 Course Description – Antimicrobial agents as

disinfectants, antiseptics and preservatives –

Definitions; types; properties; factors affecting
activities of antimicrobial agents; evaluation of the
activity of such antimicrobial agents.
 Evaluation of microbial contents of pharmaceutical
preparations and products
 Aseptic techniques – Principles and processes, etc
 Good manufacturing practice (GMP)
Dr. C. N. E. Ibezim
Learning objectives
• At the end of this class, the students should:
• Be able to understand the meaning of some commonly used terms and
what they actually stand for;
• Adequately/correctly use the terms encountered;
• Be able to evaluate a given antimicrobial agent;
• Appreciate the necessity of disinfection as it applies to pharmaceutical
manufacturing, industries and research institutes;
• Understand the intricacies of Good manufacturing practices (GMP),
aseptic techniques etc.
 Antimicrobial agents
Introduction

• Before 19th century, post surgery mortality was on increase due to

inapplicability of aseptic technique during procedures.
• However with the application of Pasteur’s Germ theory technique, a
reversal was witnessed.
• Pasteur’s Germ theory technique:
• Introduction of phenol (carboxylic acid ) spray solution by Joseph
Lister
• Mandatory personnel hand washing with chlorinated water
• Controversies arising from irritating effect of phenol during surgery
gave birth to “control of microbial growth”.
• “Control” is either by killing microorganisms or inhibition of their
growth.
• Harmful effects of microorganisms:
• Sickness
• Contamination of pharmaceutical products and its consequences
which includes loss of therapeutic activity, change in colour, odour,
taste etc
• Microbial control employs 2 methods:
• Physical methods: Heating (Dry and moist heat), filtration,
desiccation, osmotic pressure, radiation etc
• Chemical methods: Use of disinfectants, antiseptics, preservatives,
sanitizers, anti-infectives, germicides, sterilant etc.
• Chemical antimicrobial agents are generally known as non-antibiotics.
• What does it mean to be a non-antibiotic?
Definition of terms and types of antimicrobial agents
• Antimicrobial agents: Agents that interfere with growth and
metabolism of microorganisms.
• Not all antimicrobial agents are anti-infectives.
• Main classes of antimicrobial agents:
• Disinfectants- Non-selective, wide range of activity on non-living
surfaces
• Antiseptics- use on living tissue.
• Antibiotics/antibacterial – destroy microorganisms within the body.
• Anti-infectives: A general term used to describe any medicine that is
capable of inhibiting the spread of an infectious organism or by killing
the infectious organism outright. Antimicrobial agents + others = Anti-
infectives.
• Anti-infectives encompass a wide variety of pharmaceutical agents
• Disinfection: Destruction, reduction or inhibition of vegetative microbes or/and
pathogens on a non-living surface.
• Achieved either by mostly chemical method or sometimes physical
• Agents of disinfection are known as DISINFECTANTS.
• Sterilization: Outright, total, or complete destruction of all viable organisms
including spores and viruses. Agents of sterilization are known as sterilant.
• Sterilization is achieved by physical (heat, filtration, irradiation) or chemical means
• Heat is, either wet steam under pressure at 120 °C or more for at least 15 minutes,
or dry heat at 160 to 180 °C for three hours.
• Disinfection is a less absolute term than sterilization.
• Sterilization leads to sterility unlike disinfection
• With sterilization, bacterial spores are killed while disinfection does not kill
bacterial spores.
• Disinfection using disinfectants reduce microbial load to a level which is not
harmful to health or quality of perishable goods [British standard institute (BSI)].
• Germicide (microbicide):
• Agent that kills the growing forms but not necessarily the resistant spore
forms of germs of all kinds.
• Agents that kill cells are called “cidal agents” e.g Germicides, bactericides,
sporocides, virucides, fungicides.
• Agents that inhibit cell growth are called “static agents”. Examples are
fungistatic, bacteriostat.
• Biocide: a chemical substance or microorganism intended to destroy,
deter, render harmless or exert a controlling effect on any harmful
organism by chemical or biological means.
• Are non-selectively toxic. ie not only to microorganisms but also very
often to nontarget species. Eg: disinfectants, insect repellants, industrial
chemicals and material preservatives.
• Degerming: Removal of microbes from a limited area. Example is the
mechanical removal of microbes around injection site by an alcohol swab.
• Sanitization: A process of lowering microbial count (by cleaning and
disinfection) on eating and drinking utensils to a safe public health
levels.
• Examples: use of high temperature washing or dipping into chemical
disinfectants.
• Agents used in sanitization are known as sanitizers.
• Sanitizers are commonly used to control bacterial levels in:
• equipment and utensils found in dairies, other food-processing plants,
• eating and drinking establishments,
• places in which no specific pathogenic microorganisms are known to be
present and destruction of all microorganisms may not be necessary.
• Antisepsis: Destruction, reduction or inhibition of vegetative pathogens
and/or microbes on a living surface.
• Antisepsis means against sepsis ie microbial contamination or wound
infection.
• Employs chemical antimicrobials known as antiseptics.
• Antiseptics can be applied to skin, open wounds, body cavities (mouth,
nose, ear etc), mucous membrane etc.
• These agents have bactericidal and/or fungicidal activities with capacity
to prevent putrefaction
• Chemotherapy: the use of chemical compounds in the treatment of
diseases.
• Chemotherapeutic agents: originally, antimicrobial agents of synthetic
origin useful in the treatment of microbial or viral disease.
• However has been broadened to include anticancer and other drugs.
• Antibiotics: Any substance of natural origin that inhibits the growth
and replication of a bacterium or kills it outright can be called an
antibiotic.
• Substances produced synthetically, semi-synthetically or by
microorganisms which at low concentration kills or inhibits the
growth of other bacteria microorganisms.
• Antibiotics came into global prominence with the introduction of
penicillin in 1941.

• They target bacterial infections within or on the body. This

differentiates antibiotics from antiseptics and disinfectants
• They have activity against bacteria and not fungi or viruses
• Are also known as antibacterial agents.
• Brief history of antibiotics:
• 1928: Discovery of first antibiotic, penicillin by Alexander Fleming.
• 1930s: The first commercially available antibacterial was Prontosil, a
sulfonamide developed by the German biochemist Gerhard Domagk.
• 1945: Penicillin was introduced on a large scale as a treatment for
bacterial infections.
• Florey and Chain efficiently purify the antibiotic and scale-up its
production.
• 1940 – 1962: The golden era of antibiotics. Market introduction and
use of most antibiotics used today.
• Preservatives: Agents included in pharmaceutical preparations to
prevent microbial contamination and/or spoilage of products.
• Are included in non-sterile (oral and topical) products to minimize the
risk of infection by consumers
• Non-inclusion in a preparation or inadequate amount can cause:
• Changes in physicochemical properties of products such as loss of product
stability, changes in colour, pH, viscosity and formation of gas.
• Loss of therapeutic efficacy from the microbial production of enzymes in the
course of metabolization and degradation of active ingredients.
• May be included in sterile products (parenteral and ophthalmic) to kill
any contaminant introduced during use from multi-dose containers.
• Preservation is necessary in preparations containing water because
such are susceptible to microbial attack.
• Are used to describe the purpose of antimicrobial agents in protecting
medicines, pharmaceutical formulations, cosmetics against microbial
spoilage.
• Preservatives should cut across 2 main categories of contaminants:
• Spoilage contaminants: Results from water (portable, deionized, and distilled
water) containing Pseudomonas, E. coli, Xanthomonas, Achromobacterium,
Aerobacter and Flavobacterium species.
• Pathogenic and potentially harmful contaminants associated with
pharmaceutical preparations which include Salmonella, E. coli, Pseudomonas
aeruginosa, Staphylococcus aureus etc.
• Examples of antimicrobial preservatives include: Methyl, ethyl, propyl
and butyl Parabens, Sorbic acid etc
• Minimum Inhibitory Concentration (MIC): The lowest
concentration of an antimicrobial agent that will inhibit the
visible growth of a microorganism after overnight incubation.

• Minimum Bactericidal concentrations (MBC): The lowest

concentration of antimicrobial agent that will prevent the growth
(by killing) of an organism.

• Selective toxicity: ability of an agent to kill or inhibit other

microorganisms with little or no harm to the host
General ideal characteristics of antimicrobial agents
No single agent is best for the control of microorganisms for any and all
purposes. Thus in the preparation and evaluation of new
compounds/chemical agents these properties are considered.
1. Antimicrobial activity: A measure of inherent capacity of an agent to kill
or inhibit microorganisms. The chemical at a low concentration should
have a broad spectrum of antimicrobial activity.
2. Stability: changes in the substance upon standing should be minimal and
should not result in significant loss of antimicrobial activity.
3. Should be cidal in action
4. Solubility in water and other solvents
5. Selective toxicity
6. Homogeneity of active ingredient and /or its mixtures in each application.
7. Non-combination with strenuous organic materials: E.g affinity for
protein or other organic substances may result in unavailability of
agents for antimicrobial action.
8. Toxicity to microorganism at room or body temperature, not
requiring a rise in temperature for it work.
9. Capacity to penetrate: for those agents whose activity is required at
sites order than the site of application.
10. Deodourizing but non-corroding and non-staining
11. Detergent capacity
12. Available and affordable.
• Additional Specific properties of antiseptics
• Active in the presence of body fluids and organic matter (saliva, pus,
blood etc)
• Should be rapid in action/fast acting and sustained lethal action
• Non-irritating to tissues and non-corrosive
• Non-allergic and sensitizing to subjects
• Absence of systemic toxicity/ non-absorbable

• Additional Specific properties of disinfectants

• Compatible with other organic compounds
• Good penetrating power
• Non-corrosive
• Additional Specific properties of preservatives
• Compatibility with other ingredients and containers
• Non-toxic to human and animal tissues or cells
• Must have good partitioning between oil and aqueous product
components
• Must be effective at low concentration
• Readily soluble
Levels of disinfection
• Three levels of disinfection with respect to power of the disinfection, the amount and kind of
microbial killing involved are:
• High-level disinfection (HLD): will kill all organisms, except for large concentrations of
bacterial spores. Examples:
• Moist heat (75-100°C) for 30 mins
• 2% Glutaraldehyde
• 3-25% hydrogen peroxide
• Chorine dioxide (100-1000ppm for chlorine)
• Intermediate level disinfection (ILD): destruction of all vegetative bacteria including M.
tuberculosis, lipid enveloped and some non-lipid enveloped viruses. Fungi and spores are not
destroyed. Examples:
• 70-75% alcohol
• 0.4-5% phenolic compounds
• Quaternary ammonium compounds (QACs) at higher concentrations.
• Low level disinfection (LLD): destroys most vegetative bacteria, fungi and virus but not spores
and more resistant organisms. Examples:
• 0.4-1.6% QACs
• Phenolics (bisphenols and chloroxylenols)
 Microbial resistance ladder
• Enveloped viruses
• Most G-negative bacteria
• Non-enveloped viruses
Direction of resistance increase

• Vegetative helminths and protozoa

• Fungi and fungal spores
• Most G-positive bacteria
• Protozoan cysts
• Staphylococcus and Pseudomonas species (Vegetative bacteria)

• Mycobacterium
• Bacteria endospores
• Prions
Factors affecting antimicrobial activities of chemical
antimicrobial agents
Generally classified based on:
• Organisms
• Chemical agents
• Environments/host

1. Organism-based factors:
• The nature:
• G+ve and G-ve bacteria have varied sensitivities to antimicrobial agents.
• Vegetative cells more susceptible than spores.
Spores possess rigid protective structures and/or capsules, spore coats and cortex.
• Possession of unusually high lipid/waxy cell wall leading to hydrophobic nature by
mycobacteria
• Biofilm formers are more resistant than planktonic/free-living ones.
 • Vegetative forms of fungi are most susceptible than the spores e.g chlamydiospores, conidia
spores etc.
• Protozoa are resistant to most antimicrobial agents.
• Inoculum size (number): The larger the number of microbes, the more time or higher
concentration a germicide needs to destroy all of them, all other conditions remain
constant.
• E.g 10 B. atrophaeus spores were killed in 30 minutes while 100,000 Bacillus
atrophaeus spores took 3 hours.
• aggregated or clumped cells are more difficult to inactivate than monodispersed cells
• Mixture of resistant, non-resistant groups and dead cells in a mixed, large microbial
population
• Location of the organism: organisms inhabit aqueous environment just as chemical
antimicrobial agents.
• Intracellularly located microorganisms
• Presence of organic matter (pus, excreta, blood, milk etc) may impair penetration of
antimicrobial agents.
• Surface application, cracks and uneven surface application of chemical agents do not affect
deep-seated resident flora of the skin.
• Age of microorganisms:
• Susceptibility of organisms at log phase than those in other phases.
• May be due to the content of lipid, nucleic acid, protein and cell wall
permeability of the organisms.
2. Chemical agents:
• A. Concentration: Higher concentration have greater efficiency in
killing microorganism.
• Rate of kill of bacteria population varies directly with concentration of
the agent between the MIC and maximal concentration of agent.
• Below the MIC, exist only sub-inhibitory concentration of the agent at
the reactor site.
• Above the maximal concentration, no significant ↑ in the
antimicrobial action occurs.
• May be due to saturation of chemical agent at the reactors and
receptor sites or due to steric interference due to accumulation of the
agent at the reactor site.
• Plot of log death time(Time to kill a standard inoculum) against Log
concentration gives a straight line.
• Slope = concentration exponent (ⴄ).
•ⴄ=
• =
Log death time

Log concentration
 • ⴄ is a characteristic of each type of antimicrobial agent.
• It indicates the activity of the agent on dilution.
• Examples:
Concentration
Dilution Disinfection
Agent coefficient (ⴄ) factor (d.F) effect (d.f ^ⴄ) dilution effect
Mercuric Chloride 1 3 3 3 times as long

Rapid inactivation
Phenol 6 3 729 by dilution
Mercuric Chloride 1 2 2 Twice as long
Phenol 6 2 64
Ethanol 9 2 512
Ethanol 9 3 19683
formaldehyde 1 2 2
formaldehyde 1 3 3
• 1. For mercuric chloride, ⴄ = 1 means its activity will be reduced by
the power of 1. thus a 3-fold dilution implies that its disinfection
activity will be reduced by 31 which is 3 .
• Interpretation= Disinfection activity will be 3 times as long.
• 2. Phenol ⴄ = 6; activity will be reduced by power of 6. A 3-fold
dilution implies that its disinfection activity will be reduced by 36
which is 729
• Similar interpretation applies to other antimicrobial agents.
• NB: agents with low conc. Coefficient/dilution exponent cannot easily be
inactivated by dilution but require use of chemical inactivator.
• B. Solubility:
• The more aqueous soluble an agent, the higher its penetration into
microbial cells and higher the antimicrobial action.
• C. Partition coefficient:
• The ratio of concentration of a solute in 2 immiscible or slightly miscible
liquids when in equilibrium across the interface between them.
• Especially important with preservatives
• The more an antimicrobial agent partitions into an aqueous environment,
the higher its antimicrobial action.
• Preservatives that favour partition into oily phase e.g chlorocresol will
require an ↑in their concentration.
• C. Formulation: considerations in formulation include the nature of
solvent or vehicles, solubility etc.
• Some agents are more active in alcoholic solvents than aqueous
solvents.
• Low soluble agents could be prepared using soaps, hydrocarbon
carriers and protective coverings.
• Nature of solvents affects the partition coefficient of disinfectants
• Partitioning in favour of oily phase will reduce concentration of most
antimicrobial agents.
• Some Preservatives easily adsorb unto finely divided particles in
suspension thus negatively affects antimicrobial action
• Interaction between preservatives and other components of the
formulation and/or packaging containers.
• 3. ENVIRONMENT-RELATED FACTORS
• A. pH: Changes in pH can affect the growth rate of inoculum, potency of
agent and its ability to combine with the sites on the cell surface.
• Bacterial growth is optimal at pH 6-8, fungi and yeast thrive at <6 thus on
either side, growth declines.
• Effect of pH on potency of agent: antimicrobial agents demonstrate activity
in different forms viz undissociated form (phenols, Benzoic acids,
nitrophenols, salicylic and acetic acids); cationic or anionic forms (QACs e.g
cetrimide) and acridine dyes.
• Active forms are affected by the pH of the environment
• Best activity is obtained at the pH that favours the formation of active
species.
• Weakly acidic agents are effective at low pH.
• Cationic agents are active at alkaline regions .
• Non-ionic and ampholytic agents are not affected by pH changes.
• Effect of pH on cell surface:
• Bacterial cell surface have increased negatively charged ions.
• Alkaline (-ve) pH will favour interactions with positively charged ions of antimicrobial
agents whereas negatively charged (anions) will be facilitated under acidic or +ve
conditions.
• Intercation of AMA with cell surface follows adsorption process thus increased
adsorption increases activity.
• B. Presence of organic matter or interfering substances:
• Organic matter include: Pus, blood, serum, faecal matter, food residues, organic
wastes ets.
• Presence of organic matter may interfere with AMA’s efficacy via:
• Microbial Coating with increase in the viscosity of the environment and ↓diffusion of the agent
• Adsorption of and reaction with organic matter. This reduces the concentration of AMA to the
organisms.
• Interaction and degradation of organic matter releasing metabolites (may be inactive or alter
physicochemical properties of the agent).
• Such interactions include:
• Phospholipids in serum, milk and faeces will reduce the AM activity of QACs.
• Phenolic compounds with cetomacrogols will give inactive complex
• Materials like rubber, fabrics, corks and plastics adsorb AMA resulting to loss of activity.
• Other environment-related factors include:
• Temperature, packaging materials (corks, rubbers, bottles etc), surface activity etc.
• Temperature:
• An ↑ in temperature ↑s AM activity of an agent.
• Generally, as temperature ↑s arithmetically, the rate of kill ↑s geometrically.
• The effect of temperature ↑ on the rate of bactericidal activity at fixed concentration of
AMA and inoculum size is expressed quantitatively by means of temperature coefficient
(Ꝋ).

• Ꝋ = change in rate of AM action per degree rise in temperature.

 • MAJOR GROUPS OF CHEMICAL ANTIMICROBIAL AGENTS

Agents Properties Mode of action Uses limitations Examples

Poor
penetration of
as skin disinfectants; to organic matter.
Intermediate level of disinfection; Bactericidal form tinctures of They quickly Ethyl alcohol,
including mycobacterium, fungicidal, not sporicidal. denaturation of volatilize; can Ethanol,
Alcohols Activity enhanced by presence of water. Readily proteins; interference antiseptics (used with leave particle Trichlorobutanol,
available and inexpensive. Have cleansing and with metabolism; lysis acetone); cleaning of
clinical thermometers and residues not Phenyl ethanol,
detergent action leading tto mechanical removal of (dissolving of organism) general instrument disintinguishabl Isopropanol,
microorganisms. disinfection. e from other Benzylalcohol etc
undesirable
particulate
matter.
 irritation action and
carcinogenic
high level disinfectant; highly antimicrobial and chemical sterilant in possibilities with
sporicidal; 34-38% aqueous solution of equipment sterilization; in formaldehydes; little
protein inactivation; fumigation; soil and air penetrating power
formaldehyde is known as formalin; potent interaction with amino- and disinfection; viral vaccine for other organic
bactericide affecting vegetative bacteria, viruses hydroxyl groups of nucleic preparation e.g polio and materials order than Glutaraldehyde
Aldehydes and many spores (at 8%); 2% glutaraldehyde kills s;
acid, carboxyl and sulphydryl influenza,; production of serum; antimicrobial
non-sporing bacteria within 2 minutes; spores of groups in proteins leading to toxoids from toxins; dilute activity is affected formaldehydes
bacilli and clostridium in 3 hours;
cell death. solution (0.1-0.2%) of by temperature and
mycobacterium, viruses and fungi in 10 mins;
formalin may be used as humidity; toxic to
activity is not affected in the presence of serum
gargles. skin causing
hardening and
wrinkling of skin.

Ease of inactivation
by other chemicals; Hydrogen
pproduction of peroxides,
High level disinfectant; broad spectrum activity hydrogen peroxide is pungent odour; acts Peracetic acids
Peroxygenes with efficacy aginst viruses, bacteria, yeasts and Attacks the enzymes with widely used as biocides as oxidant by and ozones,
and thiol groups; affects the for disinfection, producing/releasing benzyl
permangates fungal spores; environmentally friendly with ribosomes and nucleic acids. sterilization and hydroxyl (OH-) free peroxides,
degradation into water and oxygen.
antiseptics radicals which potassium
attack cell essential permanganate
elements like s
proteins and DNA.
 Agents Properties Mode of action Uses limitations Examples

penetration and
disruption of cell
Low level membrane; exhibit
disinfectants; Active rapid cytocidal
against bacteria at action at high instrument Activity reduces by
5% and inactive concentration; cell disinfection; mucous dilution (n=6); Chlorxylenols,
Phenols and against spores; protein precipitation. membrane and skin activity is affected by Bisphenols,
fungicidal and kills At low surfaces at low presence of organic Chlorocresols,
phenolic compounds
some viruses;are concentration, they concentration; matter, increase in Hexachlorophene,
corrosive and can inactivate essential environmental pH reduces the Cresols (e.g Lysol) etc
damage tissues enzymes, disrupts surfaces. activity
causing systemic cytoplasmic
poisoning. membrane and
leakage of cellular
content.
 Agents Properties Mode of action Uses limitations Examples

mercuric compounds
Heavy metals have are used as topical
Most ancient of strong affinity for
antiseptics,
antiseptics and proteins combining preservatives and
disinfectants; mercuric with the thiol (SH)
disinfectants;
compounds have static groups leading to organomercurials are
activity against inactivation; forms used as fungicides and
vegetative bacteria complexes and renders Mercury (Hg), Silver
and molds; Phenyl them metabolically preservatives at 0.01- Toxicity to animals; (Ag), Copper (Cu),
0.02 %; cupric salts are activity reduction in
mercuric salts are inactive; high used as potent the presence of phenylmercuric nitrate
Heavy metals and their relatively non-toxic, concentration of Siver (PMN),
salts non-irritant and non- and copper fungicides and organic matter; phenylmercuric
algacides in reservoirs corrosive action;
corrosive; precipitates cell acetate (PMA),
organometallic are proteins; silver and swimming pools; possible pollution to thiomersals,
0.001% phenyl environment
effective against Gram compounds act as nitromesals etc
mercuric salts are used
positive cocci, spore- protoplasmic poison as preservatives for
forming rods, forming silver
tuberculosi, mycosis proteinates(a complex multidose injections
and some ophthalmic
and not Gram positive with sustained solutions and at
rods. antiseptic action
0.002% in sterilization
through slow release) with bactericide.
 Approximate
Agents concentration used Mode of action Uses
(percent)

denaturation of proteins; as skin disinfectants; to

interference with form tinctures of
Alcohols (e.g., ethyl alcohol)
metabolism; lysis antiseptics (used with
(dissolving of organism) acetone)

as skin disinfectants and

Cationic, surface-active
quaternary ammonium
compounds
denaturation of proteins; antiseptics; in sanitizing
inactivation of cellular
0.1–0.25 eating and drinking
metabolites; dissolving of
cell wall utensils, food-processing
equipment

as surgical scrubs (used

Bisphenols (2 phenols linked with soaps and
2–5 inhibition of cell growth
together) detergents); as
deodorants
 Approximate
Agents concentration used Mode of action Uses
(percent)

in ointment and salves as

Iodine and iodized 2–16 precipitation of cell skin antiseptics; in
compounds proteins surgical-instrument
disinfection

in disinfection of
dwellings, ships, storage
Aldehydes (e.g., general microorganism
formaldehyde) 1–5 poison houses, utensils, clothing;
in hospital-instrument
sterilization

precipitation of cell as skin antiseptic in skin

Mercurials (inorganic and
0.001–1 proteins; destruction of ointments and salves; as
organic)
enzymes preservatives for drugs

as disinfectants; in
cell-membrane
Oligodynamic metals ointments and salves; in
traces destruction; coagulation of
(silver, copper, mercury) cell materials cement (e.g., in shower
rooms)
 Approximate
Agents concentration used Mode of action Uses
(percent)
as disinfectants; in
Oligodynamic metals traces cell-membrane destruction; ointments and salves; in
(silver, copper, mercury) coagulation of cell materials cement (e.g., in shower
rooms)

in cosmetics and
deodorants;
Heavy metals 0.1–1 precipitation of cell proteins
antiperspirants; skin
antiseptics

precipitation of cell proteins; as skin antiseptics (salicylic

Acids 0.1–5 and benzoic acids); in food
destruction of cell wall
preservatives (benzoic acid)

inhibition of cell function;
Dyes (e.g., acridine) 0.1–1 combination with essential
metabolites
in dentistry as mucous
antiseptics; in laboratory
media to inhibit growth of
unwanted bacteria
 Approximate
Agents concentration used Mode of action Uses
(percent)

Antibiotics and
interference with cell in chemotherapy of
chemotherapeutic drugs
(e.g., penicillin, 0.001–1 metabolism; synergistic action in disease; in ointment and
body to counteract infection salves as skin antiseptics
sulfonamides)

as skin antiseptics in dilute

cytoplasmic poisons; disruption
Coal-tar derivatives (e.g., of cell wall; precipitation of
0.1–5
phenol, cresols) proteins; inactivation of
enzymes
solutions; as floor and wall
disinfectants, combined
with soaps; as surgical-
instrument sterilizers

as disinfectants with soaps

Aromatic oils (especially pine
oil)
effect on cell constituents;
0.1–5 mechanical effect inhibits cell
growth
for washing floors and
walls; as a deodorant on
inanimate surfaces
Evaluation of microbial contents of pharmaceutical preparations
and products Why do you undertake product evaluation?
• Sterile and non-sterile products could be evaluated
• Sterile products should be devoid of microbial presence
• For non-sterile preparations;
• only a limited level of microbes are allowed
• Certain organisms must be excluded as follows:
• Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacteria e.g
Escherichia coli → Topical preparations
• Oral dosage forms (solids or liquids) → not exceeding 103-104 aerobic
bacteria per g or ml; 102 yeasts and mold per g or ml; 102 cells per g or
ml of other enterobacteria.
• Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella spp →
should be absent in oral dosage forms.
• Prior to evaluation:
• Samples are to be held under the same storage conditions required by
the package label or insert.
• Note the nature of product by:
• Determine its solubility or miscibility in/with water;
• Determine suitable solvent/medium for sample dilution, dissolution,
suspension or emulsification
• Check the presence of an antimicrobial agent. If present, it should be
eliminated by dilution or neutralization
• All equipment, reagents and media must be sterile and the test carried
out under aseptic conditions.
• Must provide a 10% homogenate solution/suspension or emulsion of
the sample. However, 1g quantity may be used for expensive products.
• Surfactants (e.g 0.1% Tween 20 or 80) may be added to a difficult-to-
wet preparations.
• Sterile phosphate buffered sodium chloride (PBS) at pH 7.0 should be
used as diluents.
 Some antimicrobial quenching agents
Antimicrobial agents Quenching/inactivating agents

Sulphonamides Paraaminobenzoic acids (PABA)

Cetrimides/QACs Lubrol W

Penicillins Penicillinase (beta lactamases)

Benzoic acids Polysorbates
Benzylalcohols and Phenols Dilution or Tween 80
Glutaraldehyde Glycin
Halogens Sodium thiosulphate
Mercurials Thioglycollic acid
METHODS
• Qualitative and quantitative methods
• Qualitative → Morphological, biochemical, molecular.
• Quantitative methods:
• Microbial enumeration is undertaken via:
• Direct Total cell count
• Indirect total population count
• Viable cell count methods
• Direct total cell count: total number of living and dead cells including
bacteria spores or yeasts in a given volume or area.
• Can be achieved through:
• Cell observation in a specialized counting chamber under phase contrast
microscopy
• Electronically using coulter counter
• Indirect total population count can be employed quantitatively or
Qualitatively.
• Quantitative total population count uses spectrophotometer.
• Qualitative total population count compares against McFarland
standard of a known turbidity (108 cfu/ml).
• In indirect method, bacterial culture acts as a colloidal suspension
which blocks or reflects light passing through it.
• Measurement of amount of light absorbed, the number of bacteria
contained in the sample can be estimated or compared.
• The percentage of light transmitted (% transmittance) is inversely
proportional to bacteria contained;
• The percentage of light absorbed (% absorbance) is directly
proportional to bacteria population.
• VIABLE CELL COUNT
• Allows the identification of only actively dividing/growing cells in a sample.
• METHODS
1. Plate count method or counting on agar plates
• Relies on bacteria growing as a visible colony on a nutrient medium
• Effective counting/estimation can be obtained only on the dilution of original sample such
that an average of 30-300 colonies of target bacterium are grown.
• Fewer than 30 colonies makes the interpretation statistically unsound.
• Colonies greater than 300 results in overlapping and imprecision in the count.
• The procedure involves making of serial dilution of sample in sterile water,
• Cultivation of dilutions in nutrient agar or appropriate nutrient medium,
• Incubation at appropriate temperature and duration,
• Colony counting, average determination and calculation of number of cells/gram or ml.
Techniques
1. Spread plate: useful for the growth and enumeration of aerobic microorganisms from a
given sample.
- Makes use of aliquot of 0.1 ml of the dilution.
2. Pour plate: used for the analysis bacterial species that grow poorly in air. Has
the following variations:
- Single agar layer: Uses 20ml of growth medium mixed with 1ml of appropriate
dilution.
- Double agar layer: involves 15 ml of first molten agar layer on petri dish;
followed by a 5ml molten agar mixed with 1 ml of appropriate sample dilution.
- Surface drop/Miles and Misra/surface viable count: Uses single drops of
appropriate sample dilution on an over heated and over dried agar.
3. Counting on filtration membranes:
• Use for assessment of filtrable drugs such as syrups, suspensions etc using 0.45
µm nominal porosity filter membrane.
• Appropriate sample dilution is filtered while the filter membrane is aseptically
transferred onto appropriate growth medium.
• Here, the passage of nutrients through the filter during incubation facilitates
the growth of organisms in the form of colonies, on the upper surface of the
membrane
• EVALUATION OF ANTIMICROBIAL AGENTS
• Qualitative and quantitative methods
• Quantitative methods- minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC)
• Qualitative methods- spectrum of activity, culture and sensitivity
DISINFECTANTS
Categories of disinfectant evaluation methods:
Non-routine methods: depicts the practical use and the conditions of use
of disinfectants.
• Examples in-use disinfectant evaluation method.
Routine based test: Include various phenol coefficient tests and other
tests not based on phenol coefficient principles.
• PHENOL COEFFICIENT TESTS
• Used to compare the effectiveness of test disinfectant or germicide
against a standard phenol under the specified conditions of the test.
• Valuable in:
a. Routine examination of phenolic products
b. Preliminary screening/ determination of antimicrobial properties of
germicides
c. Research on new compounds and formulation.
• Modifications of phenol coefficient tests
• Rideal-Walker
• Chick-Martins
• AOAC (United states Association of official agricultural chemists) methods
• Kelsey sykes Tests
• Principle:
• The minimum concentrations of pure phenol that produces viable sub-cultures
at one time of sampling but non-viable cultures at the next is compared to the
minimum concentration of test disinfectant that produces similar results.
• This comparison is known as phenol coefficient.
• Points to consider to achieve satisfactory evaluation using phenol coefficient
test
1.Organisms: should be those to be encountered in the environment
- Should be within the young, actively dividing age (22-26 hrs old)
- Those with genetically preserved characteristics
- Typed Salmonella typhi
2. Use synthetic of semi-synthetic culture medium
3. Result interpretation: PC = 1: Same effectiveness as phenol
PC <1 shows less effective disinfectant than phenol, PC >1 indicates more
effective disinfectant than phenol.
1. Rideal-Walker Phenol coefficient test (RW PCT)
• Test reported by S. Rideal and J. T. A. Walker in 1903
• Though it may be obsolete however some basic principles remains applicable.
• Materials used and procedure:
• Salmonella Typhi
• Standard nutrient broth with pH 7.6 sterilized at 121oC for 20 mins
• Four dilutions of standard phenol and test disinfectants.
• Phenol dilutions in units of 5’s while test disinfectant is in 50’s.
• Inoculate dilutions with 0.2 ml of culture and withdraw a standard loopful of
each dilutions into 5ml volumes of sterile recovery medium (broth) at 2.5, 5
7.5 and 10 mins.
• Incubate all samples for 48 hours at 30oC and observe presence or absence of
growth in each tube.
• Calculate the phenol coefficient using the formular:
• Phenol Coefficient Test =

• Example:
Disinfectant REACTION TIME (mins)
Dilutions
2.5 5 7.5 10

Phenol + + - -
1 : 95

Test Disinfectant

1 : 200 - - - -

1 : 250 + - -

1 : 300 + - - -

1 : 350 + + - -
• Rideal Walker phenol coefficient =
= = 3.68
Interpretation: Test disinfectant is more effective disinfectant than
phenol
2. Chick- Martin PCT
• Proposed by H. Chick and Martin 5 years after RW PCT
• A modification of Rideal Walker PCT as follows:
• Addition of organic matter (5% yeast)
• Use of Staphylococcus aureus in addition to Salmonella Typhi
• Use of 30 mins as time of sub-culture from reaction mixture to
recovery medium
• Use of 10 ml recovery medium
• Chick Martin PCT is calculated with the formular:
Mean of highest concentration of phenol showing growth (in both
tubes) and lowest concentration for growth inhibition divided by the
same mean for the test disinfectant.
Example:
Phenol (%) Tubes Disinfectant (%) Tubes
1 2 1 2
2.0 - - 0.47 - -
1.8 - - 0.41 - -
1.62 + - 0.37 + -
1.45 + + 0.33 + +
1.31 + + 0.31 + +

PC = = 4.15
Limitations of Phenol coefficient test
• Used only for phenolic disinfectants
• Except for chick martin PC, it does not consider the effect of organic
matters on disinfectant efficacy.
• Use of only an organism. This does not represent a wide spectrum of
activity of phenolic disinfectants
• Use of fixed temperature which precludes the disinfectant assessment
under different temp. conditions.
• Varied disinfection times for different methods which don not consider
slow acting disinfectants that may be efficient with broad spectrum.
• A possibility of picking dead cells during sampling
• Difficulty with reproducibility due to variations in inoculum
concentration, age or metabolic states, different diluents, suspending
agents or recovery media.
 EVALUATION OF ANTISEPTICS
• Categories of antiseptics:
• Oral antiseptics: mouthwashes and gargles; lozenges, pastilles and paints.
• Skin antiseptics: used in other parts of the body as in wound cleaning and pre-surgery
procedures.
• Skin antiseptics evaluation methods:
• Skin penetration test e.g in acne where skin penetration is needed.
• Skin infection test
Skin antisepsis test: Applicable to antiseptics for bathing, hand washing and pre-surgery
procedures.
• Skin penetration test methods
i. Glove test using spread plate techniques
• Procedure:
• Apply antiseptics over two hands and cover with sterile rubber gloves for 2hrs
• Carefully remove the gloves without turning inside out
• Shake with 100 ml of sterile normal saline
• Spread plate 1 ml of the rinse and incubate for 48hours
• Check for presence or absence of growth.
• Repeat produce using control and compare the differences
ii. Skin biopsy test.
Skin infection test procedure:
- Inflict the skin with an open cut and inoculate the wound with virulent
strain of a test organism
- After the establishment of infection, clean the wound with solution of test
antiseptic
- Perform viable cell count
- Repeat procedure without antiseptics and compare count.
- Survival level is a measure of activity of the antiseptic
Evaluation method for oral antiseptics:
- Extinction time test: it determines the shortest interval required to kill a
given population of a test microorganaism.
- Procedure:
- Inoculate about 10 ml saliva with 1 ml culture of S. aureus or Strep.
pyogenes
• Withdraw a loopful of organism-saliva reaction mixture at 0 and 1
minute intervals
• Subculture into a nutrient broth and incubate at 37OC for 48 hrs
• Determine the extinction time as follows:
• Mouth washes and gargles ≤ 15 seconds
• Lozenges, pastiles and paints ≤ 15 minutes
• Skin antiseptics ≤ 5 minutes
• NB: An effective antiseptic is that which exerts lethal action against
test microorganism within the given period of time as stated.
 EVALUATION OF PRESERVATIVES
• Best undertaken in the final product container.
• Preservative efficacy is determined using the microbial challenge test
• A preserved preparation is exposed to a wide variety of organisms most
likely to encountered during production and use
• In microbial challenge test, the following is considered wit respect to the
organism:
• microbial association with the product,
• high level of resistance to preservatives,
• capacity to opportunistic contamination,
• ability to elicit a particular health hazard if present in a product
• Use of organisms at the optimal growth levels
• Incubation temp. similar to product storage and use temperature.
• Procedure: Refer to Laboratory practical manual
 Aseptic techniques – Principles and processes, etc
 Good manufacturing practice (GMP)

 Please make up the note as explained during the lecture.

 Thank you and best of luck