Pharmaceutical Microbiology

Sterilization and Disinfection II

 Objectives
• Definitions and differences
• Describe different methods of sterilisation and when they are
applicable for pharmaceuticals
• Describe and recommend antiseptics and preservation
• State use for the different antiseptics and disinfectants, required
concentrations and combinations for the respective uses
• Recommend appropriate disinfectants and sanitisers depending on
presenting circumstances
General Uses of Antiseptics/Disinfectants
• Asepsis before injections:- Views regarding alcohol swab before insulin injection

• Surgical Uses:-
• Asepsis before surgery – e.g. chlorhexidine, povidone iodine or alcohol based
• Scrubbing before surgery -
• Disinfection of instruments e.g. endoscope disinfection

• Cuts, Wounds & Lacerations: What common household antiseptic is used form minor
lacerations in the home setting?

• Disinfection of Hospital premises

• Water purify
• cation & domestic disinfection
Attributes of an IDEAL Antiseptics/Disinfectant
• High Efficacy:– Broad spectrum / sporicidal / kill all forms
• – Rapid onset / long duration action
• – Cidal not static
• – Active in presence of blood, pus, exudates and excreta
• – No Resistance
• Non Toxic:
• – Non-irritating to tissues. Should not delay healing.
• – Non absorbable / No hypersensitivity
• – Chemically stable.
• Added Advantage in particular antiseptics:-
• – Non-staining with agreeable color and odour
• – Could be added to soaps
• – Non corrosive.
 General Mechanism of Action Antiseptics,
Disinfectants
Active ingredients in antiseptics and disinfectants achieve their effects
through interactions with the microorganism cell surface followed by
penetration into the cytoplasm and action on cellular targets. In general they
have 3 main mechanisms through which they affect or kill microorganisms
E.g.
• Oxidation of Bacterial protoplasm. e.g.- KMn04, H202, Halogens

• Denaturation of Bacterial Proteins & Enzyme e.g.-Phenols, Chlorhexidine,

Alcohol, Aldehyde

• Detergent like action increasing permeability of bacterial membrane e.g.-

Soaps, Cetrimide
Factors influencing activity of Disinfectants
and Antiseptics
• TEMPERATURE: Directly proportional to temperature
• CONCENTRATION: Directly proportional to concentration up to a point – optimum
concentration. After this level no advantage in further increases in concentration e.g.
alcohol must be diluted with water for the optimum effect, as proteins are not denatured
as readily with straight alcohol.
• PERIOD OF CONTACT WITH MICROBES: Disinfectants need time to work
• NATURE OF MICROBES INVOLVED: Range of Action : Disinfectants are not equally
effective against the whole spectrum of microbes. e.g. Chlorhexidine is less active against
GNB than Gram Positive Cocci on the other hand Alcohols are generally considered to be
non-specific antimicrobials because of their many toxic effects, Hypocclorites and
Glutaryldehydes are more active against hepatitis viruses than other disinfectants
• SIZE OF INNOCULUM
• PRESENCE OF BLOOD,PUS,OR ORGANIC MATTER: May be inactivated by Dirt (organic
matter), Proteins (Pus, Blood, Mucus, Faeces) Cork and some plastics
Factors affecting choice of microbial agent
Choice of a microbial agent to use for disinfection or as antiseptic is
NOT arbitrary and should be informed by a number of factors including
• Properties of chemical agents
• Microbiological challenge
• Intended application
• Environmental factors
• Toxicity of agents
Factors affecting choice of microbial agents:
Properties of chemical agents
• May be classified based on mechanism of action and further on chemical groupings as follows
• PHENOL DERIVATIVES:- Phenol,Cresol,Hexylresorcinol,Hexachlorophene, chloroxylenol, thymol
• OXIDIZING AGENTS:-KMno4,,Hydrogen peroxide, Benzoyl peroxide
• HALOGENS:-Iodine, Iodophores, Chlorine, ChlorophoresIodine,
• ALCOHOLS:-ethanol , Isopropanol.:-ethanol , Isopropanol.
• ALDEHYDES:-:- Formaldehyde, Glutaraldehyde
• SURRFACE ACTIVE AGENTS:-FACE ACTIVE AGENTS:-
• QUATERNARY AMMONIUM (CATIONIC):- Cetrimide, Benzalkonium chloride, Dequalinium chloride.
• BIGUANIDE:- Chlorhexidine
• SOAPS:- OF sodium & Potassium 13
• METALLIC SALTS:- Silver nitrate, Silver sulfadiazine, Mild silver protein, Zinc sulfate, Calamine, Zinc oxide.
• ACIDS:- Boric acid, Acetic acid
• GASES:- Ethylene oxide
• DYES:- GENTIAN VIOLET, METHYLENE BLUE BRILIANT GREEN, ACRIFLAVIN AND PROFLAVIN
• MISCELLANEOUSISCELLANEOUS:-:- NITROFURAZONE
Special Points Pharmacists Recommendation
for antiseptics and disinfectants
• Antiseptics and disinfectants should be used at the recommended strength,
in the correct vehicle
• Prolonged continuous and indiscriminate use may lead to sensitization see
recent FDA warning.
• Medicated soaps and cleansers have limited role in controlling recurrent
skin infections – concerns with daily use
• Regular intermittent use of antiseptic e.g povidone iodine,
chlorhexidine/cetrimide are of value in contoling recurrent staphylococcus
infections.
• Dilute acetic acid(5%) or silver notrate dressings are of value for the control
of pseudomonas
General Use of antiseptics and disinfectants by class
• Alcohols – alone or in combination for skin disinfection

• Quaternary ammonium type products – skin cleansing

• Chlorinated solutions – irrigation and cleansing of offensive wounds

• Hydrogen peroxide – diluted – cleaning wounds contaminated with foreign

materials

• Organic iodine – favourable antibacterial, antifungal and antiviral efficacy

Biguanides: Chlorhexidine
• Intermediate level antibacterial activity
• Widely used antiseptic - for surgical skin
preparation and surgical hand scrubs, wound
disinfection, urethral disinfection etc
• MoA: Self study
• non-irritating, colorless, odorless antiseptics
• Spectrum of activity:- Active against gm+ve bacteria
Moderately active against –gm-ve bacteria
• Activity against spores:Data indicate that chlorhexidine
may be capable of inhibiting spore growth and
germination but that it does not kill the spores.
• Ineffective at ambient temperatures against bacterial
spores and M. tuberculosis
• Effectiveness may be dampened in the presence of
organic matter
• non-irritating, colorless, odorless antiseptics
Biguanides: Chlorhexidine
• Formulations: Savlon liquid antiseptics:- Chlorhexidine gluconate 1.5%
+ cetrimide 3% exhibits enhanced efficacy. Other chlorhexidine
formulations- lotion, Cream, Washes, Aqueous solution, mouth
washes, burn ointments and wound protectants. It has also been
incorporated into products as disinfectant.

• Available in BEML –
• Chlorhexidine 20% solution
• Chlorhexidine 4% liquid soap

• Concentration and formulation by Use:

• Preoperative skin disinfection – 0.5% aqueous solution in
70% alcohol
• Wound disinfection – 0.05% aqueous solution
• Urethral disinfection and catheter lubrication – 0.05%
solution in glycerol
• Bladder irrigation – 0.02% solution
• Obstetric cream – 1% cream
• Storage of sterile instruments – 0.05% solution
• etc
QUATERNARY AMMONIUM (CATIONIC
ANTISEPTICS): CETRIMIDE
• Intermediate level antiseptic
• Narrow spectrum: Mostly effective against G(+) agents
• wound clearing from dirt ,skin disinfectant
• Anti-plaque agent
• As antiseptics, Surgical instruments, utensil,
• Formulations: Well tolerated and non-toxic when applied to skin and
mucus they have general use in treatment of wounds and abrasions, as
preservatives in certain preps (benzylkonium chloride)
• Sometimes used in combination with chlorhexedine
QUATERNARY AMMONIUM (CATIONIC
ANTISEPTICS): CETRIMIDE
• Formulations: Well tolerated and non-toxic when applied to skin
and mucus they have general use in treatment of wounds and
abrasions, as preservatives in certain preps (benzylkonium
chloride)
• Sometimes used in combination with chlorhexidine
BEML - Cetrimide +Chlorhexidine (15+1.5)% solution
• Concentration and formulation by Use:
• Cleansing of burns and wounds – 0.1% solution. 0.5-1% may be used if
skin or wound are bacterially contaminated
• Pre-operative cleansing – 0.5% in 70% alcohol
• Shampoo – 1% solution
 PHENOL DERIVATIVES:
• Extensive list with varying uses
• General MoA
• Act by denaturing bacterial protein
• Scope:
• Mainly bactericidal and fungicidal
• Limited activity against viral particles
• Examples: chloroxylenol in (Dettol), thymol in ( Listerine).
Note final products contain combinations and therefore
scope of activity may differ from general scope depending
on final formulation.
• Use:
• What is Dettol used for?
• What is Listerine used for?
 OXIDISING AGENTS
Hydrogen peroxide
• MoA:
• Scope: Self study
• Uses-
• Mouthwash (3%)- dilute with water- stomatitis/ cavity
preparation
• Bleaching agent (for teeth stains)- 30% H2O2+ glycerine+
pumice
• Surgical use (3-6%)- wound cleaning / slough remove
• Removal of ear wax – Deodorant – Styptic
• Note: To be kept in dark bottles. High
concentrations can be caustic
• What is the reason for including hydrogen
peroxide in the alcohol based hand sanitiser?
Reflection: Hydrogen peroxide in WHO
formulation of hand sanitiser- Formulation II
To produce final concentrations of isopropyl alcohol 75% v/v, glycerol
1.45% v/v, hydrogen peroxide 0.125% v/v:
• Pour into a 1000 ml graduated flask:
1.isopropyl alcohol (with a purity of 99.8%), 751.5 ml
2.H2O2 3%, 41.7 ml
3.glycerol 98%, 14.5 ml
• What is the utility/rationale for including hydrogen peroxide in this
formulation?
Reflection
• Why is the maximum recommended concentration of IPA in hand
sanitiser not 100%?
• Choice between alcohol and hypochlorite
HALOGENS:
IODINE & I- releasing agents: • Chlorine & Cl- releasing agents:
• Rapid action • Rapid action
• Broad spectrum • Chlorine = HIGHLY REACTIVE
• Not readily inactivated by organic matter • Broad spectrum
• MICROBICIDAL AGENT, IODINATES AND OXIDIZES • readily inactivated by organic matter
MICROBIAL PROTOPLASM
• MICROBICIDAL AGENT, CHLORINATES AND
OXIDIZES MICROBIAL PROTOPLASM
• Chlorine is used as a gas or in combination with
other chemicals. Chlorine gas is used for
disinfecting municipal water supplies, swimming
pools, and sewage. Sodium hypochlorite – ordinary
household bleach- is good disinfectant.
Chloramines consist of chlorine and ammonia. They
are more stable than most chlorine
 IODINE & I- releasing agents: Iodophores
• These are complexes of Iodine. An
iodophore is a combination of iodine and
an organic molecule. Iodophores do not
stain and are less irritating than iodine.
• Slow RELEASE of FREE IODINES
• Betadine and Povidone Iodine are
examples of iodophores
• Non-irritating, non-toxic.
• USES: for boils, burns, otitis externa, ulcer,
vaginitis, surgical scrubbing, disinfection
of endoscopic instruments.
• Varied formulations: e.g. Tinea infections
(5%- cream, solution, Ointment). Dental
uses- Mouth wash/ Gargle (2%).
Chlorine & Cl- releasing agents:
• Even in very small amounts, it exhibits fast bactericidal
action. Chlorine works by oxidizing proteins, lipids and
carbohydrates. Hypochlorous acid, which is a weak acid that
forms when chlorine is dissolved in water, has the most
effect on the bacterial cell, targeting some key metabolic
enzymes and destroying the organism. Chlorine compounds
have also been shown to affect surface antigen in enveloped
viruses and (DNA) as well as structural alterations in non-
enveloped viruses.
• Very few chemicals are considered sporicidal; however,
chlorine compounds in higher concentrations have been
shown to kill bacterial spores such as Clostridium difficile (C.
diff).
Chlorine & Cl- releasing agents: Chlorophores
• COMPOUND RELEASES hypochlorous.
• TYPES:-
• 1.CHLORONATED LIME (BLEACHING
POWDER) :USES: Disinfection of drinking
water, Swimming pool • Sanitizer• Tooth
stains, Tooth stains removal 26
• 2.SODIUM HYPOCHLORITE SOLN:- USES:(4-
6%) DISINFECTANT IN DAIRIES FOR MILK
CANS, Lab: disinfectant, Biohazard Spill
management, EQUIPMENTS,ANTISEPTIC,
also used as Bleaching agent in dentistry
• 3 .EUSOL: ((chlorinated lime 1.25%+Boric
acid). USES:- Dissolving pus,Dissolving pus,
Necrotic matter, Clean infected wound
Reflection
• A client whose partner has recently been started on antiTb treatment
wants you to suggest a disinfectant that he can use to disinfect
utensils that are shared. Which of the three would you recommend
and Why?
• Chlorhexidine?
• Household bleach?
• Savlon?
ALCOHOLS
• Intermediate level antibacterial activity
• Both ethanol and isopropanol are widely used,
normally at a concentration of about 70%.
Concentrations of 60% to 90/95% are effective.
• MOA- ppts bacterial proteins (denaturation) /cidal
• They are bactericidal and fungicidal but are not
effective against endospores or non-enveloped
viruses.
• EFFECTIVE ANTISEPTIC on skin, also prior to injection
IPA. Rapid action. Surface disinfection
• CLEANSING AGENT AT 40-90% CONC.
• ADRs: BURNING SENSATION,INJURY, irritant on Muc.
membranes.
• Alcohols enhance the effectiveness of other
chemical agents
Reflection
• Why should 99/100% alcohol be diluted before use?
• What are the recommended concentration of ethanol for
• Cleaning surfaces
• Cleaning instruments
• Skin disinfection
• Anti viral activity
ALDEHYDES
• High level disinfectant

• MOA- DENATURES BACTERIAL PROTEINS. PROTOPLASMIC POISON.

• FORMALDEHYDE (Formalin):
• USES: Disinfection of surgical instruments (2-8%), Preservation of dead tissue samples
(4% aqueous Solution), fumigation, urinary antiseptic (Methenamine)
• ADRs: ECZEMATOID REACTIONS, irritates, No oral use.
ALDEHYDES: Glutaraldehyde
• Glutaraldehyde
• Broad spectrum
• Less pungent
• Less irritating
• Not inactivated by biofluids
• Activated at alkaline pH. – At pH 8 biocidal activity is greatest but stability is poor due to
polymerisation.
• Note reason why in practice it is generally supplied as an acidic 2% solution. Explain?
• Activation prior to use with suitable alkylating agent.
• What do you think is the likely impact of activation on shelf life of glutaryldehde???
• Rapid kill for vegetative bacteria but spores may require up to 3 hrs
• USES:
• 1. Cold chemical sterilization of instruments that cannot be sterilised by other means-
Disinfection of surgical instruments , Endoscopes (to be kept for 12 hrs)
• 2. Hospital disinfectant of premises / Operating theaters
Take home
• For the following classes of disinfectants/antiseptics give at least 3
examples of agents that are listed in the Essential Drug List for
government facilities and match individual agents to the following
classes of disinfectants and include mechanism of action
Take Home Reflection -
• For each of the disinfectants listed under the Essential Medicines List
• Classify based on mechanism of action
• Classify based on chemical properties
• Indicate scope of antimicrobial activity
• Indicate wether used as antiseptic or disinfectants
• Level of Disinfectant
Essential List item CHAPTER 15.0 DISINFECTANTS Classify based on Scope of antimicrobial Indicate general use i.e Level of
AND ANTISEPTICS Classify based on mechanism chemical props activity Disinfectant or Antiseptic disinfectant
Cetrimide +Chlorhexidine (15+1.5)% solution
Chlorhexidine 20% solution
Chlorhexidine 4% liquid soap
Hydrogen peroxide 3% solution
Ortho-phthalaldehyde 0.55% solution
Povidone iodine 10% solution
Povidone iodine 7.5% scrub solution

Sodium dichloroisocyanurate (presept) disinfectant

tablet
Spirit rectified 96%
Factors affecting choice of microbial agent:
Microbiological challenge - Overview
• How does microbial challenge affect outcome of chemical treatment?
• Type of microorganism present: microorganisms vary in their sensitivity to
chemical action
• Level of microbial contamination (bioburden)

• Type of Microorganism: Micro-organism

• Why is it important to know the type/s of microorganism that are being
targeted in the disinfection/sanitisation process?
Factors affecting choice of microbial agent:
Microbiological challenge – Types of Microorganism
• Vegetative bacteria
• Note that G(-) microorganisms which are the main cause of nosocomial infections are more resistant than G(+) species
• Mycobacterium tuberculosis
• Mycobacterium tuberculosis and many mycobacteria are resistant to most bacteriocides. Note that tuberculosis remains an important
public health problem ad the greatest risk is from undiagnosed patient. Therefore it is important to adequestely disinfect equipment
used for respiratory investigations to prevent transmission – See some of the agents that are effective against M.tuberculosis –
glutaryaldehyde, ethanol, IPA, Hypochloride,
• Bacterial spores
• They are the most resistant of all microbial forms. They are resistant to most antimicrobial agents except aldehydes, halogens and
peroxygens
• Fungi:
• Many disinfectants have good activity against pathogenic fungi like candida albicans
• Viruses:
• Susceptibility depends wether the virus possesses a lipid envelope. Non-lipid viruses are usually more resistant, less predicable
response and will need specific selection of specific antimicrobial agent to suite special circumstances. See concerns with handling of
articles contaminated with HIV, Hepatitis B, Covid etc. Glutaryldehyde effective disinfectant agains HIV contaminated spillage
• Protozoa:
• There were further evidences that soft contact lens wearers could be at greater risk for protozoan infection wich can cause corneal
scaring and loss of vision. Hydrogen peroxide based disinfection has been shown to be amongst the most effective, reliable, constisten
acathomamoebacidal effect
Factors affecting choice of microbial agent:
Intended application
• Is the antimicrobial agent intended as?
• Preservative
• Risk to patient
• Does preservative interact with other substances in formulation to antagonise them or
do they antagonise the preservative
• Antiseptic
• Disinfection
• Does it cause corrosion?
• Does it affect integrity of item being disinfected (contact lenses)
• Does it change texture of item being disinfected e,g. fabric
Reflection: Intended application
• HALOGENS are effective against
mycobacterium tuberculosis,
spores, and spillage of HIV
contaminated blood and body
fluids
• Would you recommend its use for
use in
• Disinfection glassware contaminated
with above?
• As ingredient in hand sanitiser in
high risk area for transmission of
Tuberculosis via hand contact?
• As disinfects of clothes with spillage
of possible HIV blood
Factors affecting choice of microbial agent:
Environmental Factors
• Organic matter and effect on antimicrobial activity
What is the general effect of organic matter affect disinfectant activity?

• Dried organic deposits may inhibit penetration of chemical agent –

where possible object to be disinfected could be thoroughly cleaned
Factors affecting choice of microbial agent:
Toxicity of agents
• Toxic volatile substances and impact on immediate environment
• Formaldehyde versus glutaryldehyde
• General limit on exposure of individuals to excessive levels of the
chemical substances