chemical control of microbes disinfectants antiseptic preservatives

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

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)


•Intermediate level disinfection

•===indicates destruction of all vegetative bacteria including T.B .

• = but may exclude viruses & fungi

• =and have little or no sporicidal activity


•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– most vegetative cells– Fungal spores– enveloped viruses– Yeast– Protozoan trophozoites

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orga

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s 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.

22

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

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 H2O + O

• nescent oxygen•==sporocidal

•==contact lenses preservative

•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

•Anionic= mainly used as sanitizers

•Cationic=are quaternary ammonium compounds• ) QUATS )

•==effective at alkaline pH

According to the ionization of the hydrophilic group of the molecule

•==bactericidal )Gm.+ve bact)

•==limited antifungal

•==non sporicidal

•==skin treatment, wound, abrasions & in certain instances as preservatives

•Benzyl cholium chloride

•Cetrimide

•==surgery•==urology & gynecology

•Detergent•Dish soap

•Household cleaners•Some mouth washes

Aqueous & alcoholic solution & as creams

•Sites of action of chemical antimicrobial agents

Sites of action of chemical antimicrobial agentsChemical 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

BiguanidesChlorhexidine =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

•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/minDeaths/minNumber of survivors

001000000

1900000100000

29000010000

390001000

4900100

59010

691

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

2 -the time during which the chemical acts

3 -the temperature at which the chemical acts

1 -the number of organism to be killed

2 -kind of organism

3 -the physiological state of the organism

4 -the environmental condition in which the killing take place

•The term concentration is applied to chemical agents-

•For 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 the cell surface more negatively charged

•--cationic biocide bind more strongly to the cell surface with consequent increase in activity

•Standardization of disinfectants

•evaluation of liquid disinfectant

Standardization of disinfectantsevaluation 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 .

• #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 .

•#Packaging materials like cardboard, plug, card liners, etc are greatly contaminated with bacterial and fungi spores

•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 Gram-positive 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

• 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

chemical control of microbes disinfectants antiseptic preservatives

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agent slide

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sporicidal activity

chemical disinfectants

chemical agent