1. CHAPTER – IV
DISINFECTION

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

3. 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.

4. 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

5. 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

6. 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…

7. 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….

8. Mechanism of action of disinfectants

9. 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…

10. 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.

11. 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.

12. 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.

13. 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

14. 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

15. 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
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

16. 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

17. Concentration B 1 6 1
Log t 1 A
Log C

18. 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

19. 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

20. 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

21. 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

22. EVALUATION OF DISINFECTANT

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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.

31. 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 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

32. 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

33. 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 =

34. Essential difference between phenol coefficient tests

35. Medium pH
Medium volume
Rxn Mix Volume
Diluent
Reaction Temp
Organism
Sampling Time (Mins)
Calculation
RWC
FDA`
CMC
AOAC
Crown Agents
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

36. 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