DISINFECTANTS

1. DISINFECTANTS

2. INTRODUCTION • Disinfection- It is nothing but the destruction of microbial organisms excluding the spores, not necessary killing all the microorganisms but reducing them to a safe level, which is not harmful to health. • Hence disinfectants are applied to inanimate objects. • Disinfectant- It is an agent used for the destruction of the organisms on the inanimate objects. They have bactericidal action.

3. INTRODUCTION • Sanitization- It is the colloquial word for the cleaning action/disinfection is termed as sanitization. • Bactericide- It is a chemical agent capable of killing bacteria (vegetative cells) but not necessarily the spores. • Bacteriostatic agent- It is a chemical agent capable of preventing the growth of bacteria. They do not kill the organisms but they prevent the growth of the organisms.

4. INTRODUCTION • Fungicide- These are the agents capable of killing fungi and fungal spores. Fungistatic agents prevent the growth of fungal spores. • Germicides- Germ refers to all pathogenic micro organisms capable of killing all forms of microorganisms. Like wise viricidal which kill viruses. • Sporicides- Kills the spores of all microbes. • Algicide- Kills algae.

5. INTRODUCTION • Antibiotics- these are the chemical substances produced by bacteria and moulds. Now-a-days the antibiotics can be prepared synthetically by altering the genetic sequence of microbes. •  Antiseptic- Agents used in case of sepsis. These are used for destruction of microbes but not all living tissue. Not necessarily killing all the micro organisms but they reduce them to a level which is not harmful to health.

6. IDEAL PROPERTIES OF DISINFECTANTS • It should have wide spectrum of activity, which is effective against all microbes. Ex-Virus, fungi, protozoa, algae and bacteria • Disinfectants should have high potency under all the conditions of use. • It should be readily soluble or miscible with water at all effective anti microbial conc. • It should have low degree of toxicity. • It should not be caustic. • It should only disinfect the surface without harmful effect on delicate tissue.

7. IDEAL PROPERTIES OF DISINFECTANTS • It should not have damaging effects on clothes, metals, even on rough surface • It should not be active in presence of water and organic matter. • It should have high penetrating power • It should be stable. • It should be compatible with other antiseptics. • It should be safe and easy to use. • It should not have any offensive odour or colour • It should produce quick action. • It should be effective in both acidic and alkaline medium.

8. PHYSICAL, CHEMICAL, BIOLOGICAL INDICATORS FOR STERILIZATION

9. PHYSICAL, CHEMICAL, BIOLOGICAL INDICATORS FOR STERILIZATION • DRY HEAT - 180oC for 2hr. • RADIO STERILIZATION - Plastic dosimeter. • FITRATION - Bubble point test. • CHEMICAL INDICATOR S – Moist heat -Browning tubes type 1 and 2 color change (Red to yellow to green) and Autoclave.

10. MODES OF ACTION OF DISINFECTANTS 1. Primary Cell Interactions - Usually disinfectants are adsorbed by the cells. • Ex: Phenol, Iodine, Chlorohexidine, basic dye, etc.

11. MODES OF ACTION OF DISINFECTANTS 2. It shows electrophoretic mobility changes i.e., movement of micro organisms can be increased or decreased or even stopped due to the change in the surface electric charges. • Ex-Cell wall of the microbes are negatively charged. Phenols, halogenated phenols which increases the rate of migration of aerobacter which is monotrichous in nature,Chlorohexidines decreases the migration rate.

12. MODES OF ACTION OF DISINFECTANTS 3. Cell Permeability Modifications /Leakage of Cellular Constituents - It is called partial lysis of microbial cells. • Ex - Hexyl resorcinol, acetyl tri methyl ammonium bromide and chlorohexidine with phenols brings about the lysis. Phenolicaldehyde, mercuric chloride and mercuric hypochlorate brings about lysis of the microbial cells.

13. MODES OF ACTION OF DISINFECTANTS 4. Protein Coagulation of Microorganisms - It brings irreversible coagulation of the cellular protein component. • Ex- Phenolic compound, acetyl trimethyl ammonium chloride, phenol chlorohexidine brings about coagulation of protein.

14. MODES OF ACTION OF DISINFECTANTS 5. It affects enzyme systems there by affecting metabolic functions. • Ex- Alcohol, phenols, etc

15. MODES OF ACTION OF DISINFECTANTS 6. Oxidation of Cell Cytoplasm - It deals with the interaction with the sulphahydral groups, brings about oxidation. • Ex-Mercuric compounds, silver, halogens, copper, etc. Still other compounds are metallic compounds, amine groups, etc. Triphenyl methane dye brings about denaturation of the cell wall components. Acid groups and acrylidine dye brings about damage to nucleic acid function of the microorganisms.

16. TYPES OF DISINFECTANTS 1. Phenols - Phenol, cresol, chlorocresol, chloroxylenol, ortho phenyl phenol, hexachlorophane are the phenolic groups used as disinfectants. 2. Alcohols - Ethanol, Methanol, Isopropanol, 2-phenoxyethanol, 2-phenyl ethyl alcohol, chlorobutanol are used as the chief source of disinfectants.

17. TYPES OF DISINFECTANTS 3. Aldehyde - Formaldehyde,glutaraldihyde, are halogenated disinfectant , 4 chlorine .Iodine, Chloramines and Hypochlorides are used as halogenated disinfectants. 5. Metallic compounds - Mercury compounds, silver and copper.

18. TYPES OF DISINFECTANTS 6. Dye - Acrylidine dye, tri phenyl methane dye, guanidine dye anidinedye,orthobromopropamadine, [propidine] chlorohexidine are the dyes used as disinfectants. • Furan derivatives nitro furasone is also used as disinfectant. Cationic surfactants, quinoline, and isoquinoline derivatives are used as disinfectants.

19. FACTORS AFFECTING DISINFECTANTS • Time of contact. • Concentration of the disinfectant. • Temperature. • Type of organisms present. • Presence of organic matter in the cells and the other activities of the cells. • Hydrogen ion concentration or pH of the disinfectant. • Surface tension.

20. FACTORS AFFECTING DISINFECTANTS • Formulation of the disinfectant. • Chemical structure of disinfectants. • Nature of the surface to be disinfected. • Penetration power of the disinfectant. • Potentiation of the organisms which includes synergism and antagonistic effect of organisms.

21. TIME OF CONTACT • The killing effect of the disinfectant is dependent on the time of contact between the organisms and the disinfectant. • This is expressed by death curve or mortality curve. It is a graphic representation or plot of viable count of organisms against the time of contact. • The death rate can be expressed using principles of first order kinetics.

22. CONCENTRATION OF DISINFECTANT • More of the concentration, higher will be the disinfectant action. The relation between the time taken to kill the organisms and the temperature is exponential. • Ex: The dilution of phenol reduces the disinfection activity.

23. TEMPERATURE • Increase in temperature increases the bactericidal activity of the organisms. As the temperature increases arithmetically the activity of the disinfectant increases geometrically. • Ex - Disinfectant like phenol, used against Ecoli, kills all of them in 62min at 20C. Hence increase in temperature increases the activity of the disinfectant.

24. TYPE OF ORGANISMS • The action of disinfectant is dependant on the type of organisms, number of organism, condition of the organisms Example • Resistance of organisms /susceptibility of organisms for the condition of the organisms. • Most of the vegetative cells of bacteria / microorganisms except acid fast bacilli are killed by the chemical disinfectants however the spores are not destroyed.

25. Most of the aldehyde is sporicides • Aldehyde with halogens together is good virucides. However many disinfectants have limited spectrum of activity because of the genetic stability of the microorganisms. • The effectiveness of the disinfectant is dependent on the initial number of organisms present. • Resistance of microbe is also an important factor which affects the disinfectant activity. The resistant capacity of the organisms varies with the age and the condition with which they are grown. • In lag phase they are susceptible, in log phase they are highly resistant, in the exponential phase they are resistant, and in the death phase they are susceptible.

26. PRESENCE OF ORGANIC MATTER • Organic matters are the extra cellular protein discharged by the micro organisms which may interfere with disinfectant activity. • The organic matter may form a protective coat over the bacterial cell wall, chemically it may interact with disinfectant to form an inactive compound thus decreasing the activity of the disinfectant. • The presence of fats and oils over the organisms may reduce the disinfectant activity of the phenols and phenolic compounds.

27. pH • The pH may alter the rate of growth of microorganisms • The degree of ionization of disinfectant may change with pH. • The absorption of chemical disinfectant on the cell surface may change. Usually microbes grow best in the pH 6.5-7.5 hence at this pH the concentration of the disinfectant is less effective. Only at higher concentration or at lower concentration is required to get the desired effect of the disinfectant. Some of the disinfectants are effective in the ionized form and the others in unionized form. • Ex- Benzoic acid, phenol, salicylic acid-unionized form

28. SURFACE TENSION AND effect of FORMULATION and preparation. • Ex-Addition of alcohol in the preparation of the disinfectant increases the effect, because, alcohol increases the penetration capacity of the disinfectants.

29. EVALUATION OF DISINFECTANTS/ EVALUATION OF BACTERIOSTATIC ACTIVITY • Serial dilution method in fluid medium. • Serial dilution method on solid medium. • Cup plate method. • Fish spine bead method. • Cylinder method. • Filter paper method/ disc diffusion method. • Gradient plate method. • Ditch plate method

30. SERIAL DILUTION METHOD IN FLUID MEDIUM • Different concentration of the disinfectants are prepared (gradient concentration of the disinfectant are added to nutrient medium) • The tubes are labeled and to each of the test tube known organisms are inoculated and incubate for 48hr at a specific temperature. • The growth is observed by minimum inhibitory concentration by turbidometric method or by UV spectrophotometric method.

31. Note: The minimum inhibitory concentration varies with the condition of the test tube with the variation in temperature; it varies with pH and type of organisms. The test results are required to be given with the condition for the test and the reagents used because MIC varies with the type of nutrient, conditions with which the test are performed, temperature and pH.

32. SERIAL DILUTION METHOD ON SOLID MEDIUM • Double strength agar medium is prepared • Mixed with equal volume of disinfectant and sterilize. • Pour it into petri dish and allow to solidify. • Keep it at 37oC for 1day so that if any contamination ,it will be enlighten. If not the plates are transparent, clear with clean surface. • Organisms are streaked over the agar / on the surface and incubate it for 3 days then observe for the growth. • Note: Different plates are used for different concentration of the disinfectants.

33. Advantages of Serial Dilution Method • This method is useful for the disinfectants that give turbidity with fluid nutrients and multiple point inoculation; different organisms can be tested in the same petri dishes. • This method is economical as different organisms can be evaluated in a single petri plate.

34. CUP-PLATE METHOD • Prepare agar nutrient medium and maintain it at 45o-50oC. • Inoculate the test organisms, and then pour it into petri dishes this method is known as pour plate method. • On the agar petri dishes cups/holes of 8mm diameter are made using sterile borer, the various antibacterial agents of (different concentration are poured into different cups) • Incubate for specific time at a particular temperature and observe the results.

35. FISH SPINE BEAD METHOD • Different concentrations of disinfectants are prepared. • Each fish spine bead is dipped in each concentration and place the beads on the agar plates. • Different concentration of the disinfectant can be evaluated.

36. CYLINDER plate METHOD • This method is very similar to cup plate method. • Cylinder should be opened at both the ends on the upper and on the basal surface of the agar that facilitates the diffusion of the disinfectants.

37. ADVANTAGE AND DISADVANTAGE OF FILTER PAPER METHOD • Advantage - Effect of different concentration of the different disinfectant on the organisms can be evaluated. • Disadvantage - This method can give false results as the zone of inhibition is dependent on the ability of the disinfectant, capacity of the organisms or the diffusion capacity of the disinfectants, through the agar medium, nature or virulence of the organisms, temperature of the particular environment.

38. GRADIENT PLATE METHOD • Here the two layers of the agar are prepared in the same petri plate .Wedge A is only agar and wedge B is agar + disinfectant. • These plates are placed overnight for the diffusion of the disinfectants. • After 48hr the concentration of the disinfectant can be more towards one side and towards the other side it is less. • Test organisms usually salmonella typhi or staphylococcus aureus is streaked in the same line of the slope of the agar, the line is known as the (line of concentration gradient).

39. GRADIENT PLATE METHOD • Keep it in the incubator and observe the growth of the organisms and record the minimum inhibitory concentration by the formula • MIC = (Conc of disinfectant x (length of the growth of in total volume) the organisms in cm) Total length of streaking/total length of the growth of organisms

40. DITCH PLATE METHOD • In this method a solid agar plates are selected and dig a ditch or through is made with the help of sterile scalpel and the particular type of the disinfectant is added up to 3/4th of the ditch. • Then the test organisms are streaked outside from the ditch towards the petri plates edge ,and keep it for incubation and observe the growth.

41. Advantages 1. Zone of inhibition can be identified within the same plate. 2. In the same plate, effect of particular disinfectant on different type of organisms can be evaluated. • Disadvantage 1. The specificity of one organism may alter the activity of another organisms or the diffusion of the disinfectant may alter with the medium, or antagonism of the adjacent organisms.

42. LABORATORY SPECIFIED TESTS/EVALUATION OF BACTERICIDAL ACTIVITY

43. PHENOL COEFFICIENT TEST • It is estimated by the evaluation of phenol co-efficient Rideal-Walker test. • The test is called Rideal-Walker coefficient test- In this test the bactericidal activity of a disinfectant is evaluated by comparing it with that of a specific concentration of phenol. • Requirements - Standard Rideal Walker broth, 24hr culture of salmonella typhi, inoculating loop, glass wares and other requirements, etc.

44. PROCEDURE • Prepare different concentration of the test disinfectants in water • Prepare phenol solution in 105ml of water as control-(American public health association has given standards) • To each of these add 0.2ml of 24hr culture of salmonella typhi. This is known as reaction mixture. • Organisms are added according to the time phase manner • keep the reaction mixture at 17.5oC [+or-0.5c] • Select the subculture and each reaction mixture and transfer it into a broth at 0min 2.5min, 5min, 7.5min and 10min. • These broth tubes are incubated at 37oC for 48-72hr and observe the growth.

45. PROCEDURE • The test is duplicated to get the accurate results. • The Rideal-Walker coefficient for phenol is 1. • Lysol is between 3-4 RWC • White phenol is between 10-11 RWC • Black phenol is between 14-15 RWC

46. ADVANTAGES • This test thoroughly describes the coefficient and thoroughly reproducible. • It can be cheaply carried out and quickly analyzable. • Effectiveness of the disinfectant can be expressed quantitatively.

47. DISADVANTAGES • Use of salmonella typhi as test organisms is not feasible. • Some organisms are not particularly resistant to the disinfectants. • Use of more resistant organisms is not feasible for the accurate results. • Some of the organisms will not respond quickly. Its effects may not be observed in intermittent time. • The test can be compared at a particular temperature. • Tissue toxicity is also one of the factors and the organic matter present in the culture media may lead to miss-results. • It is more valid for phenolic compound than other disinfectant like formaldehyde. • Chances of sampling air contamination is more. • Not suitable for gram positive organisms as they are more resistant.

48. CHICK MARTIN TEST • This test is similar to the RWC test. Instead of water, yeast suspension is used. • In this test reaction mixture is prepared, yeast suspension is used that makes the test more severe as the activity of some disinfectant is greatly reduced or depressed in the presence of organic matter like yeast extract.

49. FIXED CONCENTRATION METHOD • In this method fixed concentration of the disinfectants are used but the organisms are introduced at different time intervals and the death point is calculated.

50. PROCEDURE • A particular concentration of the disinfectant is prepared. • Test organisms are added to the reaction mixture kept at a controlled temperature. • Distribute uniformly to number of test tubes • At different time interval nutrient broth is added to all the test tubes. • Now the tubes are incubated for growth for 72hr and estimate turbidometrically and the test is repeated 4-5 times and the mean time or the extinction time is determined. • [According to standard estimation 1% of phenol solution on Ecoli shows extinction period of 75min.]