Slides describe the definition, classification, factors affecting antimicrobial properties and various methods of evaluation of disinfectants.

1. DISINFECTANTS
Mr. A.T. Sharma
Assistant Professor
Nanded Pharmacy College, Nanded

2. Disinfectants
• Definition
• Antiseptics
• Properties of an ideal disinfectant:
 Broad spectrum
 Non toxic
 Fast acting
 Odourless
 Surface compatibility
 Economical
 Easy to use
 Solubility and miscibility
 Not affected by physical factors
 Stable on storage

3. Classification
• Acids and alkalies
• Halogens
• Heavy metals
• Phenol and its derivatives
• Alcohols
• Aldehydes
• Quaternary ammonium compounds
• Dyes
• Detergents and soaps

4. Acids and Alkalies
• H+ and OH- ion concentrations
• Strong as well as weak acids
• Alakalies like NaOH, NH4OH
Halogens
• Cl, Br, I, F
Chlorine:
• Organic, inorganic, gas
• Formation of hypochlorous acid
Cl2 + H2O → HCl + HClO
2 HClO → 2HCl + O2

5. • Oxygen – Oxidising agent
• Clorine combines with proteins and enzymes
• Calcium hypochlorite and sodium hypochlorite
• Open wounds, athelete’s foot
Iodine:
o Sporicidal, fungicidal, virucidal
o Weak Iodine Solution BP, Aqueous Iodine
Solution BP
o Iodophores
o Oxidising agent – Innactivates metabolic
compounds

6. Heavy Metals
• Mercury, silver, copper
• Combine with cellular components
Enzyme
• Coagulate cytoplasmic proteins

7. Heavy Metal Compounds

8. Phenol and its derivatives
• Distillation of coal tar
• Lister (1867) – Antiseptic surgery
• Evaluation Standard
• Cresol with soap solution and an alkali (Lysol)
• Hexylresorcinol – mouth washes, gargles,
cough drops
• Disruption of cells, precipitation of cell
proteins, inactivation of enzymes, leakage of
amino acids

9. Phenol and its derivatives

10. Alcohols
• 60 to 70% v/v
• Ethanol, isopropanol – skin disinfectant
• Methanol – fungicide
• Preservatives in vaccines
• Protein denaturation – damage lipid complexes –
dehydration

11. Aldehydes
• Formaldehyde (HCHO) gas/ aqueous solution
( 34 – 38%w/w) - instruments
• Glutaraldehyde (CHO.CH2.CH2.CH2.CHO) –
sporicidal, tuberculocidal
• Less toxic, les irritant
• Urological, lensed instruments

12. Quaternary ammonium compounds
• Detergents
• Gram positive, Gram negative, fungi, protozoa
• Disruption of cell wall/ membranes
• Inactivation of enzymes, denature proteins
• Floors, walls, nursing homes, public places
• Skin antiseptics
• Sanitizer in dairy, egg, fishing industries

13. Quaternary ammonium compounds

14. Dyes
• Acridine dyes
o Acriflavine, Proflavine, Enflavine
o Effecitve against Gram positive bacteria
o Impair DNA complexes
o Burns, ophthalmic applications, bladder irrigation
• Triphenylmethane dyes
o Brilliant green, Malachite green, Crystal violet
o Inhibit Gram positive bacteria
o Crystal violet – fungicide
o Interfere with cellular oxidation processes

15. Antibacterial Dyes

16. Detergents and Soaps
• SAA, wetting agents, emulsifiers
• Cationic (SAA) – Quat. Amm. Compounds
e.g. cetrimide, benzalkonium chloride
• Anionic compounds – e.g. Sodium lauryl
sulphate, soaps
• Non-ionic detergents - not used
• Amphoteric compounds – detergents +
disinfectants
e.g. Tego Compounds ( AA of high mol. wt.)

17. Factors affecting Disinfectant Action
• Concentration of disinfectant
• Temperature
• Time of contact
• pH of environment
• Surface tension
• Formulation of disinfectant
• Chemical structure of disinfectant
• Type and number of micro-organisms
• Interfering substances
• Potentiation, synergism, antagonism of disinfectants

18. Concentration of disinfectant:
• Direct effect
• Optimum concentration
• Phenol – 1%
• Dilution coefficient

19. Temperature:
• Increases with temperature
• Temperature coefficient

20. Time of Contact
• Sufficient time
• Principle of First order kinetics

21. pH of Environment
• Change in pH – rate of growth, potency of
disinfectant, ability to combine
• pH 6-8 – optimum
Surface Tension
• Surfactant property improves contact
• Adsorption, wetting, spreading
• Soap with crude phenol

22. Formulation of Disinfectant
• Chlorhexidine and QUATS – 70% alcohol
• Iodine – alcohol/ KI
• Iodine with SAA
• Concentrated solution – convenient, economic
Chemical Structure of Disinfectant
• Substitution of an alkyl group up to 6 carbons
– para position to phenolic –OH group
• Halogenation of phenol
• Nitration of phenol

23. Type and No. of MO
• Bacterial spores
• Aldehyde – sporicidal
• Aldehydes and halogens + β-propiolactone – Virucidal
• Iodine, formaldehyde, alcohol, phenolic compounds –
acid fast bacteria
Interfering Substances
• Blood, body fluids, pus, milk, food residues,
colloidal proteins
• Adsorption/ chemical reaction/ shielding
• Oils/ fats

24. Potentiation, Synergism, Antagonism
• Potentiation : Polysorbate 80, low
concentration of non-ionic surfactants
• Synergism: P-hydroxy easters
• Antagonism: Sodium thiosulphate, lubrol W+,
lecithin

25. Evaluation of Disinfectants
• Tube dilution and agar plate method
• Filter paper and cup plate method
• Ditch-plate method
• Phenol coefficient method
• Kelsey Sykes method

26. Tube dilution and agar plate method
(Serial dilution method)
• Chemical agent added in to nutrient broth/
agar medium
• Inoculation
• Incubation
• Turbidity/ colonies
• Activity compared

27. Serial Dilution

28. Cup plate/ Cylinder plate/filter paper disc method
• Agar inoculated with MO poured in petri dish
• Solidified, holes of 9mm diameter
• Antimicrobial agent placed
• Incubated
• Diameter of zone of inhibition observed

29. Ditch-plate method
• Ditch prepared in agar plate
• Antimicrobial solution added
• Test MO streaked
• Width of zone of inhibition – relative activity

30. Phenol Coefficient Test
• Phenol as a reference
• MO added in rising dilutions of phenol and disinfectant
• U.K. – Salmonella typhi
• U.S.A. – S. typhi, Staphylococcus aureus, Pseudomonas
aeruginosa
o Rideal-Walker test (RW Test)
o Chick- Martin test (CM Test)
o United States Food and Drug Administration Test (FDA
Test)
o The US Association of Official Agricultural Chemist
Test (AQAC Test)

31. RW Test
• Rideal-Walker broth, S. typhi
• Dilutions of phenol and disinfectant prepared
• 5ml dilution inoculated with 0.5ml of culture
• TT placed in water bath (17.5⁰C)
• Transferred to 5ml sterile bath after 2.5, 5, 7.5,
10 minutes
• Incubated at 37⁰C for 48 to 72 hrs
• Examined

32. Dilutions

33. Differences between Phenol Coefficient Tests

34. Advantages:
• Inexpensive, quick
• Reproducible
• Eliminate useless products
• Standard for crude preparations
Disadvantages:
• Only one MO
• One concentration, fixed death time, temperature
• Organic matter
• Tissue toxicity
• Sampling errors
• Effect of dilution
• Phenolic disinfectants only

35. Kelsey – Sykes method
• S. aureus, P. vulgaris, E. coli, P. aeruginosa
• Clear/ dirty conditions
• Final concentration – 109/ml
Result:
o No growth occurs in 2 or more of 5 tubes of
18 min samples
o Not more than 5 colonies from 5 drops

36. Time (Min) Procedure
0
8
10
18
20
28
3ml of Disinfectant + 1ml of bact. Suspension, Shake
Transfer 1 drop to each 5 tubes with liquid medium or 5 drops
to agar plate
Add 1ml of bact. Suspension, shake
Transfer 1 drop to each 5 tubes with liquid medium or 5 drops
to agar plate
Add 1ml of bact. Suspension, shake
Transfer 1 drop to each 5 tubes with liquid medium or 5 drops
to agar plate

37. Any Doubt..???