CHAPTER – IV DISINFECTION

CONTENT OF THE CHAPTER GENERAL EVALUATION Introduction Terminologies Classification Properties of disinfectant Factors Affecting disinfectant action EVALUATION Bacteriostatic, Bacteriocidal evaluation Phenol Coefficient test

DIS - INFECTION Destruction of microorganisms, but not usually spores: not necessarily killing all microorganisms but reducing them to a level of not harmful to human health. Commercially for the treatment of inanimate objects and materilas.

Commercially Available Disinfectants And Their Applications s.no Enzyme Application(s) 01 Dettol Anti-infective 02 Lysol Floor Cleaning 03 Phenol Gargles, mouth washes 04 Chlorxylenol Active against staptococci 05 Chlorhexidine Skin disinfectant

TERMINOLOGIES Bactericide Killing bacteria Bacteriostat Preventing the growth of bacteria Fungicide Killing fungi incl. Spores Fingistat Prevents fungal proliferation Germicide Kills all microorganisms Virucide Kills virus Sporicide Kills spores Algicide Kills algae Antibiotic Produced by and act against microbes Antisepsis Destruction of m/o but not bac.spores on living tissue

Classification of Disinfectants Examples Groups of DI Phenol Phenol, xylenol, chlorphenols, chlorxylenols,,etc…. Alcohols Etanol, isopropanol,Iso propyl alcohol, amyl alcohol, etc…. Aldehydes Formaldehyde, glutaraldehyde, Dyes Acridine, triphenylmethane, acriflavine, proflavine, crystal violet, etc Surface active agents Cetremide, sodium stearate, sorbiton, tween 80, etc…

Groups of DI Examples Halogens Chlorine, bromine, iodine, etc… Metals Copper, aluminium, silver, mercury Guanidines & amidines Chlorhexidine, dibromopropamidine isothionate , etc… Furan derivatives Nitrofurazone,furan, etc… Quinolines and isoquinolines Hydroxyquinoline, dequalinium chloride, etc….

Mechanism of action of disinfectants

High conc. - Act as protoplasmic poison DISINFECTANT MECHANISM OF ACTION Phenol Low conc.- Disrupt cytoplasmic membrane. – leakage of cellular constituents High conc. - Act as protoplasmic poison Alcohols Denature the protein – 60-90% best active. Aldehydes Combine with amino group of protein Dyes Inactivate bacteria by reacting with acidic group of cell. Inhibit glutamine synthesis. Inhibit cell wall systhesis. Surface active agents +vely charged Quart.amm.comp strongly absorbed on the –velty charged bacterial surface. – damage cell membrane – leakage…

Halogenate or oxidize the vital cell wall components. Metals DISINFECTANT MECHANISM OF ACTION Halogens Halogenate or oxidize the vital cell wall components. Metals Hg, Ag, Cu – protein precipitants. Toxic part react with enzyme and affects the protein structure Guanidines & amidines Disrupt cell wall followed by destruction. Furan derivatives Inhibits H2 and electron transport chain (uncertain). Quinolines and isoquinolines Form a complex with iron , copper to form toxic complex.

Properties of Disinfectants Phenol Produced From Coaltar Distillaries.Activity Reduced By Dilution. More active in acidicpH. Organic matter – dimnishes its action Alcohols Most active against vegetative bacteria. Methanol vapour is fungicidal Aldehydes Gas or solution – sporicidal. Virus infectivity can be reduced in vaccine preparation Dyes Inhibit Staphylococci, Streptococci. Crystal violet destroy only staphylo not strepto – selective medium Surface active agents Soaps – anitbacterial, cetrimide- bactrecidal.

Treatment of water supplies, swimming pool, deodorant in sewage. DISINFECTANT PROPERTIES OF DISINFECTANT Halogens Treatment of water supplies, swimming pool, deodorant in sewage. Metals Slow in action. Corrosive in tissue and metals. Acetates and nitrates of Hg more effective antibactericidal, antifungal Guanidines & amidines Wide rage of G+ve, G-ve. Skin disinfectant – against pseudomonas, proteus Furan derivatives Antibactericidal against most vegetative bacteria. – included-ointment preparation Quinolines and isoquinolines Bactericidal, fungicidal . Lozenges, mouth washes, throat infections.

Factors Affecting Disinfectant Action Time of contact Conc. of disinfectant Temperature Type of m/o. present, its number & condition Hydrogen ion conc. Surface tension The formulation if the disinfectant The chemical structure of the disinfectant The nature of surface to be disinfected. Potentiation, Synergism and Antagonism of disinfectant

1.Time of contact Viable count Vs Time – Death curve a b c C B A LOG NO. OF SURVIVALS a b plot of graph between no. o f viable cells against time also called as mortality curve. c C B A TIME

A- Curve often obtained with high conc. DEATH CURVE A- Curve often obtained with high conc. B- Disinfection Process Obeying First Order Kinetics C- Sigmoid curve a- slow initial kill of more sensitive org. b- faster “near linear” average resistant org. c- slower death rate of more resistant org. Rate k = 1/ t log N / n 1907 - by Madsen and Nyman, Chick N- Initial number of organisms n- final number of organisms t - time for viable count to fall from N to n in the text book it was wrong marking of A, B ,C

Concentration 2. Concentration LOG NUM. OF SURVIVALS A = 0.66% w/v 0100 LOG NUM. OF SURVIVALS A = 0.66% w/v B = 0.55% w/v C = 0.50% w/v D = 0.44% w/v 10 A B C D 1.0 Relationship between concentration and the time taken to kill the 0.1 0.001 TIME - MINUTES

Concentration B 1 6 1 Log t 1 A Log C

Comparison of increased and decreased concentration on killing rates of Phenol and Formaldehyde Phenol : n = 6 0.7% kills in 100 mins. Doubling :1.4% kills in 100/2n 100/26 = 1.6 min Halving : 0.35% kills in 100X2n 100X26 = 6400mins Formaldehyde : n = 1 0.7% kills in 100 mins Doubling: 1.4% kills in 100/2n 100/21 = 50mins Halving : 0.35% kills in 100 X 2n 100X21 = 200 mins

Temperature K1, = reaction velocity at temp T1, Increase in the temp, increases the velosity, increases the bactericidal action Ө(T2-T1) = K2/K1 K1, = reaction velocity at temp T1, K2 = reaction velocity at temp T2 Reaction velocity K = 1 / extinction time (Extinction time = time at which no living cells can be detected from the sample taken) 2.5 2.0 Extinction time 1.5 1.0 0.5 temperature 10 20 30

Type Of Organism Present, Its Number, Cndn, Aldehyde + Halogen + β-Propiolactone - ANTIVIRAL Presence Of Organic Matter, Any In activators Extracellular protein – reduction in bactericidal action Oils & Fats – Decreases Phenol activity Hydrogene ion Concentration Affects the rate of growth of microbes Degree of ionization of disinfectant & potency Adsorption of disinfectant at cell surface Phenol, Benzoine, Salycilates are active in acidic pH Acridine - active in ionized from Surface tension

The formulation of disinfectant Increase in conc. of soap will increase the extinction time. Alcoholic formulation increases the activity The chemical structure of disinfectant Alkyl chain up to 6 increases the activity Alkyl chain more than 6 decreases the activity Straight alkyl chain give greater activity than branched Halogenation increases the activity Nitration increases the activity The nature of surface to be disinfected Uneven, porous, cracked surface resist chemical disinfectant Plain, clean impenetrable surface increases the activity Potentiation, Synergism and Antagonism

EVALUATION OF DISINFECTANT

Evaluation of Disinfectant Evaluation of Bacteriostatic disinfectant Serial dilution in liquid media Serial dilution in solid media Fish Spine bead method Cylinder plate method Filter paper disc method Cup and Plate method Gradient Plate technique Ditch Plate method

Serial dilution in liquid media Media + 0.2ml of Culture + Test DI Dilution control I st Dil 2nd Dil 3rd Dil 4th Dil 5th Dil

Serial dilution in solid media Solid media + culture + Test DI control Ist dilution I st Dil 2nd dilution IInd Dil control 5th dilution 3rd dilution 4th dilution 5th Dil 4th Dil 3rd Dil

Fish spine bead, Cylinder plate, Cup and plate, Filter paper disc method Cup Shape Fish Spine Bead Beaker containing Test Disinfectant Beaker containing Test Disinfectant Paper disc

Assessment of Bacteriostatic activity Zone of Inhibition Growth of organism on Agar Media 1.0 1.5 Zone of Inhibition 0.5 control 2.0 2.5 Dilution of Test disinfectant

Evaluation of Disinfectant Evaluation of Bactericidal disinfectant Phenol Coefficient test End point technique Extinction time method Rideal –Walker Coefficient method FDA Chick Martin test AOAC Crown Agent technique

Schematic Diagram of Phenol Coefficient test Broth tubes Reaction mixture 1 in 1000 0.5 1 in 1100 S.Typhi 24 hrs culture 1 in 1200 1.0 1 in 1300 1.5 2.0 Control Phenol 1 in 105 Incubate 2-3 days at 370C 2.5 5.0 7.5 10 Temp 17.5

R.W.COEFFICIENT There are two types of extinction time method In which the extinction time is fixed and the conc. Of disinfectant needed to kill in the specified time is estimated. In which the conc. of bactericide is fixed and the extinction time is estimated.

R.W.COEFFICIENT Standard: Rideal Walker Broth Organism: Salmonella Typhi Dilution of Phenol 1 in 105 Dilution of test DI = 1in 1000, 1 in 1100, 1 in 1200, 1 in 1300. Test DI + 0.2 ml of S.Typhi Reaction mixture should be at 17.50C temp. Subculture of each reaction mixture are taken and transformed to broth after 2.5, 5, 7.5 and 10 mins. Broth tubes are incubated at 370C, for 48 to 72 hrs, examined. Presence or absence of growth was noted

Observed results in table Minutes culture exposed to disinfectant 2.5 5.0 7.5 10 Dilution + - + - + - + 1 in 1000 1 in 1100 1 in 1200 1 in 1300 Test disinfectant - Phenol 1 in 105 + + - + = Growth - = No growth

R.W. Coefficient R.W. Coefficient = Dilution of test disinfectant killing in 7.5 mins, but not in 5 mins. / Dilution of Phenol killing in 7.5 mins, but not in 5 mins. 1100/105 = 10.47.

Essential difference between phenol coefficient tests

Medium pH Medium volume Rxn Mix Volume Diluent Reaction Temp Organism Sampling Time (Mins) Calculation RWC FDA` CMC AOAC Crown Agents 7.3-7.5 5.0ml Water 17.5 S.Typhi 2.5,5.0,7.5,10 Dil of test killing in 7.5 but not in 5 min / Same for phenol 6.8 10.0ml 20 5, 10, 15 Dil of test killing in 10 but not in 5 min / same for phenol Yeast Suspn. 30 Mean of high phenol conc. Inhibit and low permit growth / same for phenol 37 or 20 S.Typhi, Stph Greatest Dil of test killing in 10min / Same for phenol Sea water 19.0 10 Greatest Dil of test killing in 10min / same for phenol

Bentley's text book of Pharmaceutics REFERENCE Kindly refer the text book referred by The TN Dr. MGR Medical University Thank u I ACKNOWLEDGE MY MISTAKES. SORRY