Download Lecture 6

Chemical Control of
Microbes
Disinfectants •
Antiseptic •
Preservatives •
Chemical disinfectants,
antiseptics, & preservatives
Are chemicals which have the ability to
destroy or inhibit the growth of
microorganisms, and are used for this
purpose
3
Other terms
Sepsis = the presence of a toxin or pathogenic m.o in •
blood & tissue
Fungicidal & Fungistatic = chemicals used to kill
•
or stop the growth of the fungi
Bacteriostatic & Bactericidal = chemicals used to •
kill or stop the growth of the bacteria
•
Virocidal = chemicals used to destroy the viruses •
Sporacidal = chemicals used to destroy the spores •
preservatives
Are chemicals included in pharmaceutical •
preparations to prevent microbial spoilage
of the products
disinfection
=== is the process of removing pathogenic
m.o. from the surfaces of objects
•
=== reducing them to a level acceptable for a •
defined purpose
=== a level which is harmful neither to health •
nor to the quality of perishable products
Disinfectants levels of actions
High level disinfection===indicates •
destruction of all m. o. but not necessarily
bacterial spores
== complete killing == sterilization •
those which can kill spores are described •
as liquid sterilants =chemosterilant)
Disinfectants levels of actions
Intermediate level disinfection •
===indicates destruction of all vegetative •
bacteria including T.B.
= but may exclude viruses & fungi •
= and have little or no sporicidal activity •
Disinfectants levels of actions
Low level disinfection •
=== destroy most vegetative bacteria fungi •
& viruses
= not include spores & resistant bacteria •
antisepsis
===Destruction or inhibition of m.o. on living tissues
•
=The effect is to limit or prevent the harmful result of
infection
•
=Applied to skin & mucous membranes •
=they must be not toxic or irritating for skin •
=mostly used to reduce the microbial population on skin •
prior to surgery or on hands to help prevent the spread
of infection by this route
Preservatives
===Chemicals included in pharmaceutical •
preparations to prevent microbial spoilage of the
product
•
=& to minimize the risk of the consumer acquiring an
infection when the preparation is administered
==it must be able to limit proliferation of m.o. that •
may be introduced unavoidably during manufacture
& the use of non sterile products (oral drops &
injections)
•
Preservatives
= should kill any microbial contaminant •
introduced inadvertently during use in
sterile products (eye drops & multidose
injections)
= must be not toxic in relation to the •
intended route of administration of the
product
Factors affecting the choice of antimicrobial
agent
1-properties of the chemical agent •
2-microbial challenge •
3-intended application •
4-environmental factors •
5-toxicity of the agent •
properties of the chemical agent
The process of microbial killing or growth •
inhibition using antimicrobial agent is basically
==== chemical reaction---- which influenced by
a- concentration •
b- temperature •
c- pH •
d- formulation •
Microbial challenge
==the type of m.o. & the levels of microbial •
contamination (bioburden) have a significant
effect on the outcome of chemical treatment
•
***if the bioburden (concentration) is high •
--- long exposure time •
--- high conc. required •
m.o. vary in their sensitivity to the action of chemical
agents
***vegetative bacteria---disinfectant •
should be capable of killing most
vegetative bac. Within reasonable contact
period
==antiseptic & preservatives are also •
expected to have a broad spectrum
antimicrobial activity
Mycobacterium tuberculosis •
==equipments for respiratory investigations or •
therapy can be contaminated & must be
disinfected to safe level
Bacterial spores
== the majority of antimicrobial agents have no •
useful sporicidal action with exception of
***aldehydes •
***hypochlorites •
***some peroxygens •
Physical methods of sterilization •
Fungi- === vegetative form—sensitive •
===spores ---more resistant •
Viruses ===enveloped •
===non enveloped •
Relative resistance of microbes
• Highest resistance
– Bacterial endospores
• Moderate resistance
–
–
–
–
Pseudomonas sp.
Mycobacterium tuberculosis
Staphylococcus aureus
Protozoan cysts
• Least resistance
21
–
–
–
–
–
most vegetative cells
Fungal spores
enveloped viruses
Yeast
Protozoan trophozoites
Killing-Resistant Microorganisms
22
 Endospore formers (endospores):
Bacillus spp. & Clostridium spp. are highly
heat- and chemical-treatment resistant.
 Mycobacterium spp. are highly
chemical-treatment resistant.
 Certain Pseudomonas spp. can actually
grow in certain disinfectants (i.e., in quats
and iodophores).
 Naked viruses (i.e., those lacking lipid
envelopes) often are resistant to chemical
disinfection.
Factors affecting the choice of antimicrobial
agent
1-properties of the chemical agent •
2-microbial challenge •
3-intended application •
4-environmental factors •
5-toxicity of the agent •
intended application
==intended application of an antimicrobial •
agent whether preservative, antiseptic or
disinfectant will influence it's selection &
also affect it's performance
e.g. medical preparations ingredients in the •
formulation may antagonize preservative activity
==in disinfection of instruments the chemical •
must not have a harmful effect (corrosion,
affect clarity of lenses, or change texture)
Fabrics==adsorb QAC •
Rubber==phenol •
**the disinfectant can only act if it is in contact •
with the item
++access to all parts of an instrument is •
essential
++for small items total immersion in the •
disinfectant is required
Factors affecting the choice of antimicrobial
agent
1-properties of the chemical agent •
2-microbial challenge •
3-intended application •
4-environmental factors •
5-toxicity of the agent •
environmental factors
==organic matter can have a drastic effect on •
antimicrobial activity
# adsorption •
# chemical inactivation •
-- Reducing the conc. of active agent in solution •
-Acting as a barrier for penetration of disinfectant •
Blood, body fluids, pus, milk (reduce the effectiveness •
of antimicrobial agents even in small amounts)
toxicity of the agent
==Some agents cause irritation to skin, •
eyes,, respiratory distress
**Phenolics, formaldehyde, gluteraldehyde •
Covered containers •
Rubber gloves •
Face protection •
Types of compounds
Types of compounds
1-acids & esters •
2-alchohols •
3-aldehydes •
4-biguanides •
5-halogens •
6-heavy metals •
7-H2O2& peracid compounds •
8-phenols (carbolic acid) •
9-surface-active-agents •
acids & esters
Organic acids are considered •
Benzoic acid •
**used alone or in combination •
**used as the sodium salt (sodium benzoate) •
**it's active substance is the non-ionized acid •
**it is vital antifungal agent •
**used in combination with other agent (salicylic •
acid) in superficial fungal infection
**it is effective at relatively lower pH values •
**preserve acidic food products •
**conc. 0.5-0.1% are suitable for oral •
preparations
Sorbic acid
widely used preservative •
Potassium salt(=potassium sorbate) •
**inhibit mold growth in acidic food •
(cheese)
**its activity in decreasing pH •
**pharmaceutical preservative for gums, •
mucilage, & syrups
Sulphur dioxid, sulphites, & metabisulphites
**have extensive use as preservative in •
food
**sodium sulphite & metabisulphite or •
bisulphite have a role acting as
preservative & antioxidants
Esters of p-hydroxybenzoic acid ==
(parabens)
are derivatives of benzoic acid =neutral pH •
**methyl, ethyl, propyl, & butyl esters •
**a combination of esters is used for •
preparations which have two phases
--methyl ester == water soluble •
--propyl,& butyl ester ==give •
protection to the oil phase
**pharmaceutical preservatives •
(emulsions, creams, & lotions) •
parabens •
**inhibit mold growth in galenicala, liquid •
cosmetics, food, & shampoos
**very effective against fungi •
**less effective against bacteria •
alchohols
alchohols
Disinfectant & antiseptic •
(ethanol & isopropanol) •
**bactericidal = vegetative bact. •
**not sporicidal •
**have cleaning action= degermination of skin •
surface before injection by wiping out the m.o.
Ethyl alcohol---water is essential for activity •
95%-60%----bactericidal •
70%===disinfectant of skin, clean instrument & •
surfaces
===virucidal---enveloped viruses •
==combination with chlorhexidine or iodine === •
produce more active preparations
==ethanol is popular in pharmaceutical •
preparations & cosmetics
==propanol==bactericidal •
**pre-operative skin treatment •
**preservative for cosmetics •
Aromatic alkyl alcohols •
==preservatives (cosmetics & eye drops)
***benzyl alcohol •
***chlorbutol •
***phenyl ethanol •
***phenoxy ethanol •
***bronopol •
•
•
aldehydes
Gluteraldehyde •
Formaldehyde •
==antimicrobial •
==sporicidal •
Gluteraldehyde •
=broad spectrum •
--vegetative bac.= in minutes •
--spores = 3 hours •
==2%(w/v) aqueous sol.= Cidex •
=cold liquid chemical sterilization of medical & surgical •
materials that cannot be sterilized by other methods
Formaldehyde = Formalin (37%)
=disinfectant •
=preserve biological specimens •
=medical equipments •
=gaseous state (vapor)=toxic & •
carcinogenic
aldehydes
=highly toxic agents •
=used as sterilants •
=high-level disinfectants – kill spores •
=tissue preservatives •
=inactivate viruses during vaccine preparation •
biguanides
Chlorhexidine •
Alexidine •
=low-level disinfectant used in hospitals & dental •
offices & veterinary clinics
=skin degerming agents in preoperative scrubs •
or oral rinse (skin & mucous membrane)
=cannot kill spores or T.B. , limited antifungal •
Chlorhexidine •
=residual chemicals that remain active on •
surfaces for extended periods of time, providing
longer-lasting antimicrobial effects
==peridex •
halogens
Chlorine •
Iodine •
Bromine •
Fluorine
Chlorine
==disinfectant in the form of gas •
Or •
==in combination with other chemical •
substances
==wide spectrum (fungi & viruses) •
==high-level (high concentration)---kill •
T.B.& spores
==chlorine & chlorine compound cause the •
production of hypochlorous acid
NaOCL+H2O ==HOCL+NaOH •
---strong oxidizing agent •
---high-level •
---antimicrobial activity is •
depend on pH (5) •
Chloramines==organic chlorine compounds
---disinfectant & antiseptic
---release chlorine slowly
===water treatment
Iodine •
==skin antiseptic (surgical scrub) •
==bac. Fungi, viruses, spores, T.B. •
==insoluble in water •
--combination of iodine & an organic •
molecule (solubilizing agent=surfactant)
==iodine released gradually
iodophores •
Fluoride •
==antiseptic •
==water treatment •
==toothpaste •
Heavy metals •
Copper, zinc, mercury, silver •
==germicides •
==low-level disinfectant or antiseptic •
==not sporicidal •
==teeth fillings •
Thimerosol==vaccine preservative •
==silver nitrate eye drops •
---restricted==toxic •
H2O2& peracid compounds •
==high-level disinfectant (25%) •
==antiseptic (3%) •
==cleansing of wounds & ulcers •
catalase in human cells •
H2O2
==sporocidal •
==contact lenses preservative •
H2O + O •
nescent oxygen •
Phenols (carbolic acid) & phenolics •
==intermediate– low-level disinfectant & •
antiseptic •
==preservative •
==Bactericidal, fungicidal, virucidal •
==not sporicidal •
==active at acidic pH •
==caustic effect on skin •
==toxic -- systemic •
==the more highly substituted phenols are •
less toxic & can be used as preservative &
antiseptic
Carbolic acid- •
(1867) J. Lester •
Phenolics ==bactericidal, fungicidal, virucidal •
==not sporocidal •
Lysol=cresol ==used widely household •
==antibacterial soaps •
==washing creams •
•
surface-active-agents (surfactants) •
**anionic •
**cationic •
**non-ionic •
According to the ionization of the hydrophilic
group of the molecule
Anionic= mainly used as sanitizers •
Cationic=are quaternary ammonium compounds •
( QUATS ) •
==effective at alkaline pH •
==bactericidal (Gm.+ve bact) •
==limited antifungal •
==non sporicidal •
==skin treatment, wound, abrasions & in •
certain instances as preservatives
Benzyl cholium chloride •
Cetrimide •
==surgery •
Aqueous & alcoholic solution & as creams
==urology & gynecology •
Detergent •
Dish soap •
Household cleaners •
Some mouth washes •
Sites of action of chemical •
antimicrobial agents
Sites of action of chemical antimicrobial agents
Chemical agents of control have 3 primary sites of action: cell wall & cellular
membranes, proteins and nucleic acids (DNA or RNA).
Sites of action of chemical antimicrobial •
agents
Disinfectants can act on m.o. in 2 different ways •
## growth inhibition (bacteriostatic , fungistatic) •
or •
## lethal action (bactericidal,fungicidal, virucidal) •
Disinfectants are usually complex formulation of •
active molecules
Commonly used active ingredients in disinfectants and •
antiseptics include
organic phenolics, •
quaternary detergents, •
alcohols,
•
Iodophors= (iodine containing compounds).
•
Disinfectant and antiseptic compounds •
that persist on a treated surface are
termed residual, ie betadine,
and •
those compounds that evaporate or •
degrade rapidly are non-residual, ie
alcohols.
sometimes also containing : •
--co-solvents •
--chelating agents •
--acidic or alkaline agent •
--surface-active •
--anti-corrosive products •
It should also be noted that may be •
considerable variation in terms of
--pH •
--hardness •
--salinity •
Possible stages of the mode of action
Primary stage—characteristic of the mode •
of action
Secondary stage—only a consequence of •
action
Action on the external membrane of bacterial cell
-membrane consist of basic compounds (phospholipids &
•
lipopolysaccharides) , and is stabilized by Mg & Ca cations
--if ionised disinfecting molecules are absorbed by electrical •
charges at the initial contact & absorption stage , the means of
action will be possible
***non-polar molecules may dissolve & enter the lipid phase
•
***specific carrying system will lead other molecules through
the membrane
***other molecules will be able to disturb the organization of the
membrane by remaining bound to certain sites
•
•
Action on the bacterial wall
--bacterial cell wall is important & it differs •
between Gm.+ve & Gm.-ve,
which leads to great variation in the affinities of •
the hydrophilic disinfectants
Action on the cytoplasmic membrane
--an active molecule may penetrate the •
cytoplasmic membrane in 2 ways
**Passive diffusion (non-specific & slow) •
**Active transport (specific=enabling the •
accumulation of products in bacteria after
---binding to membrane protein •
Action on energy metabolism
--some disinfectants acting on adenosine
triphosphatas (ATP) production
Action on the cytoplasm & nucleic acid •
--disinfectant mechanism may operate the •
cytoplasm & nucleic acid at the
chromosome level
Action on bacterial spores
--spores are impermeable due to the •
presence of dipicolinic acid make them
more resistant
**the presence of highly oxidizing product •
(chlorine) can destabilize this structure in
spores
action of various disinfectants •
action of various disinfectants
Acidic & alkaline compounds •
--the efficacy of acidic & alkaline agents is linked to
the concentration of
***hydrogen (H) •
***hydroxyl (OH) ions •
H ions # destroy the a.a. bond in nucleic acid •
# modify the cytoplasmic pH •
# precipitate proteins •
•
OH ions # saponify the lipids in the enveloping •
membrane
destruction of the superficial
structure
pH increase more than 10 •
# disorganizes the structure of •
peptidoglycan
# causes hydrolysis of the •
nucleotides of viral genome
pH exceed 12 # to act on Mycobacteria
•
Chlorine & derivatives
Chlorine is electronegative •
* oxidizes peptide links •
* denatures proteins •
Hypochlorite & chloramine in water
hypochloric acid
Exposure to lethal dose of hypochloric acid •
*** Decrease in ATP production •
***Permeability of external membrane
•
Exposure to sub-lethal dose •
***inhibit respiration due to a non-specific oxidizing effect •
•
Quaternary ammonium compounds (QAC)
QAC irreversibly bind to the phospholipids & •
proteins of the membrane
impairing permeability •
--the sensitivity is depend on the capacity of the bacterial
cell to absorb such molecules
Gm.-ve.=lipoprotein & liposaccharides on the outer
membrane
Gm.+ve =wall protein •
Thus able to enter & destroy membrane
•
•
•
--in both Gm.-ve. & Gm.+ve. The •
antimicrobial activity of QAC with an alkyl
chain is related to lipophilia
QAC cause increase permeability •
loss of viability •
damage in outer membrane •
metabolic injury & modification of •
permeability
Phenolic compounds
Phenol acts specifically on the cell membrane & •
inactivates intracytoplasmic enzymes by forming
unstable complexes
--the lipophilic molecules are trapped by the •
membrane phospholipids
**in low concentration •
## nucleic acid is liberated out side •
**in high concentration •
## denaturation of protein
## lysis of the cell membrane •
Peracetic acid & hydrogen peroxide
Peracetic acid oxidises & denaturates proteins & lipids of
m.o.
Disorganization of the membrane
Biguanides
Chlorhexidine =primary site of action is cytoplasmic
membrane
modification of membrane
permeability
## this is due to electrostatic interaction of the
chlorhexidine with the acid phospholipids in the
cytoplasmic membrane
•
Iodine compounds
The activity requires to know the behavior of •
iodine in
*aqueous •
*alcoholic solution •
# Iodine containing products == antiseptic •
# iodine monochloride == disinfectant •
Iodine decrease the oxygen requirement of •
aerobic m.o = interferes at the level of the respiratory
chain of the m.o. by blocking the transport of electrons
through electrophilic reactions with the enzymes of the
respiratory chain
= interact with the proteins of the •
cytoplasmic membrane.
Aldehyde compounds
Formoldehyde === acts on •
## protein by denaturation •
## nucleic acid by alkylation •
** the action is pH-dependent===working better at •
alkaline pH
Glutaraldehyde = same mode of action •
Mode of action of virucidal disinfectants
The factors that to understand the action •
== presence of lipids in the virus •
== size of the virus •
Group A= lipid-containing viruses (enveloped) •
herpes, influenza, measles =susceptible •
Group B= small non-lipid viruses (no envelope) •
polio virus, HAV = resistance •
Group C= large non-lipid (no envelope) •
adeno virus •
## the presence of lipid is associated with •
a high degree of susceptibility to all
disinfectants
## the absence of lipid & small are •
associated with resistance to lipophilic
chemical agents
QAC, phenols, biguanides ==group A •
Chlorine, iodine, oxidizing agents, some •
aldehydes, strong acid & alkiline agents
== most viruses
86
87
Evaluation of non-antibiotic antimicrobial agents •
microbial growth •
Dynamics of disinfectants •
Evaluation of non-antibiotic
antimicrobial agents
***in the lab. & under favorable condition •
## when a fresh medium is inoculated with a given
number of bacterial cells
cell growth
Nutrients+ microbial cells
cell growth + energy + reaction products
*** by the time •
nutrients decrease & microbial growth increase •
•
•
## the population growth is monitored over a •
period of time
## plotting the data will yield a typical bacterial •
growth curve
## it is difficult to graph population changes of •
such enormous magnitude by using arithmetic
number
## this why logarithmic scales are generally •
used to graph bacterial growth
Bacterial kinetic parameters
Lag phase==cells initially adjust to the new •
medium until they can start dividing regularly by
binary fission
Exponential log phase •
Bacterial growth becomes limited
stationary phase
decrease in nutrients & increases in metabolic west product
Until eventually they show loss of viability
death phase
Bacterial growth expressed as change in the •
number of viable cells versus time
Generation time are calculated during the •
exponential phase (generation time = is the time
required for a cell to double)
The cells are dividing regularly & are growing by •
geometric progression
==== Cells divide at constant rate •
During the death phase the number of viable •
cells decreases geometrically (exponentially)
Nutrients+ microbial cells = cell growth + energy + reaction products
•
Lag phase=no change •
Growth phase •
=number of cells increase •
=nutrients decrease (due to utilization of nutrients) •
Stationary phase=accumulation of west products, toxic •
metabolite & inhibitory compounds = this effect the
condition (pH)
unfavorable environment
growth rat
death •
forces that affect the microbial growth •
What are the forces that affect the microbial growth ??
Dynamic of disinfectants •
Dynamics= study the forces acting upon a •
body
Dynamics of microbial growth=a study of •
environmental forces that act
either to promote •
Or •
to impede the growth of m.o. •
The course of the destruction of population of •
bacteria by chemical agent or other lethal agents,
can be followed by taking samples at intervals &
determining the number of survivors by making
colony count on a suitable nutrient medium
It is necessary to ensure that the action of the •
chemical should be completely stopped when the
sample is taken either by the use of neutralizing
agent or by dilution
If this not done there will be continuing action &
mortality obtained will be grater than that which
existed at the moment of sampling
•
Bacteria usually die at constant rate •
E.g. 1 million bacteria treated for 1 min. •
90% has died
100000 left •
if is treated for another min.
90% died
10000 •
time/min
Deaths/min
0
0
Number of
survivors
1000000
1
900000
100000
2
90000
10000
3
9000
1000
4
900
100
5
90
10
6
9
1
If the death curve
is plotted logarithmically,
the death rate is
constant
straight line
The factors that affect the disinfection process
** many factors must be considered in the application of
any physical or chemical agent to inhibit or destroy
microbial populations.
== they are classified into two groups
•
(1) factors related to a killing agent (biocide) •
(2) factors related to organisms to be killed •
•
Factors influencing antimicrobial action
Related to killing agent
Related to organisms to be
killed
1- the concentration of the
chemical agent
1-
2- the time during which the
chemical acts
2- kind of organism
3- the temperature at which
the chemical acts
the number of organism to
be killed
3- the physiological state of
the organism
4- the environmental
condition in which the killing
take place
The term concentration is applied to chemical •
agentsFor example 0.1.% phenol will not kill E. coli, but •
will only prevent its growth.
At 10 times this amount, (1% phenol )will kill in •
20-30 minutes.
5% phenol will kill in 2-3 minutes. •
• Sufficient time for contact for chemical reactions that
occur must be allowed.
• This is described as the
• dynamics of disinfection
• 1-The process is a gradual operation in which the
number of organisms killed in unit time is greater in the
beginning, but becomes less and less as the exposure
period is increased.
2.The log of number of the surviving organisms in unit
volume plotted against time gives a straight line curve.
•
## time is inversely proportional to the concentration •
factor,
i.e. the longer the time, the smaller the concentration •
factor
Other factors
## Temperature
## pH.
Temperature --- rise in temp. increase the •
biocidal activity of a disinfectant
--- Microbes killed more rapidly by the same •
concentration of the disinfectant at elevated
temp.
## At certain times a longer time at lesser temperature •
is preferred to prevent the deterioration of flavor,
texture, of the material
pH. •
---- during the disinfection process a charge of •
pH can effect
*** rate of growth •
•
*** potency of the agent •
•
*** ability of the disinfectant to combination •
with sites on the bacterial cell surface •
potency of the agent
•
==if the agent is acid or base it's degree of ionization will
depend on pH
•
==in some compounds the active site is the non-ionized
molecule while the ion is inactive
So- condition of pH which favor the formation of ions of •
these compounds will also reduce this activity
==in other cases activity of the compound is due to the
ionized molecule
•
ability of the disinfectant to combination
with sites on the bacterial cell surface
--before an antimicrobial agent can exert it's
•
effect in a cell it must combine with that cell
--pH affect the adsorption process •
--as increase external pH
surface more negatively charged
the cell •
--cationic biocide bind more strongly to the cell •
surface with consequent increase in activity
Standardization of disinfectants •
evaluation of liquid disinfectant •
Standardization of disinfectants
evaluation of liquid disinfectant
• To standardize antimicrobial agents, several kinds of
properties are evaluated.
•
(1) the ability to prevent growth of organisms, •
(2) the ability to kill organisms already grown,
•
(3) the spectrum of activity or the types of organisms
affected by an agent,
•
(4) the toxicity, or the ability to be used in or on living
medium
•
A number of different methods have been developed to
measure the effectiveness of disinfectants.
the most widely used method •
•
phenol coefficient.===the killing power of a disinfectant •
against a test organism, as compared to that of phenol under
identical conditions.
The conditions which must be specified are as follows : •
-- organism used in the test •
-- age of the culture, •
-- temperature of the test, •
--time of action of disinfectant, •
-- presence and amount of organic matter in the culture, •
--proportion of disinfectant to the culture, •
-- composition and reaction of the transfer medium, •
-- temperature and time of the incubation of the transfer •
medium
**A series of dilutions of phenol is prepared in tubes of sterile distilled
water.
•
**A similar series of dilutions of the disinfectant to be tested is
prepared.
**The tubes are kept in a water bath at a temperature of 20°C.
•
**To each dilution tube0.5 ml of 24-hr broth culture of the test •
organism (selected strains ofStaphylococcus aureus,orSalmonella
typhosa) is added. At intervals of 5, 10 and 15 minutes, a standard
loopful I s transferred from each dilution tube to a corresponding tube
containing 10 ml of sterile nutrient broth.
**The subculture tubes are incubated at 37°C for 24 to 48 hours,
**presence or absence of growth is noted.
•
•
•
The phenol coefficient is then calculated
•
## as the ratio of the highest dilution of the disinfectant killing
the organism in 10 minutes (no growth in subculture tube) but
not in 5 minutes (growth in subculture tube) to the
corresponding dilutions of phenol that will kill under like
conditions.
The phenol coefficient of a disinfectant is of great
value in determining the dilution at which it can be
used effectively.
Preservatives •
preservatives
Are chemicals included in pharmaceutical •
preparations to prevent microbial spoilage
of the products
***Micro-organisms can contaminate the •
pharmaceutical products during their
manufacture and this may lead to the spoilage
and rejection of the contaminated product or
may even cause impairment or death to the
consumer
***Micro-organisms like bacteria and fungi are •
the major problem of contamination
•
Pharmaceutical products consist of large variety of ingredients in a
complex physicochemical state.
•
These are used in prevention, mitigation and diagnosis of diseases.
Any pharmaceutical product has the potential to get contaminated
with bacteria, yeasts or moulds, which may affect their safety,
efficacy or acceptability to the patient or consumer.
Such products must meet •
---the requirements of the Good Manufacturing Practice
(GMP)
---should be stable •
---elegant during storage and use by addition of •
preservatives and also by product design and by product
formulation and packaging
•
•
Sources of Contamination
Microbial contamination from •
# raw materials,
#Dry powders of natural origin •
•
# the manufacturing equipments, •
#process operators,
•
# packaging materials
•
# environment •
•
# Water is the main source contamination
•
#Staff involved in manufacturing due to poor hygienic (skin,direct
contact)
#wet areas such as drains and sinks.
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# dry surfaces like floors and walls contaminate the product
# Airborne contamination may be due to the product left open
to the air.
#Containers and closures should be sterile.
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#Packaging materials like cardboard, plug, card liners, etc are
greatly contaminated with bacterial and fungi spores
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therefore it is necessary to maintain the quality at all the
stages of the process
•
Pharmaceutical product must meet the Good •
Manufacturing Practices (GMP) for stable and safe
product
Sterilization should be maintained while handling the raw
materials and the final product at every stage of
development, manufacture and use.
•
Factors which affects the Microbial Spoilage of
Pharmaceutical Products
*** Micro-organisms grow at neutral pH, therefore acidic
or alkaline formulations are less susceptible to
spoilage.
•
*** Water is the most important cause of the survival and
growth of micro-organisms.
•
*** Other factors includes osmotic pressure, nutrient and
oxygen availability, surface tension, oxidation-reduction
balance, storage temperature and to a less extent
packaging design
•
## combination of two or more preservatives are •
used to extend the range & spectrum of preservation
E.g.--- germall 115 has antibacterial activity but •
combined with parabens shoes antibacterial &
antifungal activity
## combination of antimicrobial preservatives may •
exhibit synergy == two compound exerts a greater
inhibitory effect == widen the antimicrobial spectrum
Eye drops & contact lens solutions
•
Factors affecting preservative activity
** interaction with formulation components •
** properties of the preservative (homogeneous , •
solubility)
** effect of containers •
glass---can expected to retain their •
preservative contact if the closure is airtight •
plastic---may penetrate through & interact with it
rubber---also react with many preservatives •
corks---source of spores & molds •
**type of organism •
** pH •
Sterility assurance
Sterile = no surviving organisms •
Sterility testing== it is a test which assesses whether a •
sterile pharmaceutical product is free from contaminating
m.o. by inoculation of the whole or part of that product
with a nutrient medium == direct inoculation
Random sampling is applied to products •
In the final container to test whether it is adequately •
protected against microbial spoilage (bacteria & fungus)
--nasal & ophthalmic with aqueous bases •
--injections----- multiple dose injections •
--infusions •
--implants •
--syringes •
--bandages •
--dressings •
--needles •
--surgical instruments •
Sterility assurance==efficacy test==sterility test
*****membrane filtration •
Oil, ointment, solid, soluble powder
solutions •
filters •
colony count •
*****direct inoculation •
WHAT IS THE ANTIMICROBIAL •
EFFECTIVENESS TEST?
Antimicrobial Effectiveness Test •
demonstrates the effectiveness of the •
preservative system in a product.
A product Is inoculated with a controlled quantity •
of specific microorganisms.
The test then compares the level of •
microorganisms found on a control sample
versus the test over a period of 28 days
AET=antimicrobial effectiveness test
The AET is performed by spiking a panel of •
challenge microorganisms (representing Grampositive cocci, Gram-negative bacilli, yeast, and
mold) individually into the product and
determining the log reduction of organisms at
prescribed time intervals to quantitatively
evaluate the effectiveness of the antimicrobial
preservative to prevent microbial proliferation
and/or kill the organisms
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Parenteral
Chlorocresol (0.1%)Benzylalcohol (1.0%)
Cresol (0.3%)Phenylmercuric nitrate,
Phenol (0.5%)acetate or borate (0.002%)
Chlorbutol
Opthalmic
Chlorhexidine acetate or gluconate (0.1%)Thiomersal (0.1%)
Benzalkonium chloride (0.3%)Chlorbutol (0.5%)
Oral liquids
Methyl, ethyl and propyl p-hydroxybenzoate
(parabenz) (0.3%)
Benzoic and sorbic acid (0.3-0.5%)
Chloroform (0.25%)
Bronopol (0.5%)
Creams
Parabenz (0.3%)Cetyltrimethylammonium
Chlorocresol (0.1%) bromide (1.0%)
Bronopol (0.5%)Phenylmercuric nitrate
(0.01%)
Final Exam
Good Luck
Final Exam
Good Luck •