The document discusses various disinfectants, their chemical classifications, and mechanisms of action against microorganisms. It highlights the importance of concentration, temperature, contact time, and environmental factors on disinfectant efficacy. Evaluation techniques for antimicrobial agents are also covered, including tube dilution and phenol coefficient tests.

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, H2SO4 HNO3 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.
Cl2 + H2O 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 +Hgcl2 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 = logt2-logt1
logC2-logC1
Where
n=concentration exponent or dilution coefficient for disinfectant,
t1= the death time with disinfectant concentration C1
t2= the death time with disinfectant concentration C2

18. 2. Temperature
• The lethal effect on bacterial population can be increased by increasing the
temperature.
• The effect of temperature on bactericidal activity may be expressed
quantitatively by means of a temperature coefficient.
• The temperature coefficient per degree rise in temperature is denoted by θ
where as per 100C rise in temperature is expressed by θ10or Q10 values.
Thus θ10 𝑡𝑖𝑚𝑒 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑡𝑜 𝑘𝑖𝑙𝑙 𝑎𝑡 𝑇0
𝐶
or Q10= 𝑡𝑖𝑚𝑒 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑡𝑜 𝑘𝑖𝑙𝑙 𝑎𝑡 (𝑇+100𝐶)
• The value for Q10for phenol is 4,which means that over the100C range used to
determine the Q10the 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.
• Synergistics effects are often shown by two antimicrobial agents which is
giving an increased activity.
• Antagonism effects are often shown by two antimicrobial agents which is
giving an decreased activity.

25. Evaluation of antimicrobial agent &
Disinfectants:-
1. Tube dilution and agar plate method.
2. Cup plate method or Cylinder plate method.
3. Ditch- Plate method.
4. Gradient plate technique.
5. 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
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.

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

30. 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
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

31.  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.

32.  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.

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

35. 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.

36. 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.