270 nm. The absorption of UV causes thymine to form a covalent bond with adjacent thymine forming thymine dimer. This thymine dimer interrupts with DNA replication causing mutations. Such mutated DNA synthesizes damaged and malfunctioning proteins and enzymes and hence affecting its structural and functional role. With such damaged proteins and enzymes, the microbial cell fails to carry out its vital life process and causing its death.
The X and gamma rays wavelength are in the range of 0.1 to 10 nm and less than 1 nm respectively. This wavelength empowers them with very high energy and penetrating power. Such high penetrating radiation can disrupts and damages the biomolecules causing death of microbes. The X and gamma radiations are utilised to sterilise the heat sensitive and non biological material such gloves and petri dish etc.
Chemical Methods Used for Controlling Microbial Growth –
Chemically, alcohol is defined as presence of hydroxyl group in hydrocarbon. For sterilization, ethyl or isopropyl alcohols are used. The high concentration (95%) of alcohol is bactericidal and fungicidal (kill the microbe) while low concentration (50 – 70%) is bacteriostatic (inhibit). Endospore is are resistant to alcohol.
The alcohol acts like dehydrating agent and denature the protein structure, it may also dissolve the lipid content of cell wall and membrane. To increase the efficiency of alcohol, it may be combined with different halogens and cationic or anionic detergents.
Chemically, phenols are aromatic organic compounds which consist of phenyl and hydroxyl group. Phenol was the first disinfectant and surfactant employed to control microbial growth in medical field. The very well known commercially available surfactant is Lysol; it is the combination of different phenolics. The mechanism of killing and inhibiting the microbes is denaturation of proteins and disruption of cell membrane. The most advantageous characteristic is that it persist or stays on surface for longer period.
Fluorine, chlorine, bromine, iodine and astatine are the halogen present in group VII A of periodic table. Due to its valence electron, the halogens tend to form salts with other metals. Out of the above halogen, iodine and chlorine are potential antimicrobial agents. The salts of iodine and chlorine act like strong oxidising agents and denature the microbial proteins.
Chlorine is generally used for disinfecting the swimming pool and Muncipal corporation water. Tincture of iodine is used as antiseptic.
The heavy metal combine and react with different groups of proteins and make them inactive affecting the important life process. The heavy metals like silver, mercury, copper, arsenic, cadmium and zinc acts like germicidal. Arsenic inhibit the energy production by precipitating the proteins. Mercury reacts with the sulphydryl groups of cysteine and methionine present in the proteins and enzymes. As they are very toxic they are used in very low concentration. Heavy metals are also used in synthetic pesticides.
Detergents are the synthetic organic molecules with polar and non polar groups. The presence of polar and non polar groups makes the detergent as an emulsifying agent. The mode of action of inhibiting the microbe is by decrease the surface tension causing in the alteration of the cell membrane permeability. Based on the type of charge present on detergents, they are classified as anionic, cationic, neutral or zwitterions detergents. The cationic detergents have positive charge ex. Ammonium group while the anionic detergents have negative charge ex. Sulphate. The neutral detergents do have any charge and zwitterions detergents have both the charges but they are less efficient than cationic and anionic detergents.
The low molecular weight aldehydes like formaldehyde and glutaraldehyde are used as antimicrobial agent. Such low molecular weight aldehydes are very reactive and hence they react with the biomolecules of microbial cells and inactivate it. The aldehydes are used as disinfectant and preservative for biomaterial.
The gaseous agents are used for sterilizing the heat labile inanimate objects such as plastic pipettes, catheters etc. The high penetrating power increases its efficiency of sterilization. Ethylene oxide gas is used to sterilant. Due to its explosive nature, it is mixed with inert gas such as carbon dioxide and Freon. The vapours of ethylene oxide denature the microbial proteins. The sterilization with ethylene oxide is carefully operated and monitored because of its explosive and toxic nature.
Activity – Enlist different methods of physical and chemical methods that you have learned in the article and complete the following flow chart.