12 Disinfections- Types of disinfectants & mode of action

Babita Khosla

13.1 Introduction

Our natural environment contains numerous microorganisms. Most of these present no concerns to health issues. However, some such as Vibrio cholerae, Salmonella typhi, Giardia lamblia and various enteric bacteria, viruses and protozoa present in water supplies are extremely harmful and can cause disease like typhoid, cholera, amoebic dysentery, giardiasis, viral gastroenteritis and hepatitis in humans. These disease-causing organisms are known as pathogens. Disinfection of wastewater is very important to public health because diseases can be transmitted directly and indirectly through contaminated drinking water or water used for irrigation, amusement, or food processing. Drinking water must be completely free from pathogenic microorganisms. For this, the best process in water treatment plants is disinfection.

Water disinfection is a technique designed to destroy or inactivate most microorganisms in wastewater.The technique of disinfection is a last step in water treatment, which is enforced directly to waste water for the control of many pathogens which cause harm to all living organisms. From many years chlorine and its constituents were used as disinfectants to control odors and various diseases in wastewater. Application of disinfectants inactivates the pathogens by destroying the cellular structure of microbes or disrupts their metabolism and makes them unable to grow or reproduce. In case of bacteria, inactivation means the inability of the bacteria to divide and form colonies. Inactivation makes viruses unable to form plaques in host cells. Protozoan Cryptosporidium oocysts become unable to multiply and thus prevent the spread of infection in a host cell.

There are two kinds of disinfection:

Primary disinfection: This kind of disinfection achieves the desired level of microorganism kill or inactivation.

Secondary disinfection: This kind of disinfection not only achieves the desired level of disinfection but, maintains a residual disinfectant concentration in the finished water that prevents the re-growth of microorganisms.

13.2 Disinfectants: Disinfectants are the substances which are applied to biotic or abiotic surfaces such as directly on the skin, in bathrooms, kitchens, or in production facilities, but may also be added to for example drinking water or swimming pool water.

Disinfecting agents are registered by the Environmental Protection Agency (EPA) as “antimicrobials” and are substances used to control, prevent, or destroy harmful microorganisms (i.e., bacteria, viruses, or fungi) and must provide a 99.9 percent inactivation of microbes, their cysts and enteric viruses to protect health. When a killing action is implied, the suffix –cide (e.g. biocide, bactericide, virucide, sporicide) is used, while –static (e.g. bacteriostatic, virostatic, sporostatic) is added when an organism’s growth is merely inhibited or it is prevented from multiplying.

13.2.1 Properties of Disinfectants

Disinfectants should have wide spectrum of activity, competent to destroy microbes, should be operating in the presence of organic matter, effective in any pH, have high penetrating power, non-toxic, non-allergenic, non-irritative or non-corrosive, should not leave non-volatile residue or stain, efficacy should not be lost on reasonable dilution, should not be expensive and must be available easily.

13.2.2 Factors affecting the efficiency of disinfectants are:

Nature of the disinfectant Concentration of the disinfectant Length of contact time between the disinfectant and the microbe Temperature Type and concentration of microorganism pH and ionic strength.

Relationship of Disinfection efficiency and contact time: The relationship between kill efficiency and contact time, was developed by Harriet Chick as shown in the graph below. Chick’s Law predicts the decrease in the survival ratio of microorganisms (No/N) exponentially with increasing contact time between the disinfectant and the water, whereas No represents the initial number of organisms and N is the number of organisms at time t.

Later, Watson modified Chick’s equation to account for varying types of disinfectants and developed coefficients of specific lethality (lambda) that better represented the strength of the disinfectant as well as the pH of the water. Watson’s modification of Chick’s equation is shown below.

Ln N/N0 =λ Cn t

Where:

N/N0 is the survival ratio for the microorganisms being killed λ is the Chick-Watson coefficient of specific lethality C is the concentration of the disinfectant (typically in mg/L) n is the coefficient of dilution, frequently assumed to be 1 t is the contact time (typically in minutes or seconds)

13.2.3 Measurements of effectiveness: The effectiveness of the disinfectants is calculated by comparing it against a known disinfectant. Phenol is the standard disinfectant, and the corresponding rating system is called the “Phenol coefficient”. The disinfectant to be tested is compared with phenol on a standard microbe (usually Salmonella typhi or Staphylococcus aureus). Disinfectants that are more effective than phenol have a coefficient > 1. Those that are less effective have a coefficient < 1.

An alternative assessment is to measure the Minimum inhibitory concentrations (MICs) of disinfectants against selected (and representative) microbial species, such as through the use of micro-broth dilution testing.

13.3 Types of disinfectants

Disinfectants are characterized into 3 activity levels according to classification given by United States Environmental Protection Agency (EPA):

High-Level Disinfectants – These types of disinfectants kills vegetative microorganisms and inactivates viruses, but not necessarily high numbers of bacterial spores. Such disinfectants are capable of sterilization when the contact time is relatively long (6 to 10 hours). As high-level disinfectants, they are used for relatively short periods of time (10 to 30 minutes). These chemical germicides are potent sporicides and in the United States, are classified by the FDA as sterilant/disinfectants. They are formulated for use on medical devices, but not on environmental surfaces such as laboratory benches or floors.

Intermediate-Level Disinfectants – These disinfectants kill vegetative microorganisms, including Mycobacterium tuberculosis, all fungi, and inactivate most viruses. Chemical germicides used in this procedure often correspond to Environmental Protection Agency (EPA- approved “hospital disinfectants” that are also “tuberculocidal.” They are used commonly in laboratories for disinfection of laboratory benches and as part of detergent germicides used for housekeeping purposes.

Low-Level Disinfectants – These types of disinfectants kill most vegetative bacteria except M. tuberculosis, some fungi, and inactivates some viruses. The EPA approves chemical germicides used in this procedure in the US as “hospital disinfectants” or “sanitizers.

13.4.1 PHYSICAL DISINFECTION

Physical methods of disinfection mainly includes

Heat: Heat is the most common physical agent used for the decontamination of pathogens. “Boiling does not necessarily kill all microorganisms and/or pathogens, but it may be used as the minimum processing for disinfection where other methods (chemical disinfection or decontamination, autoclaving) are not applicable or available.

Moist Heat Sterilization: “Moist” heat is most effective in achieving sterilization. Water at high pressure level (15 lb/inch2 pressure) corresponding to a temperature of 121oC is used in an instrument called

Autoclave, which is filled with hot steam. All organisms and endospores are killed within 15 minutes. The temperature of the steam in thismethod is lower when compared with dry heat sterilization, but the high pressure helps with effective sterilization to take place. The structural proteins and the organism’s enzymes are destroyed through moist heat, resulting in the death of the organisms. Moist heat method is used for heat sensitive materials and materials through which steam is permeable. Through moist heat sterilization, the most resistant of the spores require a temperature of 121°C for around half an hour. It is a more effective method when compared with dry heat sterilization. This can be supported by the fact that through moist heat, sterilization can be achieved at lower temperatures in a shorter duration.

Dry Heat Sterilization: In dry heat sterilization, dry heat is used for disinfecting different materials which can withstand temperatures of 160°C or higher for 2-4 hours. Heated air or fire is used in this process. Burning or incineration is also a form of dry heat. As compared to the moist heat sterilization, the temperature in this method is higher. The temperature is usually higher than 180 °C. Dry heat helps kill the organisms using the destructive oxidation method. This helps destroy large contaminating bio-molecules such as proteins. The essential cell constituents are destroyed and the organism dies. The temperature is maintained for almost an hour to kill the most difficult of the resistant spores. Things such as glassware, metal instruments, paper wrapped things and syringes made up of heat resistant material are effectively sterilized through dry heat.

• Ø Radiations: Three types of radiations kill microbes:

Ionizing Radiation: These include Gamma rays, X- rays, electron beams or higher energy rays. They have short wavelengths (less than 1 nanometer) and mainly used to sterilize pharmaceuticals and disposable medical supplies and food industry.

Nonionizing Radiation (Ultraviolet light): These radiations cause maximum destruction in the range ofwavelength from 250 – 265 nm.These radiations are produced by a low pressure mercury lamp constructed of quartz or special glass. Maximum disinfection is achieved with adequate intensity and time of exposure of UV rays when water is free from suspended and colloidal substances and is flowing in the form of thin sheets. The advantage of this method is that the exposure is for short period and no taste and odour is produced and complete destruction of microorganisms is achieved.

Microwave Radiation: These radiations of wavelength (ranges from 1 millimeter

to 1 meter) help in killing of vegetative cells in the presence of moisture. Heat is absorbed by water molecules, but bacterial endospores, which do not contain water, are not damaged by microwave radiation.

Ultra-sonic Sound: Waves of frequency 400 KHz have been testified to provide complete sterilization in 60 minutes. These waves are effective for water disinfection. There is no germicidal effect by these waves. Large reduction in bacterial number is observed within 2 seconds.

13.4.2 CHEMICAL DISINFECTION

Chemical disinfection is achieved by using chemical disinfectants, which are the antimicrobial agents that kill or inhibit the growth of microorganisms.

The exact mode of disinfection by antimicrobial agents depends upon the type and concentration of disinfectant and the type of pathogen being targeted.

• Ø Alcohols: Alcohols are the commonly used disinfectants; widely used products are ethyl alcohol, isopropyl alcohol and n-propanol. They show broad spectrum antimicrobial activity against gram positive and negative bacteria and shielded viruses, but cause no effect on bacterial spores and non- shielded viruses. Due to the lack of sporocidal activity they are widely used for hard surface disinfection and skin antiseptic, but not for sterilization.

Ethanol or isopropanol in concentrations of 70% – 95% are good general-use disinfectants. They are most effective against lipophilic viruses, less effective against non-lipid viruses, and ineffective against bacterial spores. Because of their quick evaporation rate, it may be difficult to achieve sufficient contact time. Generally isopropyl alcohol having greater lipophilic properties than ethyl alcohol is effective against bacteria and is less active against hydrophilic viruses (e.g., poliovirus) while ethyl alcohol is efficacious against viruses. The mode of action by alcohol is through protein coagulation/denaturation and the associated disruptions of cytoplasmic integrity, cell lysis and interference with cellular metabolism. Proteins get denatured easily when alcohol is used with water. Absolute ethanol a dehydrating agent is less bactericidal than a mixture of alcohol and water. Alcohol induced coagulation of proteins occurs at the cell wall, cytoplasmic membrane and among the various plasma proteins. Coagulation leads to loss of cellular functions. Alcohols target the bacterial cell wall which results in the lysis of cellular membrane and release   of cellular contents. The researchers concluded that the bacteriostatic action was due to the inhibition of the production of metabolities essential for rapid cell division.

• Ø Phenols: Phenol solutions have been used as antiseptic, disinfectant or as preservatives for many years as a disinfectant. They are considered as ‘general protoplasmic poisons’. Phenol acts only at the point of separation of pairs of daughter cells, so young bacterial cells are more sensitive than older cells to phenol. It also coagulates the cytoplasmic constituents at higher concentrations causing irreversible cellular damage. It possesses both antifungal and antiviral properties. In antifungal infection plasma membrane got damaged resulting in leakage of intracellular constituents.

The usefulness of phenols in laboratories is limited, because they leave a sticky residue on surfaces following treatment. Concentrated phenol is a highly toxic, corrosive substance that is easily absorbed through the skin. Use of appropriate personal protective equipment is essential.

• Ø Aldehydes

Glutaraldehydes: It is an important dialdehyde closely related to formaldehyde but seem to be more biologically active. Glutaraldehydes possessed high antimicrobial activity and are effective against all types of bacteria, fungi, viruses and with sufficient contact time they even kill bacterial spores. Glutaraldehyde and act as a disinfectant at low temperature and helps in sterilization of endoscopes and surgical equipment and as a fixative in electron microscopy. While glutaraldehyde vapors are less irritating than formaldehyde (formalin), they remain irritating to the eyes, mucous membranes, and upper respiratory tract. Exposures should be minimized by confining use to a properly functioning chemical fume hood. Glutaraldehyde is more active at alkaline than at acidic pH. As the external pH is changed from acidic to alkaline, more reactive sites will be formed at the cell surface, leading to a more rapid bactericidal effect.

Formaldehyde: Formalin is a 37% solution of formaldehyde gas in water. Diluted to 5% formaldehyde it is an effective disinfectant; at 0.2% – 0.4% it can inactivate bacteria and viruses. Unlike chlorine, formalin does not corrode stainless steel. It has a pungent, irritating odor; exposures must be limited due to its toxicity and carcinogenicity. It acts as a mutagenic agent and as an alkylating agent by reaction with carboxyl, sulfhydryl, and hydroxyl groups. It reacts with nucleic acids and also inhibits DNA synthesis. Its mechanism of action is not well known but its interactive and crosslinking properties plays a great role in this activity.

o-Phthalaldehyde: It is a new high level disinfectant used in place of glutareldehyde in endoscope disinfection. It is an aromatic compound with two aldehyde groups. It is potent for bactericidal and sporicidal activity. It is uptaken by the outer layers of mycobacteria and gram negative bacteria. It kills spores by blocking the spore germination process.

Anilides: Triclocarban is used mostly in soaps and deodrants. They are more active against gram positive bacteria than gram negative bacteria and fungi. They lead to cell death by absorbing and destroying the semipermeable nature of the cytoplasmic membrane.

Chlorhexidineis a bactericidal agent and basically used in hand-wash and oral products, due to its broad spectrum efficacy and low irritation. It is pH dependent product and its activity is reduced in the presence of organic matter. It damages the outer cell layers and then crosses the cell wall or outer membrane, presumably by passive diffusion, and subsequently attacks the bacterial cytoplasmic or inner membrane or the yeast plasma membrane, but it is insufficient to induce lysis or cell death. Damage of the semipermeable membrane leads to leakage of intracellular constituents, which can be measured by appropriate techniques.

• Ø Oxidizing agents

Hydrogen Peroxide: It is widely used disinfectant, sterilizant and antiseptic. It is environment friendly colourless liquid that is commercially available in a variety of concentrations ranging from 3 to 90%. After its action it can rapidly degrade into water and oxygen products. H2O2 determines broad spectrum efficacy against viruses, bacteria, yeasts, and bacterial spores. Generally greater activity is seen against gram + than gram – bacteria; however, the presence of catalase or other peroxidases in these organisms can increase tolerance in the presence of lower concentrations. Higher concentrations of H2O2 (10 to 30%) and longer contact times are required for sporicidal activity although this activity is significantly increased in the gaseous phase. H2O2 acts as an oxidant by producing hydroxyl free radicals (OH) which attack essential cell components, including lipids, proteins, and DNA. It has been proposed that exposed sulfhydryl groups and double bonds are particularly targeted.

Peracetic acid (PAA): It is sporicidal, bactericidal, virucidal, and fungicidal at low concentrations (<0.3%) and considered a more potent biocide than hydrogen peroxide. End products of PAA are (acetic acid and oxygen) and it remains active in the presence of organic loads. Like H2O2, PAA possibly denatures proteins and enzymes and increases cell wall permeability by disrupting sulfhydryl (-SH) and disulphide (S-S) bonds.

Chlorine compounds: Chlorine-containing solutions (household bleach – 5.25% sodium hypochlorite) have universal disinfectant activity. With proper concentration and sufficient contact times, hypochlorite solutions can be considered chemical sterilants since they will inactivate bacterial spores. The downside is that chlorine compounds are quickly inactivated by excess organic materials and are corrosive to metals and tissues. Consequently their use in labs has some limitations. In solutions of 50-500 ppm available chlorine, they are effective against vegetative bacteria and most viruses. Bacterial spores require concentrations of 2500 ppm with extended exposure time. Household bleach (5.25% sodium hypochlorite) diluted 1:100 with water yields a disinfectant solution containing to 525 ppm available chlorine; a 1:10 dilution yields 5000 ppm available chlorine. Since the free chlorine is inactivated by light and air, disinfectant chlorine solutions are best made fresh before use.

Other Halogens: Halogens like bromine and iodine also possess good disinfection property. They are good oxidizing agents and available in the form of pills and tablets of tetraglycerine-hypo-peroxide and solution of Povidone-iodine and poloxamer-iodine. But the use of these halogens is costlier than chlorine. Iodine act as disinfectant by penetrating the cell wall of microorganisms quickly, and disrupts the synthesis and structure of proteins and nucleic acids.

The quantity of bromine & iodine should not exceed 8 ppm & they can kill bacteria in a contact time of 5 minutes. They are mainly used for the disinfection of swimming pools.

Electrolyzed water: The electrolysed water/anolyte is an oxidizing, acidic hypochlorite solution made by electrolysis of sodium chloride into sodium hypochlorite and hypochlorous acid. Anolyte has an oxidation-reduction potential of +600 to +1200 mV and a typical pH range of 3.5––8.5, but the most potent solution is produced at a controlled pH 5.0–6.3 where the predominant oxychlorine species is hypochlorous acid.

Potassium Permanganate: Potassium Permanganate is a strong oxidizing agent which possesses germicidal properties. It is generally used for iron and manganese removal and for the removal of odour. It oxidizes the organic matter present in water. It is used for six hours in the amount of 1-2 mg/l. It is considered as a costly disinfectant.

Quaternary ammonium compounds (QA): These disinfectants have wide germicidal range, noncorrosive nature and low toxicity. They are effective against gram positive and negative bacteria, and enveloped viruses, but ineffective against non-enveloped viruses, fungi and bacterial spores. The area which is to be disinfected by QA must be cleaned and rinsed free of soap. Extremely hard water also deactivates QA disinfectants. QA compounds are generally low in toxicity, but prolonged contact can be irritating

Metallic Ions: Some metals like silver, copper, iron and mercury ions also posseses disinfection property. Silver ion kills bacteria by passing current through 15v DC battery in contact time of 15-180 minutes at a dose of 0.05- 0.1 mg/l. This method is not practiced at large scale as it is very costly.

                           Table 1: The antimicrobial action of various chemical disinfectants

13.4.3 MEMBRANE PROCESS

These processes help in the removal of microorganisms and also remove taste, colour and desalination. The principle of micro filtration and ultra- filtration is pressure-dependent physical separation. The extent to which dissolved solids, turbidity and microrganisms are removed is determined by the size of the pores in the membranes. The substances that are larger than the pores in the membranes are fully removed and substances smaller than the pores of the membranes are partially removed, depending on the construction of a refuse layer on the membrane.

• Ø Micro filtration (MF) Micro filtration is a membrane separation process in which a semi-permeable membrane with a pore size of 0.03 – 10 microns and a relatively low feed water operating pressure of approximately 100 to 400 kPa (15 to 60 psi). The representative materials removed by MF include sand, silt, clays, Giardia lamblia and Cryptosporidium cysts, algae, and some bacterial species. MF is not an absolute barrier to viruses; only part of the viral contamination is caught up in the process, even though viruses are smaller than the pores of a micro filtration membrane. This is because viruses can attach themselves to bacterial biofilm. Micro filtration can be implemented in many different water treatment processes when particles with a diameter greater than 0.1 mm need to be removed from a liquid.

The primary impetus for use of MF has been the increasingly stringent requirements for removing particles and microorganisms from drinking water supplies. Additionally, there is a growing emphasis on limiting the concentrations and number of chemicals that are applied during water treatment and membrane filtration can significantly reduce chemical addition to the water.

• Ø Ultra filtration (UF) Ultra F involves the pressure-driven separation of materials from water using a membrane pore size of approximately 0.001 to 0.1 µm and an operating pressure of approximately 200 to 700 kPa (30 to 100 psi). UF will remove all microbiological species removed by MF (partial removal of bacteria), as well as some viruses (but not an absolute barrier to viruses) and humic materials. Disinfection can provide a second barrier to contamination and is therefore recommended.

The primary advantages of low-pressure UF membrane processes compared with conventional clarification and disinfection (post-chlorination) processes are:

No need for chemicals (coagulants, flocculants, disinfectants, pH adjustment) Size-exclusion filtration as opposed to media depth filtration Good and constant quality of the treated water in terms of particle and microbial removal Process and plant compactness Simple automation.

• Ø Reverse Osmosis (RO) Reverse Osmosis can effectively remove nearly all inorganic contaminants from water. It is normally used for desalination, but also for disinfection of waste water. RO can also effectively remove radium, natural organic substances, pesticides, cysts, bacteria, and viruses. RO is  particularly effective  when used in series.  Water passing through multiple units can achieve near zero effluent contaminant concentrations. Disinfection is also recommended to ensure the safety of water. Fouling is the limiting phenomenon responsible for most difficulties encountered in membrane technology for water treatment. To prevent plugging or damaging of membranes by hard and sharp particles from the feed water, water needs to be pre-filtered before micro filtration or ultra filtration processes take place. The pores of the pre-filtration unit need to be between 0.5 and 1.0 mm, depending on the composition of the wastewater. The pre-treatment of water is very important when these filtration techniques are applied, because membrane fouling can easily disturb the purification process.

Reference:

McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clinical Microbiology Reviews 1999;12(1):147–179.