KLA210/437/200: Lecture Synopses Lecture 6

Lecture 6: Inactivation of Microbes

Introduction and Definitions
Microorganisms do a large number of things that we dont like. They:
cause diseases of people, plants and animals;
spoil food;
cause rotting and decay of organic-based objects (inc. wood, fabrics, rubber etc.)
can accelerate degradation of inorganic materials;
cause fouling of pipes, boat hulls, nets, etc.
The deleterious activities of microorganisms are intimately linked to their metabolism but, more
importantly to their numbers. Thus, there is a lot of practical interest in methods for killing microbes to
reduce their numbers, or to stop them from reaching large numbers where their combined action can
cause overt damage.
Sterilisation describes the complete elimination of cells and (endo)spores from an object, or product or
environment. The challenge for sterilisation is to eliminate endospores, which are extremely resistant to
heat, radiation, and chemicals.
Decontamination refers to the treatment of an object to make it safe to handle (i.e. to render it unlikely
to spread disease). Disinfection refers to the elimination of microorganisms from inanimate objects
while an antiseptic is an agent that kills or eliminates microbes but that is safe to use on living tissue
(e.g. skin).
The suffix -cidal indicates the death of microbes, i.e. bactericidal, fungicidal. virucidal, etc. The
suffix -static indicates the prevention of growth (but not necessarily the inactivation) of microbes. The
suffix -lytic indicates that not only are cells killed, they are lysed and disintegrate.

Methods for Microbial Inactivation

Methods for elimination of microbes can be categorised roughly into physical and chemical processes.
Physical methods involve:
heat
radiation
physical removal (e.g. filter sterilisation)
high pressure, electroporation, sonication
A range of chemicals are used to disinfect inanimate objects or as antiseptics.

Magic Bullets?
Many of the methods available to inactivate microorganisms are just as damaging to any living tissue, or
organic matter. Thus, damage can also occur to the object being disinfected/sterilised by the treatment
applied. The ideal is to find a method that has selective toxicity, i.e. that only attacks the microbial
contaminants. This need was identified by Ehrlich in the early years of the 20th Century and described
by him as a magic bullet, i.e. something that only damages the desired target. Currently, there is
much research interest in the use of nano-particles of silver. Silver has long been known to have
antimicrobial properties, and silver compounds were used to treat infections prior to the advent of
antibiotics, after which they fell out of favour. Products like antimicrobial gels for fighting human
infections, antimicrobial paints, and antimicrobial food packaging films that are based on the inclusion
of silver nano-particles are being developed or already available commercially. Thus, the magic bullet
might turn out to be a silver bullet! (see short article by Edward-Jones (2009), a copy of which is
available on the MyLO page).
KLA210/437/200: Lecture Synopses Lecture 6

Physical Methods: Heat

All organisms have an upper temperature limit for growth and when that temperature is exceeded
denaturation of macromolecules occurs. If the macromolecules cannot be replaced, microbial death
ensues, sooner or later. Thus, heat can be used to kill microorganisms, but is not selective in its action.
Methods of heat sterilisation include:
autoclaving involves treatment with steam at 121C, which can only be achieved in a pressurised
vessel (called an autoclave); the usual treatment time is 15 minutes at this temperature.
Autoclaving is appropriate for most heat resistant items such as glassware, surgical instruments,
wound dressings, some plastics, microbiological media, etc. In the autoclave the energy in the steam
is efficiently transferred to the material being heated when steam condenses onto the object or
material.
dry heat treatment at 170C for 3 hours is considered a sterilising treatment. This process takes
longer (than autoclaving) because the energy of the molecules in the air is not as efficiently
transferred to the objects being heated. Dry heat sterilisation is not often used, but is appropriate
for sterilisation of paper, powders etc. that could absorb moisture.
Tyndallisation involves a series of mild heating steps, and can be used for heat sensitive materials, e.g.
blood products, some amino acids in solution, some sugars in solution. As stated above, the
challenge in sterilisation is to eliminate endospores that may be present, and that could
subsequently germinate, and start to grow. The trick in tyndallisation is to encourage any
endospores present in the product to germinate, so that they are less resistant to heat. Thus,
tyndallisation involves heating to 100C for 30 minutes on three consecutive days. After the first
heat treatment spores should germinate, to become vegetative cells that are then eliminated by the
heat treatment on the second day. The third heat treatment is included for additional certainty.
Pasteurisation was originally undertaken at 63 - 66C for 30 minutes. It is not a sterilisation procedure,
but does eliminate most vegetative bacteria, but particularly pathogenic bacteria (Mycobacterium
tuberculosis, Brucella). Those not killed are inhibited from outgrowth in the product by refrigeration.
(UHT treatment is a variation involving much higher temperature, that eliminates even more bacteria
and does not require refrigeration to prevent outgrowth of survivors).

Physical Methods: Irradiation

Relies on electromagnetic radiations of different energies, e.g. microwave, ultraviolet, & ionising
radiations
microwave radiations are translated into heat i.e. water, fat and other molecules absorb microwave
energies and vibrate/oscillate rapidly. (this is called dielectric heating and occurs because the molecules
are polar and oscillate in the alternating magnetic field induced by the microwaves). In doing so they get
hotter and that heat is transferred to the product.
ultraviolet light
(220 - 300 nm, ~260nm is most lethal)
useful for surfaces, air, water - but UV light has poor penetrating power
causes pyridine dimers in DNA, i.e., adjacent cytosine or thymine nucleotides bond to each other,
causes DNA reading errors,
damage can be repaired using the enzyme photolyase, (n.b. mammals no longer have this
enyzme).
ionising irradiation
EM radiation (photons/particles) of sufficient energy to ionise molecules, and penetrate many
materials
KLA210/437/200: Lecture Synopses Lecture 6

types include X-rays , gamma rays (usual sources are Cobalt 60, Caesium 137), electrons (cathode
ray tubes)
this generates highly reactive species (e-, OH, H) that interact with, and denature, macromolecules
in the cell
used for disposable plasticware, food, etc
killing dose - that which achieves 12D (i.e. 1012) reduction
dose is measured in rads (100 erg/g) or Grays (100 rad)
e.g. 40,000 Gray for Clostridium botulinum; 2400 for Salmonella, lethal dose for humans is 10 Gray
called radurisation when applied to food

Physical Methods: Filtration

membrane filters can be made with very small pore sizes and can be used to sterilize heat labile
solutions, esp. culture media, blood products
pore size must match organisms of concern (0.22 m pore size is common and small enough to trap
most bacteria; a 0.10m is also often used to ensure that smaller bacteria, e.g, mycoplasmas which
are wall-less and are intracellular parasites, are eliminated particularly for cell culture).
HEPA (High Efficiency Particulate Air) filters are another form of filtration and can eliminate
microorganisms in air, they are to filter air in clean rooms (e.g. tissue culture work, some food
preparation areas), laminar flow cabinets, etc.

Other Physical Methods

A variety of other techniques are being experimented with, particularly for food products so as to
preserve the natural attributes of the food while still removing bacteria that could cause disease or
cause food spoilage.
Sonication is a method that uses ultrasound to kill cells in solution. The probe of the ultrasound device
is inserted into the solution. It generates very large pressure differentials close to the probes tip,
that can cause cells to rupture (once this happens most cells will die). There is also a heating effect
however, and its important when using ultrasound (sonicators) to cool the liquid being treated so as
not to denature molecules that you are trying to release from the cell.
Pulsed electric fields are used in the food industry for some liquid foods. They depolarise cell
membranes, leading to formation of holes or large pores in the cell membrane. The advantage is
that it is only effective against intact cells, and does no damage to cells that are already dead, such as
those of foods that have already been processed. The same process is used to get DNA into cells in
molecular biology procedures, in which case it is called electroporation.
high pressure processing is a new technique in the food industry. It involves pressures that are 3000 to
6000 times greater than normal atmospheric pressure. This pressure is applied evenly throughout
the food, so does not crush the food, but can eliminate many vegetative bacterial and fungal cells,
and some viruses. The advantage is that it does not greatly affect the food.
Other methods being explored include pulsed light, high intensity light, plasma (ionised gas) discharges
but are experimental at this time.

Kinetics of Inactivation
Microbial inactivation kinetics (i.e. rates of death) are usually considered to be (essentially) exponential
decline, i.e. when subjected to a lethal treatment, the number of cells declines as an exponential
function of treatment time. For example, if 90% of cells are killed in the first 5 minutes, then 90% of
the surviving cells are killed in the next five minutes, and 90% of those surviving after 10 minutes are
killed in the next five minutes.
KLA210/437/200: Lecture Synopses Lecture 6

Thus, the lethality of a process is characterised as the time taken for a 10-fold reduction in cell numbers
(this is the same as saying the time taken to eliminate 90% of cells). This time is called the D value.
The D value depends on:
organism (species and strain)
temperature/radiation intensity
suspending medium (i.e. the system that the cell is in)
and some other factors.
Because the D value depends on temperature, it is often written as, for example, D55C, D63C, D72C etc. to
denote the conditions that it relates to.
D value is calculated by plotting log10(cfu) vs time, and is the slope of this plot.
z value is another measure used to characterise the thermal tolerance of cells. It is the change in
temperature that will lead to a 10-fold change in D value. z-values are typically in the range 5 - 10C,
and can be calculated by plotting log10(Dvalue) for different temperatures against the temperature.
The z-value is the slope of the resultant plot.
thermal death time is the time required under defined conditions to eliminate all contaminants. As
such, the thermal death time depends on how many cells are present in the sample to start with. It
can be calculated as the [log10(number of cells) + 1] times the Dvalue. This calculation predicts the time
taken to get to less than one cell in the sample. Usually, we want more confidence that no cells
remain, and so increase the number of Dvalues required, e.g. [log10(number of cells) + 4] times the Dvalue
should give 99.9% confidence that no sample contains a viable cell (alternatively, that less than 1 in a
1000 units contain a viable cell).

Chemical Methods: general

Chemical antimicrobials are natural or synthetic compounds that kill or inhibit the growth of
microorganisms.
sterilants (=sporicides) destroy cells and (endo)spores. Examples include ethylene oxide,
formaldehyde, hydrogen peroxide, chlorine, peroxyacetic acid.
disinfectants destroy cells (but not necessarily spores), and are intended for use on inanimate objects
sanitisers destroy cells (but not necessarily spores), and are to be used on surfaces that may contact
living tissue or that may be ingested
antiseptics (germicides) destroy cells (but not necessarily spores), and can be used on living tissue

Chemical Methods: modes of action

There are a variety of chemicals and modes of action for killing cells, or inhibiting their growth. These
include:
lipid solvents (which affect the cell membrane). Examples include: ethanol, detergents, quaternary
amines
strong oxidants (which affect all organic molecules). Examples include: hydrogen peroxide,
chlorine/hypochlorite
protein denaturant/precipitant. Examples include: copper sulphate, alcohol, silver nitrate, phenolics,
alkylating agents (which interfere with DNA, protein). Examples include: formaldehyde, glutaraldehyde,
ethylene oxide
iodine compounds (iodophors). These react with tyrosine residues in proteins.
KLA210/437/200: Lecture Synopses Lecture 6

Its hard to predict how effective a chemical will be against any specific microbe. Effectiveness depends
on:
contact time
temperature
concentration of chemical
other environmental conditions (pH etc)
presence of other organic matter (which can compete for the active ingredients)
EPS layers, biofilm formation, because these can impede access of the chemical to the cell. Also,
cells in biofilms switch on resistance mechanisms.

Chemical Methods: antibiotics-NOTE THAT MORE DETAILED INFORMATION ON ANTIBIOTICS

AND CHEMOTHERAPEUTIC AGENTS WILL BE DELIVERED LATER IN THE SEMESTER BY THE
MEDICAL MICROBIOLOGY STAFF
Most antibiotics originate from microorganisms, i.e. they are naturally occurring compounds, but these
can be modified chemically to make them more effective these are called semi-synthetic
antimicrobials. Some antibiotics are fully synthetic, i.e. man-made compounds that are active against
microbes. Broad-spectrum refers to antibiotics that are active against both Gram positive and Gram
negative cells. (Conversely, a narrow-spectrum antibiotic is active against only some Gram positive or
Gram negative cells).
Among synthetic antimicrobials, modes of action include:
growth factor analogues such as sulfa drugs (analogue of PABA, part of folic acid, required for
nucleic acid synthesis), and isoniazid (nicotinamide analogue)
nucleic acid analogues which result in blockage of DNA replication, non-sense transcripts,
mutations; and include the compounds fluorouracil (like uracil), bromouracil (like thymine)
quinolones interact with DNA gyrase, prevent DNA supercoiling/packing and are based on nalidixic
acid. Ciprafloxacin is an example. Quinalones are broad spectrum.
Among antibiotics, modes of action include:
protein synthesis blockers
initiation blockers e.g. streptomycin
elongation blockers e.g. puromycin, chloramphenicol, cycloheximide, tetracycline
these also affect mitochondria (i.e. have side effects in eukaryotes, such as people)
transcription inhibitors
inhibit RNA synthesis
also affect mitochondria (i.e. have side effects in eukaryotes, such as people)
actinomycin is an example
cell wall interference
penicillins and derivatives (called -lactams)
stop peptidoglycan cross-linking by binding to transpeptidases
cephalosporins are similar but are more resistant to -lactamases, have a broad spectrum,
e.g. ceftriaxone

Antibacterial antibiotics: modes of action

aminoglycosides
inhibit protein synthesis at 30S ribosomal sub-unit
e.g. neomycin, gentamycin, streptomycin
effective against Gram negatives
macrolides
protein synthesis inhibitor at 50S ribosomal sub-unit
KLA210/437/200: Lecture Synopses Lecture 6

e.g. erythromycin
11% of all antibiotics used; useful alternative to penicillins
tetracyclines
inhibit protein synthesis at 30S ribosomal sub-unit
broad spectrum
e.g. chlortetracycline, oxytetracycline (natural) but also semi-synthetic derivatives
very wide usage, including as nutritional supplements for farmed animals

Antiviral, anti-parasitic and anti-fungal drugs

Parasites and fungi are eukaryotic cells. Viruses utilise the metabolism of the host cell to replicate. As
such, its harder to find a magic bullet antibiotic because these pathogens are much more similar to the
hosts they attack than are bacteria. This means that these drugs often affect the host, leading to side
effects. Accordingly, there is a quite limited range of antiparasitic, antifungal and antiviral drugs
available. There are some specific antibiotics for retroviruses, causes of four diseases in humans
including AIDS. There are novel anti-influenza drugs that inhibit virus uncoating or virus release from
infected cells. Interferon is a natural compound, produced by humans in response to viral infections,
that induce other host cells to be able to resist viral infection,

Summary
there are many reasons to want to kill or remove microbes or inhibit their growth
however, its often hard to target the microbes without also harming the material to be
decontaminated, or infected host
there are a wide range of physical and chemical methods available, some more specific for the
target than others
for inanimate, inorganic, materials, can use strong chemicals/treatments but many materials are
easily damaged or denatured
selective toxicity describes the desire to find a treatment that only damages the target cell
many selectively toxic compounds exist, but its hard to predict how well they will work against a
particular microbe due to unique physiology/biochemistry (e.g. EPS, resistance plasmids)

READING
Chapter 26 of Madigan, Martinko, Dunlap and Clark Brock Biology of Microorganisms, 13th Edn.