Disinfection, Definition, classification,Mode of action, factors affecting & Evaluation of disinfectant

Disinfection, Definition, classification,Mode of action, factors affecting & Evaluation of disinfectant as per bacteriostatic & Bacteriocidal action
Department of Pharmaceutics,PRCOP,Loni

1. Disinfection By S.D.Mankar Assistant Professor Pravra Rural College of Pharmacy,Loni

2. Disinfection is the process of destruction or removal of microorganism and reducing them to a level not harmful to health. Generally kills vegetative cells but not heat resistant endospores . If the object is inanimate e.g working areas,dishes , benches etc. the chemical agent is known as disinfectant.

3. Chemical classification Acid & Alkali: - The germicidal activity of acid is proportional to H + ion concentration of there solution. Mainly HCL, H 2 SO 4 HNO 3 and phosphorous acid act as generator for H + ions. Similarly germicidal action of alkalis is depends on dissociation & release of OH - ions in solution.

4. Halogens:- Chlorine, iodine,bromine& fluorine in the free state as well as there compounds strongly act germicidal. Chlorine & Iodine are effective antimicrobial agent. Iodine is one of the oldest & Most effective antiseptics. It is effective against all kind of bacteria, endospore , fungi, & some viruses. Also skin disinfectant & for cold sterilization of surgical sutures.

5. It impairs protein synthesis & alters cell membrane Iodine is available as a tincture . E.g. betadine ,( Povidone -iodine) Chlorine as gas or in combination with other chemicals. Its germicidal action is caused by the hypochlorous acid(HOCL) that forms when chlorine is added in water. Cl 2 + H 2 O H + + cl - + HOCL HOCL is strong oxidizing agent that prevents much of cellularenzyme system from functioning.

6. Heavy Metals:- Most widely used are mercury, silver, & copper. They are act antimicrobially by combining with cellular proteins. Enzyme SH SH +Hgcl 2 enzyme Hg +2 HCl when metal ions combine with sulfhydral groups on cellular proteins, denaturation results.

7. Phenol & Its derivatives:- These are obtained by distillation of coal tar & have powerful microbiocidal action. Many derivatives of phenols are more effective and less costly such as cresol are more germicidal than phenol when they are emulsified in liquid soaps & alkalies . Phenolic compound mainly destroy M.O. by process of disruption of cells, ppt. pf cell protein & inactivation of enzyme.

8. 5. Alcohol:- They have rapid bacteriocidal action against vegetative bacteria when dil. To conc. To 60 to 70 % v/v with water. Ethanol & isopropanaol are used as skin disinfectants while methanol used as fungicidal. Alcohols are protein denaturants & damage lipid complex in cell membrane.

9. 6. Aldehyde:- formaldehyde is main aldehyde use for disinfectant action. It is effective as gas or as an aq.sol . Containing 34-38% w/w of H.CHO. Glutaraldehyde is a dialdehyde with rapid sporicidal and tuberculocidal action. It is used 2 % aq.solution .

10. 7. Quaternary Ammonium compounds:- Most of cationic detergents are . Quaternary Ammonium compounds and they are highly active against gram + ve bacteria ant quite active against gram – Ve bacteria. There mode of action is disruption of cell wall and membranes and also denaturation of proteins & inactivation of enzymes . Are widely used for control of M.O.on floors, walls, nursing homes & other public places

11. Also used as skin antiseptic and as sanitizing agent in dairy, egg & fishing industries.

12. 8. Dyes:- Number of dyes have been uses to inhibit the bacterial growth. Basic dyes are more effective bacteriocides than acidic dyes. E.g.Triphenyl methane, acridine . 9.Dteregent and soaps:- They are widely used as Surface active agents, wetting agents, and emulsifiers.

13. They are mainly classified in four classes. 1. Anionic 2.Catioinic 3.Non-ionic 4.Amphoteric most imp. Antimicrobial agent are cationic surfactants. E.g.benzylammonium chloride.

14. The most important antibacterial agents are the cationic surface active agents. Eg : cetrimide , benzalkonium chloride etc.  Soaps and sodium lauryl sulfate are anionic compounds. Soaps prepared from saturated fatty acids are more effective against gram negative bacilli while those prepared from unsaturated acids have greater action against gram positive.

15.  Nonionic detergents are not ionized. However these substances do not posses significient anti-microbial activity . Amphoteric compounds have the detergent properties of anionic surfactants combined with disinfectant properties of cationic surfactants. Eg : Tego compounds.

16. Factors affecting on Disinfectant action:- 1.conc. Of disinfectant 2.temp. 3. time of exposure 4. pH of environment 5.Surface tension 6.Formulation of disinfectant 7.Chemical structure of disinfectant 8. types an no of M.O. present 9.interfering sub.in environment 10.potentiation, synergism, antagonism.

17. 1. Concentration of disinfectants The lethal effect of bacterial population is increased by increasing the concentration of disinfectant. However, the effectiveness is generally related to the concentration exponentially, not linearly. There is optimum concentration of phenol at about 1%. Beyond this concentration, the disinfecting effectiveness becomes less . The dilution coefficient can be calculated from the following equation: n = logt 2 -logt 1 logC 2 -logC 1 Where n=concentration exponent or dilution coefficient for disinfectant, t 1 = the death time with disinfectant concentration C 1 t 2 = the death time with disinfectant concentration C 2

18. 2. Temperature The lethal effect on bacterial population can be increased by increasing the temperature. Th e e f f e c t of t empe r a tu r e on bac t er i ci d al act i v i ty m a y b e e x p r essed quantitatively by means of a temperature coefficient. The temperature coefficient per degree rise in temperature is denoted by θ where as per 10 C rise in temperature is expressed by θ 10 or Q 10 values. Thus θ 10 𝑡𝑖𝑚𝑒 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑡𝑜 𝑘𝑖𝑙𝑙 𝑎𝑡 𝑇 𝐶 or Q 10 = 𝑡𝑖𝑚𝑒 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑡𝑜 𝑘𝑖𝑙𝑙 𝑎𝑡 (𝑇+100𝐶) The value for Q 10 for phenol is 4,which means that over the10 C range used to determine the Q 10 the activity will be increased by factor 4.

19. 3. Time of contact Sufficient time of contact must be allowed for the disinfectant to exert its action. 4 . pH of the environment A change of pH during the disinfection process can affect the rate of growth inoculum. A pH of 6-8 is optimal for the growth of many bacteria and the rate of growth declines on either side of the range. Phenolic and acidic antimicrobial agents usually have greatest activity in acidic conditions. Acridine dyes and quaternary ammonium compounds are usually more active in alkaline then in acidic solutions. Amphoteric antimicrobials (Tego compounds) have optimum activities at widely differing pH values.

20. 5. Chemical structure of disinfectant Chemical structures of compounds affects the disinfectant activity. Substitution of an alkyl chain upto 6 carbons in length in para position to phenolic –OH group increases activity but greater than 6 carbons in length decreases water solubility and disinfectant activity. Generally, halogenation increases the antibacterial activity of phenol but nitration increases antibacterial activity and systematic toxicity also.

21. 6 . Formulation of Disinfectant Formulation may be imp. Of the effective use of disinfectant. E.g.chlorhexadine shows 70 % more effectiveness in alcohol than aq. Solution.

22. 7 . Types and number of micro-organisms present The efficiency of disinfection greatly depends on the nature and the number of contaminating microorganisms and especially on the presence and absence of bacterial spores. It can be seen that most vegetative bacteria are rapidly killed by most chemical disinfectants. Bacterial spores are difficult to destroy but some disinfectants e.g aldehyde are sporicidal .

23. 8 . Interfering substances in the env . Material such as blood, body fluids, pus, milk, food residues or colloidal proteins may reduce the effectiveness of disinfectant if present in small amounts. The presence of oil and fat markedly reduces the disinfecting ability of phenolics .

24. 9 . Potentiation, synergism and antagonism of disinfactants Potentiation of a disinfectant leads to enhanced antimicrobial activity. S yne r gi s t i c s e f f ects a r e of t en s h o wn b y t w o a n tim i c r o b i al a g e n ts w h ich is giving an increased activity. A n t a g o n i s m e f f ects a r e of t en s h o wn b y t w o a n timic r o b i al a g e n ts w h ich is giving an decreased activity.

25. Evaluation of antimicrobial agent & Disinfectants :- Tube dilution and agar plate method. Cup plate method or Cylinder plate method. Ditch- Plate method. Gradient plate technique. Phenol coefficient method ( Rideal -Walker test)

26. Evaluation of antimicrobial agent & Disinfectants:- 1.Tube dilution & Agar plate method:- Chemical agent is incorporated into nutrient broth or agar medium and inoculated with test M.O. This tube are incubated at 30-35 C for 2-3 days. Observed result for turbidity of colonies.

27. 2. filter paper, cup plate & cylindrical plate method:- Agar is melted, cooled at 45 C – inoculated with test M.O. and poured into sterile Petri plate. Cup plate method- make holes with cork borer and antimicrobial agent is directly placed in to holes. Filter paper or cylindrical method spread antimicrobial agent over solidified agar medium. Observe zone of inhibition 30-35 C for 2-3 days .

28. 3. Ditch plate method:- A solution of antimicrobial agent is carefully run into ditch which is prepared into agar plate. A loopful of each M.O. is then stricked from the ditch on agar surface. The width of zone of inhibition indicates activity.

29. 4. Phenol- coefficient test:- In this method test chemical is rated for its microbial property with reference to phenol under identical condition. In this test similar quantity of M.O are added to rising dilution of phenol and of the disinfectant to be tested. The phenol coefficient test includes 1.Rideal- walker test 2.Chick martin test 3.USFDA test 4.USAPOAC test

30. this test is suitable for testing disinfectant miscible with water and exert there antimicrobial action in a manner similar to that phenol. The phenol coefficient of test disinfectant may be calculated by radial walker test, that use radial walker broth and salmonella typhi as a sensitive M.O . Different dilution of test disinfectant and phenol are prepared and 5 ml of each dilution is inoculated with 0.5 ml of 24 hrs of broth culture of organisms.

31. all tubes are placed in 17.5 C in water bath. Subcultured of each reaction mixture are taken an transferred to 5 ml of sterile broth after 2.5, 5, 7.5, 10 mins . The broth culture tubes are incubated at 37 C for 48-72 hrs and are examined for presence or absence of growth.

32. RW coefficient=Dil. Of disinfectant killing in 7.5 but not 5 min ___________________________ Dil . Of Phenol killing in 7.5 but not 5 min

33. If the phenol coefficient of the test disinfectant is one means that disinfectant has effectiveness as phenol. If less than one that means less effective & more than one means more effective than phenol. Advantages:- Inexpensive & easily performed Reproducible result can be possible Valuable to eliminate useless product and supply std. for crude prpn .

34. Disadvantages:- Choice of test organism There is no indication of the activity of disinfectant in the presence of organic matter. No information related to tissue toxicity. Sampling errors are large.