The Control of

Microbial Growth
-Dian Natasya-

Mengapa farmasis perlu

mempelajari ttg pengendalian m.o?
Produk
terkontaminasi?

Kerusakan produk degradasi kimiawi,

perubahan aspek fisikokimia
Product recall
Infeksi penyebaran penyakit

References
Tortora GJ, Funke BR, Case CL, 2013, Microbiology an
Introduction, 11th edition, Benjamin Cummings, San
Francisco, USA
Denyer SP, Hodges NA, Gorman SP, 2011, Hugo and
Russells: Pharmaceutical Microbiology, 8th edition,
Blackwell Science Ltd., Blackwell Publishing
Company, USA

Terminology

Copyright 2013 Pearson Education Inc.

The Rate of Microbial Death

Copyright 2013 Pearson Education Inc.

The Rate of Microbial Death

Copyright 2013 Pearson Education Inc.

Factors that influence effectiveness

Number of microbes microbial density
Environmental influences presence of extraneous
organic material
Disinfectant concentration and exposure time
Microbial characteristics innate (natural) resistance
Physical and chemical factor : temperature, pH, &
mineral content of water

Targets of Microbial Control Agents

Cell membrane
Damage to the lipids or proteins of the plasma membrane
causes cellular contents to leak into the surrounding medium
and interferes with the growth of the cell
Enzyme & Protein
Some of chemical bonds (hydrogen or covalent bonds) are
susceptible to breakage by heat or certain chemicals
denaturation
DNA & RNA
Damage to these nucleic acid by heat, radiation, or chemical is
frequently lethal to the cell; no longer replicate, cannot carry
out the normal metabolic functions

Methods of Microbial Control

Physical

Chemical

Physical method of microbial control

vegetative forms of bacteria and fungi, along with
the larger viruses, showing a greater sensitivity to
sterilization processes than small viruses and
bacterial or fungal spores.
D value : the time taken at a fixed temperature
or the radiation dose required to achieve a 90%
reduction in viable cells

Heat

Heat is the most reliable and widely used means of sterilization, affording
its antimicrobial activity through destruction of enzymes and other
essential cell constituents
Heat resistance varies among different microbes
The lethal events proceed most rapidly in a fully hydrated state, thus
requiring a lower heat input (temperature and time) under conditions of
high humidity where denaturation and hydrolysis reactions predominate,
rather than in the dry state where oxidative changes take place.

Moist heat
sterilization

Dry heat
sterilization

Heat resistance varies among different microbes

Thermal Death Point (TDP)
Lowest temp. at which all microorganism in a liquid culture are killed in 10
minutes
Thermal Death Time (TDT)
Minimum length of time in which all microorganisms in a liquid culture are
killed at a given temperature
Decimal Reduction Time (DRT or D value)
Z value
The increase in tem-perature needed to reduce the D - value of an
organism by 90% (i.e. 1 log cycle reduction; Figure 21.2 B)

Copyright 2011 Blackwell Publishing Ltd

Moist Heat
Primarily kill microorganisms by the coagulation of protein denaturation
1.

Boiling Water
kills vegetative bacterial cells, Fungi and many viruses 10 minutes
not effective for endospores and some viruses
Hepatitis viruses (can survive up to 30 min of boiling)
Some spores may survive boiling water for up to 20 hours

2.

Autoclave (steam under pressure)

preferred method of sterilization
The higher the pressure in autoclave, the higher the temperature
Water boils at 100 C
1 atm (15 psi) 121 C

Moist Heat

Most effective: the organisms are either contacted by the steam directly
or are contained in a small volume of aqueous (primarily water) liquid
Care must be taken to ensure that steam contacts all surfaces
At 1 atm (15 psi) , 121 C in 15 minutes
Culture media, instruments, dressings, syringes, etc
The air must completely exhausted expulsion of the air
Equilibrium time, autoclave 121 C in 15 min121
Volume
(mL)

Equilibrium time
(minutes)

501-1.000

20

251-500

15

101-250

10

Pressure-temperature relationship

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Indicator for sterilization

Spores of Bacillus stearothermophilus
fermentable sugar
pH indicator
basic - red
acid - yellow

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Dry Heat
Kills by oxidation effect
1. Direct Flaming
Inoculating Loop and Needle 100% effective
2. Incineration
disposable wastes (paper cups, bags, dressings)
3. Hot Air Sterilization
Oven ( 170C, 1 hour ; 160C, 2 hours )
used on substances that would be damaged by moist heat
sterilization
dressings or powders

Pasteurization
Disinfection - not sterilization (removes unwanted organisms)
Milk Pasteurization:
to eliminate pathogenic microbes & lowers microbial numbers, which
prolongs milks good quality under refrigeration.
63 C for 30 minutes
72 C for 15 seconds (High-temperature short-time (HTST)
pasteurization)
Thermoduric bacteria
able to survive high temp.
unlikely to cause disease or cause refrigerated milk to spoil

UHT
Ultra-high-temperature (UHT) treatments
The milk falls in a thin film through a chamber of superheated steam and
reaches 140 C < 1 second. Its held for 3 seconds in a holding tube and
then cooled in a vacuum chamber, where the steam flashes off.
in < 5 seconds the milk temp. : 74 C 140 C 74 C

Filtration
Removes microorganisms from solutions that might be damaged by heat
High-efficiency particulate air (HEPA) filters: remove almost all
microorganisms > 0.3 m in diameter
Membrane filters: 0.1 mm thick, diameter 0.22 m and 0.45 m

culture media
enzymes
vaccines
Antibiotics

Low temperature

Ordinary refrigerators (0-7 C ): the metabolic rates of most microbes

reduce bacteriostatic effect
Deep-freezing: pure culture is placed in a suspending liquid and quickfrozen at -50to -95C
Lyophilization (freeze-drying): frozen (-54 to -72C) and dehydrated in a
vacuum (sublimation)

Desiccation
Absence of water cannot grow or reproduce but can
remain viable for years
Lyophilization
The resistance of vegetative cells to desiccation varies with
the species and the organisms environment.
Gonorrhea bacterium: withstand dryness only about 1 hour
Tuberculosis bacterium: viable for months
Viruses are generally resistant to desiccation

Osmotic Pressure
Molds and yeasts more capable than bacteria of growing in
materials with high osmotic pressure

Radiation
Depends on its wavelength, intensity, and duration
Ionizing Radiation
gamma rays & x-rays
penetrates most substances
Used on substances that could be damaged by heat
plastic petri dishes
plastic syringes
catheters
surgical glove

 Non-Ionizing Radiation
UV Light
does not penetrate plastic, glass or proteinaceous
matter
Used to reduce microbial populations
hospital rooms
nurseries
operating room
UV lights damages the DNA cell by causing bonds to form
between adjacent thymines in DNA chains (thymine
dimers) inhibit correct replication of the DNA

Methods of Microbial Control

Physical

Chemical

Pemilihan agen kimiawi sbg antimikroba

Karakteristik agen kimia : temperatur, pH, konsentrasi, formula

Jenis mikroorganisme target
sel vegetatif
Mycobacterium tuberculosis
Endospora bakteri
fungi
virus
protozoa
prion
Tujuan penggunaan : pengawetan, desinfektan, antiseptik
Kondisi lingkungan
Toksisitas agen

Copyright 2011 Blackwell Publishing Ltd

Copyright 2011 Blackwell Publishing Ltd

Copyright 2011 Blackwell Publishing Ltd

Phenol and Phenolics

Phenols are a group of caustic chemical compounds that denature

proteins and disrupt cell membrane
irritating to the skin and have limited uses as an antiseptic
Derivates of phenol: Phenolic
Combination of phenolics and soaps or detergents: exert antimicrobial
activity by injuring lipid-containing plasma membrane leakage of
cellular contents.
Advantages of phenolics: remain active in the presence of organic
compounds, stable, persists for long periods after application
Suitable agents for disinfecting pus, saliva, and feces.
Lysol,contains O-phenylphenol

Bisphenols
Derivates of phenol, contains two phenolic groups connected by a bridge

Hexachlorophene, pHisoHex, used for

surgical and hospital microbial control
procedures
Hexachlorophene especially effective in
removing gram-positive cocci and prevents
skin infections in newborns. (Note:
excessive use can lead to neurological
damage)
Triclosan, in hand soap, dishwashing soap,
and toothpaste
The widespread use of triclosan has
resulted in a population of microbes that
are resistant to the actions of triclosan

Biguanides

Chlorhexidine, strong afinity for binding to the skin or mucous membranes,

low toxicity
Used as skin degerming agents for preoperative scrubs, skin cleaning
Biocidal against most vegetative bacteria and fungi

Halogens

Iodine and Chlorine

Iodine, most effective antiseptics, against all kinds of bacteria, many
endospores, various fungi, some viruses.
Oxydize protein inactivation of enzyme
Tincture iodine and iodophor
Betadine, povidone-iodines
Chlorine, as a gas or in combination with other chemicals
Germicidal action is caused by hypochlorous acid (HOCl)
Calcium hypochlorite, Ca(OCl)2, used to disinfect dairy equipment and
restaurant eating utensils
Sodium hypochlorite, (NaOCl)

Alcohols

denature proteins, disrupt membranes and dissolve lipids

Kill bacteria and fungi but not endospores and nonenveloped virus
Ethanol and isopropanol
The addition of water allows for the denaturation of proteins within the
microbe
Recommended optimum concentration: ethanol 70%, biocidal action 10
seconds.
Ethanol 60-95% seems to kill as fast

Heavy metals and their compounds

Silver, mercury, copper, zinc

Oligodynamic action
When the metal ions combine with the sulfhydryl groups on cellular
protein, denaturation results
1% silver nitrate solution: antiseptic
Mercuric chloride: disinfectans (toxic, corrosive)
copper sulfate: algicide in reservoirs, swimming pools
Zinc chloride: mouthwashes

surfactants

Decrease surface tension among molecules of a liquid

Soap and detergents
Soaps helps to remove microbes through scrubbing. They have little to no
true antiseptic activity.
Detergents are organic surfactants such as quaternary ammonium
compounds (quats) that disrupt cellular membranes.
Quaternary ammonium compounds are bactericidal. They effectively
target gram-positive bacteria and are less active against gram-negative
bacteria

Chemical food preservatives

Simple organic acid, or salts of organic acids: Sodium benzoate, sorbic
acid, calcium propionate
typically reduce the pH of the food product
Calcium propionate: interefere the molds metabolism or the integrity of
the plasma membrane

Antibiotics
a substance produced by microorganisms that in small amounts inhibits
another m.o

Aldehyde

Inactivate proteins by forming covalent cross-links with several organic

functional groups on proteins (-NH2, -OH, -COOH, -SH)
Formaldehyde (formalin: 37% aqueous solution of formaldehyde gas)
Glutaraldehyde, less irritating and more effective than formaldehyde
Glutaraldehyde is used to disinfect hospital instrument, include
respiratory-therapy equipment
When used in a 2% solution (Cidex), it is bactericidal, tuberculocidal, and
virucidal in 10 minutes and sporicidal in 3 to 10 hours. Glutaraldehyde is
one of the few liquid chemical disinfectants that can be considered a
sterilizing agent.

Gaseous Chemosterilizers

Gaseous Chemosterilizers: Etylene oxide, Denaturation of protein

suspected carcinogens
Peroxygens (Oxidizing agents)
Oxidizing cellular components of the treated microbes.
Hydrogen peroxide, Benzoyl peroxide

Evaluating a Disinfectant

Use Dilution Test

Phenol coefficient
The Disk Diffusion method
In-Use Test

Microbial Characteristics and Microbial Control

Resistant to
chemical biocides