microbiology

1. Antiseptics, Disinfectants
and
Preservatives
Lecture -8-
An iodine antiseptic being applied to the skin

2. Introduction
• Chemical biocides are used as antiseptics, preservatives and disinfectants.
• They differ from antibiotics in being entirely chemically synthesized and
also causing more general damage to cells.
• The rate and extent of activity is influenced by concentration, temperature,
solubility, pH and other factors.
• Antimicrobial activity can be measured using qualitative or quantitative
methods comprising broth:
• Broth dilution,
• Agar diffusion and
• Kill curve techniques.

3. Antiseptics, Disinfectants and Preservatives
• 1- Antiseptics
• These are agents that have a broad spectrum of antimicrobial activity but are
sufficiently nontoxic to allow them to be used on broken skin or mucosal surfaces.
• 2- Disinfectants
• Again, these agents will possess broad antimicrobial activity but will have toxicity
issues limiting their use to inanimate surfaces such as worktops, floors, sinks and
drains.
• 3- Preservatives
• These are broad spectrum antimicrobial agents incorporated into pharmaceutical
and other products to prevent the growth of contaminant microorganisms which
might arise during use.
• There is a common misconception that preservatives merely stop contaminant
microorganisms from growing, but preserved formulations do actually bring about a
reduction in the number of viable cells used to challenge the product

4. Mechanisms of Action
• Their principal mechanisms of action are:
• 1- Disruption of cell wall
• 2- Cell membrane structure and function,
• 3- Intracellular coagulation and chemical modification of cellular
proteins and nucleic acids.
• For this reason resistance to biocides tends to occur less readily than
is found with antibiotics.

5. Antiseptics, Disinfectants and Preservatives
• Factors influencing the activity of biocidal agents
1. Temperature
2. Concentration and Potency of Disinfectants.
3. pH
4. Solubility
5. Interaction with excipients and packaging materials
6. Number and Location of Microorganisms

6. Biocide group Examples Mechanisms of Action Spectrum of Activity Formulation Issues Commercial use
Alcohols Ethanol,
isopropyl
Benzyl alcohol
Disrupts cell membrane
(lipids)
G+ & G - & fungi
Not sporicidal
High concentration.
Inactivated by organic material
Flammable
Widely used as antiseptic and
preservatives
Quaternary
ammonium
Benzalkonium
chloride,
Cepacol
Cetrimide
Disrupts cell membrane
(lipids)
B.S, More active against
G+ & G –(LESS) & fungi,
Enveloped viruses. Not
sporicidal
Pseudomonas strains that are
resistant and can grow in presence of
Quats are a big concern in hospitals.
Inactivated by organic material
Widely used surface active agents,
(Antiseptic Disinfectant)
Biguanides Chlorhexidine Disrupts cell membrane Good activity against
G+ but less G - & fungi
Incompatible with negatively charged
excipients in formulation
Widely used as medical antiseptics
Aldehydes Glutaraldehyd
e - (Cidex);
Formaldehyde
Denature proteins Good activity against
G+ & G - & endospores,
fungi, viruses
Relatively high toxicity (glutaraldehyde) Used as disinfectant of medical equipment
Esters Methyl, ethyl,
butyl, benzyl
parabens
Disrupts membrane
transport, Inhibit nucleic
acid
Mainly G+ & fungi, but
less G -
Activity increase with alkyl chain length Widely used as preservatives in
pharmaceutical industry
Halogens Chlorine
Hypochlorites
Iodine -
Iodophor
Cause enzyme and protein
damage
Broad Antimicrobial
Cidal activity
Spectrum Sporicidal
Can be irritant,
Staining
Used in skin disinfectant and as general
disinfectant
Heavy Metals Copper,
Mercury Silver
Interacted with protein
and enzyme
Activity against G+ & G - &
fungi. Not sporicidal
Toxicity problems with Mercury Limited use as preservatives
Silver Nitrate used as topical antiseptic
Phenolics Phenol
Chlorocresol
Triclosan
Disrupts cell membrane Cidal activity against G+ &
G -. Fungus, viruses Slowly
against spores & acid fast
bacteria
High concentration.
Skin irritant, Has strong odor
Antiseptic & disinfectant and
preservatives
Peroxygens Hydrogen
peroxide
Denature proteins Broad Antimicrobial
Spectrum Sporicidal
Hydrogen peroxide unstable Antiseptic & disinfectant

7. Evaluating a Disinfectant
Chlorine
Chlorine Chlorine
Staphylococcus aureus Escherichia coli
Pseudomonas aeruginosa

8. An ideal antiseptic or disinfectant should
have the following
• Wide spectrum of activity.
• Able to destroy microbes within practical period of time.
• Active in the presence of organic matter.
• Effective contact.
• Active in any pH.
• Stable.
• Long shelf life.
• High penetrating power.
• Non-toxic, non-allergenic, non-irritative or non-corrosive.
• Not leave non-volatile residue or stain.
• Not be lost on reasonable dilution.
• Not be expensive and must be available easily.

9. Measurement of
antibacterial activity
• Antimicrobial activity can be measured in a variety of ways each
with their own advantages and disadvantages.
• While chemical assays can determine the concentration of an agent
in solution, they cannot give information about the antimicrobial
efficacy of the biocide and will not be considered further here.
• For information on antimicrobial efficacy it is necessary to evaluate
the inhibitory effect of the formulation on live cultures.
1. Broth dilution methods
2. Agar diffusion methods

10. Broth dilution methods
• If a bactericidal concentration of antimicrobial agent is diluted it will eventually reach a
concentration at which it has no effect on a population of cells.
• Some of the cells may be killed but there will not be enough molecules of agent to
damage the population as a whole.
• The end point of the dilution process can therefore be titrated to work out the lowest
concentration of biocide which just inhibits the growth of a population of cells.
• This is termed the Minimum Inhibitory Concentration or MIC
• MIC values are often quoted as if they are constants but they are not.
• The values will vary markedly depending upon the:
• 1- Incubation conditions,
• 2- Growth medium,
• 3- Strain of culture used,
• 4- Its growth history etc.
• In addition, the biocide dilution sequence occurs in steps, with sometimes a factor of 2
or even 10 between successive concentrations in the series.

11. Broth dilution methods
• A weakness of the diffusion method is that it doesn’t determine whether a drug is bactericidal and not just
bacteriostatic
• The MIC is determined by making a sequence of decreasing concentrations of the drug in a broth, which is then
inoculated with the test bacteria.
• The wells that don’t show growth (higher concentration than the MIC) can be cultured in broth or on agar plates
free of the drug.
• If growth occurs in this broth, the drug was not bactericidal, and the MBC can be determined.
• The MIC test demonstrates the lowest level of antimicrobial agent that greatly
inhibits growth, the MBC demonstrates the lowest level of antimicrobial agent
resulting in microbial death.
• Determining the MIC and MBC is important because it avoids the excessive or erroneous use of expensive
antibiotics and minimizes the chance of toxic reactions that larger-than-necessary doses might cause.
• Dilution tests are often highly automated.
• 1- The drugs are purchased already diluted into broth in wells formed in a plastic tray.
• 2- A suspension of the test organism is prepared and inoculated into all the wells simultaneously by a special
inoculating device.
• 3- After incubation, the turbidity may be read visually, although clinical laboratories with high workloads may
read the trays with spectrophotometers that enter the data into a computer that provides a printout of the MIC.

12. Turbidity Estimation of
Bacterial Numbers
The amount of light striking the light-
sensitive detector on the spectrophotometer
is inversely proportional to the number of
bacteria under standardized conditions.
The less light transmitted, the more bacteria
in the sample.
The turbidity of the sample could be
reported as either 20% transmittance or 0.7
absorbance. Readings in absorbance are a
logarithmic function and are sometimes
useful in plotting data.
More than a million cells per milliliter must be present for
the first traces of turbidity to be visible.
About 10 million to 100 million cells per milliliter are needed to make a
suspension turbid enough to be read on a spectrophotometer.
Therefore, turbidity is not a useful measure of contamination of liquids by
relatively small numbers of bacteria.

13. Decreasing concentration of drug
Doxycycline
(Growth in all wells, resistant)
Sulfamethoxazole
(Trailing end point; usually read where there
is an estimated 80% reduction in growth)
Streptomycin
(No growth in any well; sensitive at all
concentrations)
Ethambutol (Growth in fourth wells;
equally sensitive to
ethambutol and
kanamycin
Kanamycin
A microdilution, or microtiter,
plate used for testing for minimal
inhibitory concentration (MIC) of
antibiotics.
Such plates contain as many as 96
shallow wells that contain
measured concentrations of
antibiotics.
They are usually purchased frozen
or freeze dried.
The test microbe is added
simultaneously, with a special
dispenser, to all the wells in a row
of test antibiotics.
To ensure that the microbe is
capable of growth in the absence
of the antibiotic, wells that
contain no antibiotic are also
inoculated (positive control).
To ensure against contamination
by unwanted microbes, wells that
contain nutrient broth but no
antibiotics or inoculum are
included (negative control)

14. Determination of the minimum inhibitory
concentration (MIC) of a biocide
Low conc. Of
antimicrobial
high conc. Of
antimicrobial
Growth of micro. Inhibition of growth

15. Agar diffusion methods
• This technique is commonly used to assay antibiotics and it is also used as a means of
determining the sensitivity of clinical isolates to a particular antibiotic prior to treatment.
• It is also used as a means of assessing the activity of chemical biocides.
• When a filter paper disc impregnated with a known concentrations of chemotherapeutic
agents is placed upon an agar surface the biocide will dissolve in the water component of
the agar and diffuse away from the disc.
• A concentration gradient will be established (the profile of which will change over time),
being highest close to the disc and progressively less as we move towards the edge of the
plate.
• While the biocide is diffusing, the bacteria within the agar will start to grow.
• Those bacteria close to the disc will experience high concentrations of biocide whereas those
further away will initially not see any biocide molecules at all.
• As the concentration gradient is established, there will effectively an inhibitory front moving
through the agar preventing growth of bacteria as it does so.
• Also known as the Kirby-Bauer test

16. Agar diffusion methods
• The farther the agent diffuses from the disk, the lower its concentration.
If the chemotherapeutic agent is effective, a zone of inhibition forms
around the disk after a standardized incubation.
• The diameter of the zone can be measured; in general, the larger the
zone, the more sensitive the microbe is to the antibiotic.
• For a drug with poor solubility, however, the zone of inhibition indicating
that the microbe is sensitive will be smaller than for another drug that is
more soluble and has diffused more widely.
• The zone diameter is compared to a standard table for that drug and
concentration, and the organism is reported as sensitive, intermediate, or
resistant.
• Results obtained by the disk-diffusion method are often inadequate for
many clinical purposes.
• However, the test is simple and inexpensive and is most often used when
more sophisticated laboratory facilities aren’t available

17. Agar diffusion methods
• The formation of biocide diffusion zones is therefore a dynamic process involving diffusion of
biocide and a growth of bacteria.
• Eventually, the inhibitory front will reach a point in the agar where the numbers of
• If the starting concentration of cells in the agar is higher, then it will take less time for this
critical concentration to be achieved.
• Hence the inhibitory front will not have progressed so far and so the zone size will be smaller.
• Similarly, if the biocide concentration is higher, the inhibitory front will have reached further
into the agar by the time the critical concentration has been reached and so the zones of
inhibition will be larger.
• If everything else is kept constant, the size of the zone of inhibition will be dependent upon the
biocide concentration and so this method can be used to assay the biocide.
• Agar diffusion data are often misinterpreted due to the erroneous belief that zones of the same
size mean that two biocides are equally effective, and to a lack of appreciation of the effects of
solubility, diffusion coefficients and concentration exponents.

18. Agar Disk Diffusion Method (Kirby-Bauer test)

19. Determination of biocide activity using zones of
inhibition.

20. E test
• A more advanced diffusion method, the E test, enables a lab technician to
estimate the minimal inhibitory concentration (MIC),
• The lowest antibiotic concentration that prevents visible bacterial growth.
• A plastic-coated strip contains a gradient of antibiotic concentrations, and
the MIC can be read from a scale printed on the strip
The E test (for epsilometer), a gradient diffusion method that
determines antibiotic sensitivity and estimates minimal
inhibitory concentration (MIC). The plastic strip, which is
placed on an agar surface inoculated with test bacteria,
contains an increasing gradient of the antibiotic.
The MIC in μg/ml is clearly shown.

21. Kill curves
• Whilst MIC determinations and agar diffusion techniques give qualitative
or semiquantitative information about the antimicrobial activity of a
biocide they do not give any quantitative details on the rate at which the
agent kills cells and how this might be influenced by environmental
factors.
• It is necessary to inoculate a biocide solution with a known concentration
of viable cells and then take samples at intervals of time to determine the
number of surviving cells at each time point.
• Of importance, when a sample is taken the antimicrobial agent must be
neutralized so that when placed upon agar to evaluate survival there is
no residual activity to inhibit cell growth.
• Options for neutralization depending upon the nature of the biocide;
these may include dilution (for those with high concentration exponents),
specific chemical inactivation or general neutralization with a
combination of lecithin and Tween 80.

22. Kill curves

23. The use of specific chemical preservatives
• For the majority of multiple-use pharmaceutical products the only way to
prevent microbial growth is by the use of specific chemical preservatives.
• Most important point to make, is that the inclusion of a preservative into a
formulation should form part of the original design process and not just be
added in at the end as an afterthought.
• An ideal preservative should have the following properties:
• 1- Broad spectrum of antimicrobial activity;
• 2- Rapid antimicrobial action;
• 3- Chemically stable and effective under all pH conditions;
• 4- Compatible with excipients and packaging materials;
• 5- Safe;
• 6- Cost effective.

24. Preservative efficacy
testing
• The preservative efficacy test is essentially run along the same lines as the kill-
curve methodology explained above.
• The product is tested in its final container and different samples are inoculated
with a range of different cultures.
• The final concentration of cells in the test sample and the volume ratio of
product to inoculum are all described in detail.
• The organisms used (including specified strain numbers) are generally the same
for the main pharmacopoeias and include:
• Pseudomonas aeruginosa
• Candida albicans
• Staphylococcus aureus
• Aspergillus braziliensis
• Escherichia coli
• Zygosaccharomyces

25. Preservative efficacy
testing
• After inoculation of the products in their final containers they are stored under specified conditions for 28
days.
• During this period samples are removed at intervals of time and neutralized before enumerating the
survivors using plate counts.
• Bacteria are incubated at 30–35 C for 18–24 hours;
• Candida and Zygosaccharomyces at 20–25 C for 48 hours and Aspergillus at 20– 25 C for one week.
• The pharmacopoeias define the performance criteria required for different product types and for each of
the various microorganisms which have challenged the product.
• Although the term ‘preservative’ implies that this will simply prevent growth, the system must be able to
kill those challenge organisms.
• Note that there are two sets of acceptance criteria for parenteral/ophthalmic and topical products.
• The A criteria are more demanding and are the ones generally applied.
• However, there may be some circumstances when the less stringent B criteria may be acceptable, for
instance if there is a risk of toxicity if the concentration of preservative is too high.
• From the table it can be seen that in order to pass the preservative efficacy test, a topical product, for
example, would have to bring about a 2-log reduction in viable count of all the challenge bacteria within
48 hours; a 3-log

26. Disinfectant testing
• Most people will know what they mean by the term disinfectant but in
fact there is no universally accepted definition.
• Clearly, there is a need for a disinfectant to exert a much greater
antimicrobial effect than a preservative and this generally implies the
reduction of microbial load to a level where it no longer poses any threat.
• Note that it does not imply sterility.
• As disinfectants are intended for use within diverse environments there is
a requirement that they possess bactericidal, sporicidal, fungicidal and
veridical properties.
• Disinfectant testing is by no means straightforward
• As with the preservative efficacy test, the basic approach is to add
microorganisms to a disinfectant and remove samples at intervals of
time, neutralize the biocide and assess the survivors.
•