Chemical disinfectants, antiseptics and preservatives. 4th Lecture: Chemical disinfectants, antiseptics and preservatives.

Disinfectants: Disinfectants are chemical agents which are used to destroy microorganisms on inanimate (dead) objects. Disinfectant does not necessarily kill all microorganisms, but it reduces them to a level which is acceptable for a define purpose. The term disinfectant can be used to include antiseptics, in a wider sense ( but British Standards Institution consider antiseptic is not a synonym for disinfectant). Disinfectants on the other hand are non selective, irritant, corrosive or toxic to be applied to the skin or to tissues.

Disinfection levels: 1. High Germicidal: (p:286) Killed all microorganisms unless extreme challenge or resistance exhibited. M. surviving ( challenge of resistant bacterial spore, prions) Bactericidal, Sporocidal, Fungicidal & Virucidal e.g. Ethylene oxide, Formaldehyde, Glutaraldehyde. 2. Intermediate Germicidal: Killed most vegetative bacteria including M. tuberculosis, most viruses including hepatitis B virus (HBV), most fungi. M. surviving (bacterial spores, prions) Bactericidal, Non- sporocidal, Fungicidal &Virucidal e.g. Alcohol, Chlorine compounds & Iodine solution. 3. Low Germicidal: Killed most vegetative bacteria, some viruses, some fungi. M. surviving (M. tuberculosis, bacterial spore, some viruses, prions) Bactericidal, Fungicidal & kill lipid viruses only e.g. Chloroxylenoles, Thiomersal, QACs, Chlorhexidine, Iodophors.

Levels of disinfection attainable Disinfection level High Intermediate Low All microorganisms unless extreme challenge or resistance exhibited Most vegetative bacteria including M. tuberculosis Most viruses including hepatitis B virus (HBV) Most fungi Most vegetative bacteria Some viruses Some fungi Microorganisms killed Extreme challenge of resistant bacterial spores Prions Bacterial spores M. Tuberculosis Some viruses and prions Microorganisms surviving 4

Some high level disinfectants have good sporicidal activity and have been ascribed the name ‘liquid chemical sterilant’ or ‘chemosterilant’ to indicate that they can effect a complete kill of all microorganisms, as in sterilization. 5

Disinfectants 6

Antiseptics: Antiseptics are chemical agents which are used to destroy or inhibition of microorganisms on living tissues. Antiseptics have more selectivity in their action toward the bacteria than the living tissues, at the recommended concentration ,therefore antiseptics usually exert a bacteriostatic or low bactericidal effect at the recommended concentration. Antiseptics must not be toxic or irritating to skin, and they are often lower concentrations of the agents used for disinfection.

Antiseptics 8

Preservatives: Preservatives: These are included in pharmaceutical preparations to prevent microbial spoilage of the product and to minimize the risk of the consumer acquiring an infection when the preparation is administered. Preservatives must be able to limit proliferation of microorganisms that may be introduced unavoidably into non- sterile products( oral& topical medications) during their manufacture and use, while in sterile products(eye drops and multi- dose injections) preservatives should kill any microbial contaminants introduced inadvertently during use.

Preservative is not toxic, and employed at lower concentrations, and levels of antimicrobial action lower order than for disinfectants or antiseptics(European Pharmacopoeia ). There are around 250 chemicals that have been identified as active components of microbiocidal products in the European Union.

Preservatives 11

Factors affecting choice of antimicrobial agent:(P:286) Properties of the chemical agent. Microbiological challenge. Intended application. Environmental factors. Toxicity of the agent.

Properties of the chemical agent The process of killing or inhibiting the growth of microorganisms using an antimicrobial agent is basically that of a chemical reaction and the rate and extent of this reaction will be influenced by the factors of concentration of chemical, temperature, pH and formulation. 13

Properties of the chemical agent Tissue toxicity influences whether a chemical can be used as an antiseptic or preservative, and this limits the range of chemicals for these applications or necessitates the use of lower concentrations of the chemical.

Microbiological challenge The types of microorganism present and the levels of microbial contamination (the bioburden) both have a significant effect on the outcome of chemical treatment. If the bioburden is high, long exposure times or higher concentrations of antimicrobial may be required. 15

Microbiological challenge Microorganisms vary in their sensitivity to the action of chemical agents. Some organisms, either because of their resistance to disinfection or because of their significance in cross-infection or nosocomial, merit attention. 16

Microbiological challenge of particular concern is the significant increase in resistance to disinfectants resulting from microbial growth in biofilm form rather than free suspension. Microbial biofilms form readily on available surfaces, posing a serious problem for Hospital Infection Control Committees in advising suitable disinfectants for use in such situations. 17

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Vegetative bacteria At in-use concentrations, chemicals used for disinfection should be capable of killing most vegetative bacteria within a reasonable contact period. This includes ‘problem’ organisms such as listeria, campylobacter, legionella, vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA).. 19

Vegetative bacteria CHROM agar Listeria listeria 20

Vegetative bacteria campylobacter 21

Legionella on BCYE agar plate 22

Vegetative bacteria Antiseptics and preservatives are also expected to have a broad spectrum of antimicrobial activity but at the in-use concentrations, after exerting an initial biocidal effect, their main function may be biostatic. Gramnegative bacilli, which are the main causes of nosocomial infections, are often more resistant than Gram-positive species. 23

Vegetative bacteria Pseudomonas aeruginosa, an opportunist pathogen has gained a reputation as the most resistant of the Gram-negative organisms. However, problems mainly arise when a number of additional factors such as heavily soiled articles or diluted or degraded solutions are involved. 24

Pseudomonas aeruginosa 25

Mycobacterium tuberculosis M. tuberculosis and other mycobacteria are resistant to many bactericides. Resistance is either (a) intrinsic, mainly due to reduced cellular permeability or (b) acquired, due to mutation or the acquisition of plasmids. Tuberculosis remains an important public health hazard, and indeed the annual number of tuberculosis cases is rising in many countries. 26

Mycobacterium tuberculosis The greatest risk of acquiring infection is from the undiagnosed patient. Equipment used for respiratory investigations can become contaminated with mycobacteria if the patient is a carrier of this organism. It is important to be able to disinfect the equipment to a safe level to prevent transmission of infection to other patients 27

Mycobacterium tuberculosis 28

Bacterial spores Of the conventional microorganisms, bacterial spores are the most resistant to chemical treatment. The majority of antimicrobial agents have no useful sporicidal action in a pharmaceutical context. However, certain aldehydes, halogens and peroxygen compounds have excellent activity under controlled conditions and are sometimes used as an alternative to physical methods for sterilization of heat-sensitive equipment. In these circumstances, correct usage of the agent is of paramount importance, as safety margins are lower in comparison with physical methods of sterilization 29

Bacterial spores 30

Fungi The vegetative fungal form is often as sensitive as vegetative bacteria to antimicrobial agents. Fungal spores (conidia and chlamydospores) may be more resistant, but this resistance is of much lesser magnitude than for bacterial spores. The ability to rapidly destroy pathogenic fungi such as the important nosocomial pathogen, Candida albicans , filamentous fungi such as Trichophyton mentagrophytes, and spores of common spoilage moulds such as Aspergillus niger is put to advantage in many applications of use. 31

Fungi Many disinfectants have good activity against these fungi. In addition, ethanol (70%) is rapid and reliable against Candida species. 32

Candida albicans 33

Trichophyton mentagrophytes 34

Antifungal activity of disinfectants and antiseptics. 35

Viruses Susceptibility of viruses to antimicrobial agents can depend on whether the viruses possess a lipid envelope. Non-lipid viruses are frequently more resistant to disinfectants and it is also likely that such viruses cannot be readily categorized with respect to their sensitivities to antimicrobial agents. These viruses are responsible for many nosocomial infections, 36

Viruses e.g. rotaviruses, picornaviruses and adenoviruses and it may be necessary to select an antiseptic or disinfectant to suit specific circumstances. Certain viruses, such as Ebola and Marburg, which cause haemorrhagic fevers, are highly infectious and their safe destruction by disinfectants is of paramount importance. Hepatitis A is an enterovirus considered to be one of the most resistant viruses to disinfection. 37

Viruses There is much concern for the safety of personnel handling articles contaminated with pathogenic viruses such as hepatitis B virus (HBV) and human immunodeficiency virus (HIV) which causes AIDS (acquired immune deficiency syndrome). Disinfectants must be able to treat rapidly and reliably accidental spills of blood, body fluids or secretions from HIV-infected patients. Such spills may contain levels of HIV as high as 104 infectious units/ml. Fortunately, HIV is inactivated by most chemicals at in-use concentrations. 38

Viruses However, the recommendation is to use high level disinfectants (Table 17.2) for decontamination of HIV- or HBV-infected reusable medical equipment. For patient-care areas cleaning and disinfection with intermediate level disinfectants is satisfactory. Flooding with a liquid germicide is only required when large spills of cultured or concentrated infectious agents have to be dealt with. 39

Protozoa Acanthamoeba spp. can cause acanthamoeba keratitis with associated corneal scarring and loss of vision in soft contact lens wearers. The cysts of this protozoan present a particular problem in respect of lens disinfection. The chlorine-generating systems in use are generally inadequate. Although polyhexamethylene biguanide shows promise as an acanthamoebicide, only hydrogen peroxide-based disinfection is considered completely reliable and consistent in producing an acanthamoebicidal effect. 40 40

Acanthamoebas 41 41

Prions Prions are generally considered to be the infectious agents most resistant to chemical disinfectants and sterilization processes; strictly speaking, however, they are not microorganisms because they have no cellular structure nor do they contain nucleic acids. Prions (small proteinaceous infectious particles) are a unique class of infectious agent causing spongiform encephalopathies such as bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt–Jakob disease (CJD) in humans. There is considerable concern about the transmission of these agents from infected animals or patients. 42

Prions Risk of infectivity is highest in brain, spinal cord and eye tissues. There are still many unknown factors regarding destruction of prions . And they are considered resistant to most disinfectant procedures. For heat-resistant medical instruments that come into contact with high infectivity tissues or high-risk contacts, immersion in sodium hydroxide (1 N) or sodium hypochlorite (20 000 ppm available chlorine) for 1 hour is advised in WHO guidelines and this must be followed by further treatment including autoclaving, cleaning and routine sterilization. 43

Prions 44

Intended application The intended application of the antimicrobial agent, whether for preservation, antisepsis or disinfection, will influence its selection and also affect its performance. For example, in medicinal preparations the ingredients in the formulation may antagonize preservative activity. The risk to the patient will depend on whether the antimicrobial is in close contact with a break in the skin or mucous membranes or is introduced into a sterile area of the body. 45

Intended application In disinfection of instruments, the chemicals used must not adversely affect the instruments, e.g. cause corrosion of metals, affect clarity or integrity of lenses, or change the texture of synthetic polymers. Many materials such as fabrics, rubber and plastics are capable of adsorbing certain disinfectants, e.g. quaternary ammonium compounds (QACs) are adsorbed by fabrics, while phenolics are adsorbed by rubber, the consequence of this being a reduction in the concentration of active compound. A disinfectant can only exert its effect if it is in contact with the item being treated. Therefore access to all parts of an instrument or piece of equipment is essential. For small items, total immersion in the disinfectant must also be ensured 46

Environmental factors Organic matter can have a drastic effect on antimicrobial capacity either by adsorption or chemical inactivation, thus reducing the concentration of active agent in solution or by acting as a barrier to the penetration of the disinfectant. Blood, body fluids, pus, milk, food residues or colloidal proteins, even present in small amounts, all reduce the effectiveness of antimicrobial agents to varying degrees, and some are seriously affected 47

Environmental factors In their normal habitats, microorganisms have a tendency to adhere to surfaces and are thus less accessible to the chemical agent. Some organisms are specific to certain environments and their destruction will be of paramount importance in the selection of a suitable agent, e.g. Legionella in cooling towers and nonpotable water supply systems, Listeria in the dairy and food industry and HBV in blood-contaminated articles. Dried organic deposits may inhibit penetration of the chemical agent. Where possible, objects to be disinfected should be thoroughly cleaned. The presence of ions in water can also affect activity of antimicrobial agents, thus water for testing biocidal activity can be made artificially ‘hard’ by addition of ions. 48

Toxicity of the agent In choosing an antimicrobial agent for a particular application some consideration must be given to its toxicity. Increasing concern for health and safety is reflected in the Control of Substances Hazardous to Health (COSHH) Regulations (1999) that specify the precautions required in handling toxic or potentially toxic agents. In respect of disinfectants these regulations affect, particularly, the use of phenolics, formaldehyde and glutaraldehyde. Toxic volatile substances, in general, should be kept in covered containers to reduce the level of exposure to irritant vapour and they should be used with an extractor facility. 49

Toxicity of the agent Limits governing the exposure of individuals to such substances are now listed, e.g. 0.7 mg/m3 (0.2 ppm) glutaraldehyde for both short- and longterm exposure. Many disinfectants including the aldehydes, glutaraldehyde less so than formaldehyde, may affect the eyes, skin (causing contact dermatitis) and induce respiratory distress. Face protection and impermeable nitrile rubber gloves should be worn when using these agents. Table 17.4 lists the toxicity of many of the disinfectants in use and other concerns of toxicity are described below for individual agents. 50

Toxicity of the agent Where the atmosphere of a workplace is likely to be contaminated, sampling and analysis of the atmosphere may need to be carried out on a periodic basis with a frequency determined by conditions. 51

Properties of commonly used disinfectants and antiseptics 52

Properties of commonly used disinfectants and antiseptics 53

Types of compound Acids and esters Antimicrobial activity, within a pharmaceutical context, is generally found only in the organic acids. These are weak acids and will therefore dissociate incompletely to give the three entities HA, H+ and A- in solution. As the undissociated form, HA, is the active antimicrobial agent, the ionization constant, Ka, is important and the pKa of the acid must be considered, especially in formulation of the agent. 54

Types of compound Benzoic acid This is an organic acid, C6H5COOH, which is included, alone or in combination with other preservatives, in many pharmaceuticals. Although the compound is often used as the sodium salt, the nonionized acid is the active substance. A limitation on its use is imposed by the pH of the final product as the pKa of benzoic acid is 4.2 at which pH 50% of the acid is ionized. It is advisable to limit use of the acid to preservation of pharmaceuticals with a maximum final pH of 5.0 and if possible < 4.0. Concentrations of 0.05–0.1% are suitable for oral preparations. A disadvantage of the compound is the development of resistance by some organisms, in some cases involving metabolism of the acid resulting in complete loss of activity. Benzoic acid also has some use in combination with other agents, salicylic acid for example, in the treatment of superficial fungal infections. 55

Types of compound Sorbic acid This compound is a widely used preservative as the acid or its potassium salt. The pKa is 4.8 and, as with benzoic acid, activity decreases with increasing pH and ionization. It is most effective at pH 4 or below. Pharmaceutical products such as gums, mucilages and syrups are usefully preserved with this agent. 56

Sulphur dioxide, sulphites and metabisulphites Types of compound Sulphur dioxide, sulphites and metabisulphites Sulphur dioxide has extensive use as a preservative in the food and beverage industries. In a pharmaceutical context, sodium sulphite and metabisulphite or bisulphite have a dual role acting as preservatives and anti-oxidants. 57

Types of compound Esters of p-hydroxybenzoic acid (parabens) A series of alkyl esters of p-hydroxybenzoic acid was originally prepared to overcome the marked pH dependence on activity of the acids. These parabens, the methyl, ethyl, propyl and butyl esters, are less readily ionized, having pKa values in the range 8–8.5, and exhibit good preservative activity even at pH levels of 7–8, although optimum activity is again displayed in acidic solutions. This broader pH range allows extensive and successful use of the parabens as pharmaceutical preservatives. They are active against a wide range of fungi but are less so against bacteria, especially the pseudomonads, which may utilize the parabens as a carbon source.. 58

p-Hydroxybenzoates (R is methyl, ethyl, propyl, butyl, or benzyl). Types of compound p-Hydroxybenzoates (R is methyl, ethyl, propyl, butyl, or benzyl). 59

Types of compound They are frequently used as preservatives of emulsions, creams and lotions where two phases exist. Combinations of esters are most successful for this type of product in that the more water-soluble methyl ester (0.25%) protects the aqueous phase, whereas the propyl or butyl esters (0.02%) give protection to the oil phase. Such combinations are also considered to extend the range of activity. As inactivation of parabens occurs with non-ionic surfactants due care should be taken in formulation with both materials. 60

the main antimicrobial groups as antiseptics, disinfectants and preservatives 61

†Several forms available having x%chlorhexidine and 10x%cetrimide. the main antimicrobial groups as antiseptics, disinfectants and preservatives Also used in combination with other agents, e.g. chlorhexidine, iodine. †Several forms available having x%chlorhexidine and 10x%cetrimide. QAC, quaternary ammonium compound. 62

Alcohols used for disinfection and antisepsis The aliphatic alcohols, notably ethanol and isopropanol, are used for disinfection and antisepsis. They are bactericidal against vegetative forms, including Mycobacterium species, but are not sporicidal. Cidal activity drops sharply below 50% concentration. Alcohols have poor penetration of organic matter and their use is therefore restricted to clean conditions. They possess properties such as a cleansing action and volatility, are able to achieve a rapid and large reduction in skin flora and have been widely used for skin preparation before injection or other surgical procedures. The risk of transmission of infection due to poor hand hygiene has been attributed to lack of compliance with handwashing procedures. An alcohol hand-rub offers a rapid easy-to-use alternative that is more acceptable to personnel and is increasingly being recommended for routine use. However, the contact time of an alcohol-soaked swab with the skin prior to venepuncture is so brief that it is thought to be of doubtful value. 63

Ethanol (CH3CH2OH) is widely used as a disinfectant and antiseptic Ethanol (CH3CH2OH) is widely used as a disinfectant and antiseptic. The presence of water is essential for activity, hence 100% ethanol is ineffective. Concentrations between 60% and 95% are bactericidal and a 70% solution is usually employed for the disinfection of skin, clean instruments or surfaces. At higher concentrations, e.g. 90%, ethanol is also active against most viruses, including HIV. Ethanol is also a popular choice in pharmaceutical preparations and cosmetic products as a solvent and preservative. Isopropyl alcohol (isopropanol, CH3. CHOH.CH3) has slightly greater bactericidal activity than ethanol but is also about twice as toxic. It is less active against viruses, particularly nonenveloped viruses, and should be considered a limited-spectrum virucide. Used at concentrations of 60–70%, it is an acceptable alternative to ethanol for preoperative skin treatment and is also employed as a preservative for cosmetics. 64

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Alcohols as preservatives: The aralkyl alcohols and more highly substituted aliphatic alcohols, are used mostly as preservatives. These include: 1- Benzyl alcohol. This has antimicrobial and weak local anaesthetic properties, used as preservative at concentration of 2% in cosmetics. 2- Chlorbutol(chlorobutanol; trichlorobutanol; trichloro-t-butanol). Used in concentration 0.5% as preservatives in injections& eye-drops. It is unstable, decomposition occurring at acid pH during autoclaving, while alkaline solution are unstable at room temperature. 3- Phenylethanol (phenylethyl alcohol; 2-phenylethanol). Used in concentration 0.25-0.5% , active against G-ve organisms, and employed in conjunction with another agent. 4- Phenoxyethanol( 2- phenoxyethanol). Used in concentration 0.1%,active against Ps.aeroginosa, used in combination with hydroxybenzoates to broaden the spectrum of antimicrobial activity. 5- Bronopol(2-bromo-2-nitropropan-1,3-diol). Used in concentration 0.01-0.1%, it has broad spectrum. Limitation is, when exposed to light at alkaline pH, especially if accompanied by an increase in temperature, solution become yellow or brown. A number of decomposition products including formaldehyde are produced, nitrite ions may be produced and react with secondary and tertiary amines present forming nitrosamines( carcinogenic).

Aldehydes: Gluteraldehyde: It has a broad spectrum activity( high germicidal level), not affected by organic matter. The gluteraldehyde molecule possesses two aldehyde groupings which are highly reactive and their presence is an important component of biocidal activity. At a pH of 8, biocidal activity is greatest but stability is poor due to polymerization. In practice, gluteraldehyde is generally supplied as an acidic 2% or greater aqueous solution, which stable on prolonged storage, then activated by addition of suitable alkalizing agent to bring the pH of the solution to its optimum for activity, these solution have a limited shelf-life( 2 weeks). Glutrealdehyde is employed for the cold, liquid chemical sterilization of medical and surgical materials that cannot be sterilized by other methods.

Aldehydes: Ortho-phthaldehyde: OPA is a recent addition to the aldehyde group of high level disinfectants. This agent has excellent activity in vitro studies, showing superior mycobactericidal activity compared with gluteraldehyde, it requires no activation, is not a known irritant to the eyes or nasal passages and has excellent stability over the pH range 3-9. It is used for disinfection of endoscopes appears promising.

Formaldehyde: Formaldehyed used in liquid or gaseous state for disinfection. In the vapour phase it has been used for decontamination of isolators, safety cabinets and rooms; recent trends have been to combine formaldehyde vapour with low temperature steam (LTSF) for the sterilization of heat-sensitive items. Formaldehyde vapour is highly toxic, carcinogenic if inhaled, thus its use must be carefully controlled. It is not very active at 200C and below, and requires a relative humidity of at least 70%.The agent is not supplied as a gas but either as a solid polymer, paraformaldehyde, or a liquid, formalin, which is a 34-38% aqueous solution. 4% formaldehyde, used for disinfecting surfaces.

Aldehydes: Formaldehyde- releasing agents: Various formaldehyde condensates have been developed to reduce the irritancy associated with formaldehyde while maintaining activity, and these are described as formaldehyde- releasing agents or masked- formaldehyde compounds. Noxythiolin (N-hydroxy N- methylthiourea): It has extensive antibacterial and antifungal properties and is used both topically and in accessible body cavities as an irrigation solution and in the treatment of peritonitis. Polynoxylin (poly[ methylenedi( hydroxymethyl) urea]): It is a similar compound available in gel and lozenge formulations. Taurolidine ( bis[1,1-dioxoperhydro-1,2,4-thiadiazinyl-4]metane) : It is a condensate of two molecules of the amino acid taurine and three molecules of formaldehyde. It is more stable than noxythiolin in solution and has similar uses. The activity of taurolidine is greater than that of formaldehyde.

Biguanides: 1- Chlorhexidine and alexidine: Chlorhexidine is a compound related to the biguanide antimalarial proguanil. Compounds containing the biguanide structure could be expected to have good antibacterial effects, thus the major part of the proguanil structure is found in chlohexidine. A related compound is the bisbiguanide alexidine, which has use as an oral antiseptic and anti-plaque agent. Chlorhexidine base is not readily soluble in water, therefore the freely soluble salts, acetate, gluconate, and hydrochloride are used in formulation. Chlorhexidine exhibits the greatest antibacterial activity at pH 7-8 where it exists exclusively as a dication. The cationic nature of the compound results in activity being reduced by anionic compounds including soap due to the formation of insoluble salts.

Factors affecting the activity of antimicrobial agents: 1- Temperature: In general, as the temperature increased in arithmetical progression, the rate of kill increase geometrically. The effect of temperature increase on the rate of bactericidal activity at a fixed concentration and inoculum size is expressed quantitatively as a temperature coefficient, usually in θ10 value: which is the change in activity per 100C rise in temperature, e.g. rising the temperature of phenol from 200C to 300C increased the killing activity by a factor of 4. θ10 value may be calculated by determining the extinction time at two temperature differing exactly by 100C. θ10 =Time require to kill at T0 Time require to kill at(T+10)0 * This value is constant for each compound. Compounds with high θ10 are more effective with increases in temperature than those with low θ10.

θ10 Compound 30- 50 Aliphatic alcohol 3- 5 Phenols 1.5 Formaldehyde

2- Concentration: The rate of kill of bacterial population is directly affects with concentration or dilution, therefore slightly increase or decrease in the concentration of certain agents can increase or decrease their bactericidal effect. The graph plotting the log 10 of a death time(i.e. the time required to kill a standard inoculums) against the log 10of the concentration is usually a straight line, the slope of which is the concentration exponent(η) η= concentration exponent (dilution coefficient),which is measure the effect of changes in concentration(or dilution) on cell death rate η=log t2 – log t1 log c1 – log c2 It is constant for each compound, and the dilution does not affect the cidal attributes of all disinfectants in a similar manner. Therefore compounds with low η are less affected by dilution, but those with high η will be readily inactivated.

Reduce the activity (the time required) No. of dilution η Compound 21 double the time 2 1 Mercuric chloride 31 three times 3 Formaldehyde 26 64 (the time) 36 726 (the time) ½ 1/3 6 Phenol

:3- PH Changes in PH of the medium affect both bacterial cell and disinfectant activity. Bacterial growth is optimums at PH 6- 8, any change in this PH will decrease the bacterial growth as well as change the physicochemical state of their surface. The degree of ionization of acidic or basic disinfectant will obviously depend on the PH.

Evaluation of liquid disinfectants: 1. Suspension Tests: Essentially are tests of sterility a. Phenol Coefficient Test: * Rideal – Walker (RW) test * Chick – Martin (CM) test b. Capacity use – dilution test Kelsey – Sykes (SK) test 2. Quantitative suspension test

The Rideal- Walker Test: 1903 Briefly, dilutions of the disinfectant are compared with standard dilution of phenol( from 1 in 95 to 1 in 115) for their lethal activity against Salmonella typhi NCTC786 To each 5 ml volume of disinfectant or phenol solution in distilled water held at 17-18oC is added 0.2 ml of 24 hour culture. At intervals of 2.5,5,7.5 and 10 minutes, subcultures using a standard loop are made into 5 ml volume of broth; these are than incubated for 48- 72 hours at 37oC after which presence or absence of growth in each broth is recorded.

Contact time(min.) of culture and disinfectant Dilution Disinfectant 10 7.5 5 2.5 _ + 1 in 250 X 1 in 300 1 in 350 1 in 400 1 in 100 Phenol

The phenol coefficient of the disinfectant(X) is calculated by dividing the dilution of X which allows survival of test organism at 2.5 and 5 but not at 7.5 and 10 minutes by dilution 0f phenol giving the same response. The Rideal- Walker phenol coefficient(RW) of disinfectant X is: RW of X =300 = 3 100

The Chick- Martin Test: 1908 Chick & Martin, realizing that disinfectants usually had to act in the presence of organic material, suggested the inclusion of 3% dried human feces in the test. They also considered the 10 minutes disinfection time allowed in the Rideal- Walker test was too short and they introduced a 30 minutes contact time with subcultures in duplicate at the end of this. A different strain of Salmonella typhi from that of (RW) test was used. The human fecal suspension was later replaced by dead yeast cells and the Chick-Martin test become the subject of British Standard Specification BS 808: 1938.

Series of dilutions of the unknown disinfectant and of phenol are made in distilled water in regular diminishing stages of 10%. To 2.5 ml volume of these held at 200C are added 2.5 ml of the culture- yeast suspension(2 ml of a 24 hours Salmonella typhi culture + 48 ml of 5% dry weight yeast suspension). After a contact time of 30 minutes, a standard loopful of disinfectant- culture- yeast mixture is transferred in duplicate to 10 ml of broth. These broth tubes are incubated at 370C for 48 hours, then the presence or absence of growth is recorded. The Chick- Martin Coefficient (CM) is calculated by dividing the mean of the highest concentration of phenol permitting growth in both subcultures and the lowest concentration showing absence of growth in both subcultures by the corresponding mean concentration of the unknown disinfectant.

Subculture tube Disinfectant X(%) Phenol(%) 2 1 _ 1.00 2.00 0.90 1.80 + 0.81 1. 62 0.73 1.46

Mean of phenol concentration is: ½ ( 1.80+ 1.62)=1.71 Mean of disinfectant X concentration is: ½ (0.90+ 0.81)=0.85 Chick- Martin Coefficient of X= 1.71 = 2.0 0.85 If growth occurs in one, but not in the other, of the a pair of subculture tubes the concentration value corresponding to that pair is used. Chick- Martin Coefficient of X = 1.62 = 2.0 0.81

Subculture tube Disinfectant X(%) Phenol(%) 2 1 _ 1.00 2.oo 0.90 1.80 + 0.81 1.62 o.73 1.46

Capacity use- dilution tests: There are four test organisms, Pseudomonas aeroginosa, Proteus vulgaris, E.coli and Staphylococcus aureus; the bacteria are suspended in standard hard water for the test under clean conditions, and in a yeast suspension for the test under dirty conditions, the disinfectant is diluted in hard water. To 3 ml of each disinfectant dilution add 1 ml of the bacterial suspension prepared in broth, yeast, or serum as required, and shake gently. After 8 minutes, remove the sample of the mixture with a dropper, pipette and transfer one drop to each of five tubes of the liquid recovery medium.

Alternatively, five drops may be placed separately on a nutrient agar plate. Two minutes later, i.e. 10 min. after the first inoculation, re-inoculate the disinfectant mixture with a further 1 ml of bacterial suspension, and 8 min. later subculture as before. A further 2 min. later ,i.e. 20 min. after the first inoculation, repeat the process again. The initial test is carried out at 20 to 22o C, and all subcultures are incubated at about 32o C for 48 hours. Record the number of tubes showing growth in the liquid medium or the number of colonies growing on the surface plate cultures. A capacity test (the Kelsey- Sykes): use- dilution 1.o%

Number of subculture broth ( out of five) showing growth after Disinfectant Concentration (%) 28 min. 18 min. 8 min. 3 2.0 5 2 1.0 4 1 0.5 o.25

Quantitative suspension tests: The number of survivors expression as the percentage remaining viable at the end of a giving time may be determined by viable counts & this parameter is often used in assessing bactericidal. After exposure of bacterial cells to the disinfectant, surviving organisms can be counted by two techniques , either by direct culture or by membrane filtration. The basic principle of the quantitative suspension tests using direct culture is as follows: after contact with the disinfectant, a sample of the reaction mixture is inoculated on a solid nutrient medium; after incubation the number of survivors is counted and compared with initial inoculums size.

The pour- plate technique as well as surface plates may also be used for subculturing. The decimal reduction rate, or Microbial Effect(ME) can be calculated using the formula: ME= log NC- log ND ( NC: CFU in control, ND: CFU after exposure to disinfectant). ME= ( 4+ 2.04) – (1+ 1.94) = 3.10 ( after 5 min.)

No. of CFU Dilution of subculture in disinfectant in control _ tntc 100 1.94 88 10-1 0.78 6 10-2 o 10-3 2.04 110 10-4

Methods which measure only growth inhibition( bacteriostasis) are: 1. Serial dilution: Graded doses of the test substance are incorporated into broth and the tubes inoculated with the test organisms and incubated. The point at which no growth occurs is taken as the bacteriostatic concentration( Minimum Inhibitory Concentration, MIC). It is essential when performing these tests to determine the size of the inoculum at the position of the endpoint varies considerably with inoculum size, which should always be defined in any description of results.

Tube contents for determining the MIC of phenol The test is carried out in practice by mixing the appropriate volume of the solution under test with double- strength broth and making it up to volume with water as illustrated in table below: Tube contents for determining the MIC of phenol Final volume 5 Double-strength broth 4 3 2 1 0.5% phenol solution Sterile distilled water 0.25 0.2 0.15 0.1 0.05 Final con. phenol (%w/v)

2. Ditch- plate technique: The test solution is placed in a ditch cut in nutrient agar contained in a petridish, or it may be mixed with a little agar before pouring into the ditch. The test organisms( as many as six may be tested) are streaked up to the ditch. The plate is then incubated. 3. Cup- plate technique: The solution is placed in contact with agar, which is already inoculated with the test organism and after incubation, zones of inhibition observed. A method used widely in antibiotic assays. The solution may be placed in a small amount in a well cut from agar with a sterile cork- borer.

4. Disc tests: These are modification of the earlier cup or ditch- plate procedures where filter-paper discs impregnated with the antimicrobial agents. For disc tests, standard suspensions are prepared and inoculated onto the surface of appropriate agar plates. Commercially available filter-paper discs containing known concentrations of antimicrobial agent. 5. E-tests: The most presently accepted method of determining bacterial MICs, however, is the E(Epsilometer)-test. Basically this is performed a similar manner to the disc test except that nylon strips that have a linear gradient of antimicrobial lyophilized on one side and the on the other side of the nylon strip are a series of lines and figures denoted MIC values.

Cup- plate Disc- plate

E- Test:

6. Solid dilution method: In this method the dilutions of the substance under test are made in agar instead of broth. The agar containing the substance under test is subsequently poured onto a petridish. It has the advantage that for any one concentration of the test substance, several organisms, may be tested. 7. Gradient- plate technique: In this technique the concentration of a drug in an agar plate may be varied infinitely between zero and a given maximum. To perform the test, nutrient agar is molted, the solution under test added, and the mixture poured into a sterile petridish and allowed to set in the form of a wedge(A). A second amount of agar is then poured onto the wedge and allowed to set with the petridish flat on the bench.

8. NCCLS: Regularly updated guidelines have been provided by the National Committee for Clinical Laboratory Standards (NCCLS) and are widely used in many countries.