Download Topic guide 2.3: Methods for controlling microbial

Unit 2: Industrial Microbiology
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Methods for
controlling microbial
contamination
Why could contamination of culture media or equipment in industrial
microbiological processes by other microorganisms be a problem?
Before microbial cultures are set up, all equipment is sterilised, so that no
contaminants remain. The equipment also has filters to prevent entry of
contaminants during the fermentation process. This is because contaminants
may:
•• produce other metabolites that could be toxic or cause spoilage
•• compete with the required microorganisms for nutrients or oxygen and
reduce their growth and hence the product yield
•• infect the required microorganisms, for instance, bacteriophage viruses
infect and kill bacteria.
If contamination occurs then considerable time and money is wasted while
the fermentation process is halted, fermenters are emptied and all equipment
is sterilised and then cooled to allow a new batch to be set up.
On successful completion of this topic you will:
•• understand the methods for the control of microbial contamination (LO3).
To achieve a Pass in this unit you will need to show that you can:
•• assess the effectiveness of physical and chemical methods of reducing
microbial growth (3.1)
•• assess the effectiveness of the types of chemicals used (3.2).
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Unit 2: Industrial Microbiology
1 Physical and chemical methods for
controlling microbial growth
Physical methods
Table 2.3.1: Features of some physical
methods of microbial control.
Method
dry heat
•• direct flaming
•• incineration
•• hot air sterilisation
moist heat
•• autoclaving
•• boiling of freeflowing steam
In industrial microbiology heat and filtration are the most widely-used physical
methods, although in some cases radiation may be used. Other methods such as
cold temperatures, osmotic effect and desiccation are used in the food industry.
Table 2.3.1 summarises the features of some physical methods of microbial
control.
Mode of action
Effectiveness
Some examples of use
oxidation – reduces contaminants to
ashes and carbon dioxide
oxidation – can use an oven at
170 °C for 2 hours
all give very effective sterilisation
inoculating loops
waste materials
glassware and instruments
denatures proteins
uses steam under pressure to
give a temperature of 121 °C for
15–20 minutes
denatures proteins
kills vegetative bacterial cells and
endospores
microbial nutrient media, dressings,
overalls, equipment
kills bacterial, viral and fungal
contaminants within 10 minutes
less effective for bacterial
endospores
this kills endospores, some of which
can withstand temperatures of
100 °C for 20 hours
glass and metal equipment and
pipes
•• tyndallisation
(fractional
sterilisation)
denatures proteins
intermittent sterilisation, involves 3 x
heatings at 100 °C for 30 minutes,
interspersed with a day at 30 °C, to
allow endospores to germinate so
the next heat treatment can kill the
resulting vegetative cells
pasteurisation
kills by denaturation
involves heating to 63 °C for
30 minutes or to 72 °C for
15–20 seconds
kills only pathogenic bacteria
used for milk, cream, milk products
(such as cheese and yoghurt), wine
and beer
filtration
membrane filters made of cellulose
acetate or nitrocellulose have small
pores to mechanically remove
microorganisms
pore sizes of 0.22–0.40 µm exclude
bacteria (and larger microorganisms
such as protoctists and fungi)
pore sizes of 0.01 µm exclude viruses
inlets on fermenters
air inlets of laminar flow cabinets
drinking water may be filtered
damage or destroy DNA
effective and do not produce heat
sterilise by producing heat and may
also damage DNA
radiation is not very penetrating
can be used for plastic and other
thermolabile substances
used to sterilise pharmaceuticals
and medical and dental supplies
and plastic Petri dishes
UV light used inside laminar flow
cabinets
radiation
•• ionising radiation,
such as X-rays and
gamma rays
•• UV radiation
2.3: Methods for controlling microbial contamination
used in the food industry and may
be used to sterilise nutrient media if
no autoclave is available
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Unit 2: Industrial Microbiology
Key terms
Sterilisation: Removal or destruction of all forms of microbial life, including endospores, on an
object or material.
Vegetative cells: Metabolically-active bacterial cells, not containing endospores.
Endospores: Dormant state in some bacteria to overcome adverse conditions. The cell contents
become metabolically inactive and are enclosed in a protective coat, inside the cell wall. These can
survive high heat for long duration.
Figure 2.3.1 shows an industrial autoclave used to sterilise instruments and
Figure 2.3.2 shows milk processing. Milk is filtered to remove solid impurities and
pasteurised to kill pathogenic microorganisms.
Figure 2.3.1: Industrial autoclave
used to sterilise instruments.
Figure 2.3.2: Milk processing.
Milk is filtered to remove solid
impurities and pasteurised to kill
pathogenic microorganisms.
2.3: Methods for controlling microbial contamination
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Unit 2: Industrial Microbiology
Chemical methods
Antiseptics and disinfectants are chemical substances used to prevent
contamination. Several factors influence the efficacy of these chemicals, such as:
•• concentration
•• length of exposure of microorganisms to it
•• environmental temperature at which they are used
•• type of microorganism on which they are used – spore-forming bacteria and
bacteria with slime capsules are particularly resistant to chemicals (as they are
to heat)
•• surface on which the microorganism is living – chemicals may combine with
an organic matrix and their efficiency is then reduced.
Table 2.3.2: Features of some chemical
methods of microbial control.
Method
Table 2.3.2 summarises the features of some chemical methods of microbial
control.
Mode of action
gases
•• ethylene oxide
denatures proteins
•• formaldehyde gas
inactivates proteins
•• ozone
oxidation
Effectiveness
Some examples of use
very effective sterilising agent,
especially where heat cannot be
used
very effective sterilising agent,
especially where heat cannot be
used
corrosive and irritating but kills
viruses and bacteria in a short
time and prevents regrowth of
microorganisms
used to sterilise spacecraft such as
for moon landings
denature proteins and dissolve lipids
of cell membranes
70% ethanol is effective for killing
bacteria and viruses but does not kill
endospores
•• aldehydes
inactivates proteins
effective sterilising agents
•• dyes
interfere with nucleic acids
effective at killing bacteria
•• halogens
oxidising agents and denature
proteins
effective sterilising agents
cleans skin before injections
although most microbes are
removed by the wiping action;
cleans work surfaces – often used
in combination with another
antiseptic
formalin (solution of
formaldehyde) used in embalming
glutaraldehyde used to sterilise
medical equipment
crystal violet (aniline dye)
flavines (acridine dyes) used in
antiseptics
iodine used as antiseptic for
livestock and wounds; chlorine gas
used to disinfect water; chlorine
compounds (e.g. bleach) used to
disinfect dairy equipment, eating
utensils and glassware
liquids
•• alcohols
fumigation
disinfects waste water
more effective than chlorination
Continued on next page
2.3: Methods for controlling microbial contamination
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Unit 2: Industrial Microbiology
Method
liquids continued
•• phenolics, e.g.
carbolic acid, cresol
(Lysol®)
•• hexachlorophene
•• chlorohexidene
•• thymol
•• hydrogen peroxide
•• surfactants – soaps
and detergents
solids
•• salts of heavy
metals such as
silver, zinc, copper
and mercury
Mode of action
disrupt cell membranes and
denature enzymes
Effectiveness
very effective microbicide
disrupts cell membranes and
denatures enzymes
disrupts cell membranes
disrupts cell membranes
oxidation
reduce surface tension, disrupt cell
membranes, inactivate enzymes
coagulate proteins
Key term
Fungicide: Agent that kills fungi.
effective and persistent bactericide
fungicide
effective as disinfectant for surfaces
but poor antiseptic as blood
contains the enzyme catalase that
breaks down hydrogen peroxide to
water and oxygen
bactericidal, virucidal, fungicidal,
fast acting, non-corrosive and nontoxic
effective against bacteria but limited
effectiveness against fungi
Some examples of use
carbolic acid used by Lister to
pioneer antiseptic surgery but
causes skin irritation
all chemical sterilising agents are
compared to phenol to describe
their effectiveness
surgical scrubs
mouthwashes
disinfectant
skin cleansing, sanitising equipment
in food and dairy industries;
antiseptics for skin, instruments and
utensils and rubber goods
used in wound dressings
nanoparticles of silver in bandages
are small enough to enter bacteria
and are very effective bactericides
Figure 2.3.3 shows some pipes in a brewery being inspected. They carry liquids
from the fermenting vats to the bottling line and must be sterilised between
batches.
Figure 2.3.3: These pipes in a
brewery are being inspected.
2.3: Methods for controlling microbial contamination
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Unit 2: Industrial Microbiology
Take it further
Key term
Phenol coefficient: A measure of
how effectively a chemical compound
destroys bacteria in relation to
phenol.
Although phenol is no longer widely used as a disinfectant it is still the standard used for
comparison of the effectiveness of other disinfectants. Each chemical is given a rating called the
phenol coefficient, which indicates its effectiveness.
Three test organisms are used: Staphylococcus aureus (Gram positive), Salmonella typhi (Gram
negative) and Pseudomonas aeruginosa (Gram negative and resistant to antimicrobials) are grown
in broth cultures under standard conditions. They are then exposed to the test chemical for a
specified time, before being plated onto nutrient culture media plates and incubated to see if there
is any microbial growth.
If the test chemical has to be used at a greater concentration or a longer time than phenol to get
the same results, then the phenol coefficient is less than 1. It is less active than phenol.
If the test chemical is effective at a lower concentration or needs to be used for a shorter time than
phenol to kill the microbes, it has a phenol coefficient of greater than 1 and is more active than
phenol.
For example: Disinfectant Brand X at a dilution of 1:100 has the same effectiveness as phenol at a
dilution of 1:50 for killing bacteria on a work surface.
The phenol coefficient of Brand X is 100/50 = 2. It is twice as effective as phenol.
Disinfectant Brand Y, diluted 1:20, is as effective as phenol diluted 1:50.
Calculate the phenol coefficient of Brand Y.
Which brand, X or Y, is best to use to disinfect this work surface?
Activity: Flow chart
Make a large flow chart to give
an overview of the physical and
chemical methods used to control the
growth of microorganisms.
Activity: Plan an investigation to compare heat treatment
times and temperatures to kill three species of microorganism
You have access to test tubes, water baths set at a range of temperatures, Petri dishes with nutrient
agar, Bunsen burner, inoculating loop, incubator, colony counter and cultures of Escherichia coli
(E. coli, G negative bacterium), Staphylococcus albus (G positive bacterium) and Saccharomyces
cerevisiae (yeast).
Plan an investigation to find out the temperature and length of duration of exposure that is
needed to kill each of these organisms. List the equipment you will need. State your aim and a
hypothesis to test. State the independent variables and the dependent variables. Describe the
confounding variables and explain why and how you can control them.
If possible, carry out your investigation and write a report of your findings.
2.3: Methods for controlling microbial contamination
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Unit 2: Industrial Microbiology
Case study: University Researcher
Anna Jansen carries out research into antibacterial properties of substances such as essential oils
at a university in the Netherlands. Her findings are published in the International Journal of Food
Microbiology. Her work is important in finding new antibacterial substances that can be safely
added to food.
It has been estimated that up to 30 per cent of people in developed countries suffer from a foodborne disease each year. With reductions of salt in processed foods and the preference of many
people for not having additives in food, more ‘green’ products that are considered acceptable may
be needed to restrict bacterial growth in food products.
Essential oils are aromatic liquids from various parts of plants, usually obtained by steam
distillation. Of the 3000 essential oils known, about 300 are commercially important – mainly
for flavouring. However, many of them (as well as garlic and many spices, e.g. turmeric) have
antibacterial properties. Some are used in dental root canal sealers, antiseptics and in feed
supplements for lactating sows and weaned piglets. Some ‘natural’ food preservatives are already
marketed and contain oils of sage, rosemary and citrus. Extracts from coriander, mint, mustard,
cinnamon, oregano, eucalyptus, cloves, sage, rosemary and thyme have antibacterial chemicals
such as thymol, cymene, carvacrol, citral, geraniol and terpinene.
Anna uses the disc diffusion method, agar well method and time-kill analysis to investigate
antibacterial properties of essential oils that she extracts from plants such as tea tree. She tests
the extracts on bacteria such as E. coli, Salmonella typhimurium, Bacillus cereus, Listeria
monocytogenes and Staphylococcus aureus.
Once antibacterial action has been shown in vitro, the oil needs to be tested when in food, where
a high fat content can reduce the antibacterial effect as the oils dissolve in the food fats and do not
reach any bacteria present. Therefore the required dose in vivo may be greater than the in vitro
dose. The storage conditions of the food can also affect effectiveness of the essential oils, so many
variables are involved in this research.
Essential oils may act by damaging cell membranes and their proteins, breaking down cell walls
causing leakage of cell contents, and disabling the proton-motive force used by motile bacteria.
Sometimes two oils can work synergistically and sometimes they may work antagonistically.
What does ‘working synergistically’ mean?
Activity: Investigating antibacterial properties
Plan and carry out an investigation to compare the effectiveness of bleach, hydrogen peroxide, a
skin antiseptic, and a disinfectant at preventing the growth of three types of bacteria.
Alternatively, extract one or more essential oils from plant matter and investigate their
antibacterial properties.
You can use the well method in solid nutrient agar or the disc diffusion method and measure
zones of inhibition, or the time to kill exposure method, involving making serial dilutions of each
disinfectant and exposing bacteria to them for 10 minutes, then plating them out, incubating and
counting colonies to measure growth.
Write your plan and include: clear statement of the aim of the investigation; equipment needed
and justification for its use; clear method; risk assessment and precautions to be taken; how you
will collect and analyse the data.
2.3: Methods for controlling microbial contamination
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Unit 2: Industrial Microbiology
Checklist
In this topic you should now be familiar with the following ideas:
 processes involving making products by utilising microorganisms provide suitable conditions
for contaminant organisms to grow and spoil the product or pose a threat to the health of
personnel or consumers of the product
 there are strict guidelines about contamination control (see Topic guide 2.5)
 there are physical and chemical methods to control microbial growth
 physical methods include moist and dry heat, pasteurisation, filtration and exposure to short
wavelength ionising radiation
 chemical methods include gases such as ozone and formaldehyde; liquids such as ethanol,
phenol, halogens, hydrogen peroxide and surfactants; and solids such as salts of heavy
metals.
Further reading
Annets, F. (2010) BTEC Level 3 National Applied Science Student Book, London: Pearson Education.
Cappuccino, J. and Sherman, N. (2013) Microbiology: A Laboratory Manual (10th edition), Benjamin
Cummings.
Case, C. Funke, B. and Tortora, G. (2012) Microbiology: An Introduction (11th edition), London:
Pearson.
Prescott, L., Harley, J. and Klein, D. (2004) Microbiology (6th edition), McGraw-Hill Higher Education.
Taylor, J. (1990) Microorganisms and Biotechnology, Nelson Thorne.
Acknowledgements
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2.3: Methods for controlling microbial contamination
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