Sterilisation and Containment

Topic Outline
1. Definition of sterilization and containment

2. Sterilization and containment equipment
3. Advantages and disadvantages of sterilization and
containment
4. Sterilization kinetics
5. Del factor
6. Arrhenius plot
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Sterilisation
• Means the absence of any detectable viable organism.
• Only the desired organism is detectably present.
Importance of Sterilization
•To avoid loss of productivity - contaminated product will overweigh

the desire product
•To avoid contamination of the final product
•Some contaminants are troublesome during downstream
processing
•Contaminants may cause degradation of product
•Contaminants can cause lysis of culture e.g bacteriophages
•The medium would be consumed unnecessarily to support growth
of contaminating organism
Importance of Sterilization
• To overcome the above problems is to carry out sterilization using any of these methods:
1. sterilization of medium
2. employing as pure inoculum as possible
3. sterilization of fermenter
4. sterilization of pipes, valves, etc. which come in contact with the fermentation process
5. sterilization of all materials to be added to fermenter
6. sterilization of air
7. disinfecting the fermenter and contact parts with non-toxic disinfectant
8. maintaining aseptic conditions in the fermenter during fermentation
9. maintaining the optimum/desired pH which discourages the growth of certain
contaminants/undesired organisms.
Steps to avoid contamination
• Use pure inoculum

• Media sterilization
• Fermenter sterilization
• Sterilize all raw materials
• Maintain aseptic conditions during process
Methods of Sterilization
• Filtration,
• Radiation,
• Ultrasonic treatment,
• Chemical treatment
• Heat
• Out of these methods, heat or steam is the most useful method for
the sterilization of fermentation media.
• Heat sterilization is the most widely used and reliable method of
sterilization.
• However can only be applied to thermo-stable products.
• Thermal methods include incineration, hot air oven, autoclaving(use
of dry saturated steam), boiling
• In bioprocessing moist steam methods are most common and
sterilization occurs at 121ᵒC
• Radiation – Many types of radiation are used for sterilization which
include electromagnatic radiation eg Gamma rays and UV rays),
particulate radiation eg accelerated electrons
• This method of sterilization is important for heat sensitive products.
• UV light which is much lower energy and poor penetrability is used for
sterilization of air, or for surface sterilization for treatment of
manufacturing grade water.
• Filtration process does not destroy but removes the microorganisms.
• It is used for clarification and steriliastion of liquids and gases and is
capable for preventing the passage of both viable and non-viable
particles.
• Chemical sterilization can be used to avoid damage of heat-sensitive
materials such as biological materials, and many plastics.
• In these situations chemicals, either in a gaseous or liquid form, can
be used as sterilants. Eg ethylene oxide, formaldehyde
• While the use of gas and liquid chemical sterilants avoids the problem
of heat damage, users must ensure that the article to be sterilized is
chemically compatible with the sterilant being used.
• In addition, the use of chemical sterilants poses new challenges
for workplace safety as the properties that make chemicals effective
sterilants usually make them harmful to humans.
Types of Sterilization
• Sterilization can be carried out in two different configurations ie
batch and continuous
• For a batch method–items to be sterilized are loaded in a sterilizer
(autoclave) and steam is injected according to the desired programme
and later discharge upon completion for further utilization
• It is the popular technique use in most biotech processes.
• For Continuous Sterilization - There will be continuous inflow and
outflow of material–best if capacity of operation is high
Continuous and Batch Sterilization
ADVANTAGES OF CONTINUOUS
• Reduction of sterilization cycle time
• Ease of scale-up
• Superior maintenance of medium quality (less destruction)
• Reduced surge capacity for steam (more efficient plant use)
ADVANTAGES OF BATCH
• Lower capital cost (fermenter used as autoclave)
• Lower contamination risk (less transfers of liquids)
• Presence of solids (particles) less of a problem
• Easier to use with media having a high amount of solid material
Sterilization of Air
• Air is important as a source of oxygen in aerobic fermentation
• For large scale fermentation, air need to be efficiently sterilized
• Methods of air sterilization include:
• Heating
• UV rays or electromagnetic waves
• germicidal spray
• filtration
Sterilization of Air
• Heating air is possible but not economical–due to its poor thermo-physical
properties.
• UV ray is an effective technique in killing air-borne microbes though is only
applicable in small area
• Germicide can also reduce the amount of bacteria via spraying with phenol,
ethylene oxide or formalin–can sterile air in a small size room.
• Filtration is an effective method and practical, a filter is used to remove
microorganisms form the air provided that pores of a filter need to be
smaller than the size of microbes or there is use of absolute filters • pore
size is bigger than the size of microbes–fibrous filter (cotton, glass-wool,
slag, steel-wool etc.)
Factors affecting sterilization
1. Population size
2. Population composition
3. Concentration of the antimicrobial agent or intensity of the
treatment
4. Period of exposure to the lethal agent
5. Temperature
6. Environmental conditions
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Design Sterilization
• Moist heat treatment carried out at 121oC for 15 mins for the
sterilization of culture media, vessels and connecting pipework
• Microorganisms are not killed instantly on exposure to a lethal agent
• Population decreases exponentially by a constant fraction at constant

intervals
• Several factors influence the effectiveness of any antimicrobial

treatment
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STERILISATION KINETICS OF
DESTRUCTION
The destruction of microorganisms by steam may be described as
a first order reaction
First-order reaction— A chemical reaction involving only one
chemical species, in which the rate of decrease of the
concentration of the reactant is directly proportional to its
concentration.
Represented by the following equation -dN / dt = k .N
where N = number of viable organisms present

t = time of the sterilisation treatment
k = reaction rate constant
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BASIC KINETICS
Integration gives Nt / N0 = e -kt
Where
N0 = initial number of viable microorganisms,
Nt = no. of viable m-organisms present after treatment period t
t = time,
k = destruction coefficient (B. stearothermophilus)
On taking natural logs, equation is reduced to

ln (Nt / N0) = -kt
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Graphical Representation of the Arrhenius Plot
Nt Nt Slope = -k
ln
No No
Time Time
Plots of the proportion of survivors and the natural logarithm of the

proportion of survivors in a population of microorganisms subjected to a
lethal temperature over a period of time
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Sterilization Kinetics
• This kinetic description makes two predictions which contradict each other
i. An infinite time is required to achieve sterile conditions
ii. After a certain time there will be less than one viable cell remaining
• Thus a value of Nt of less than one organism remaining is considered in

terms of the probability of an organism surviving a treatment
• e.g If a treatment reduced the population to 0.1 of a viable organism,
implies a probability of one in ten batchs becoming contaminated
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Arrhenius Plot
• As with any first order reaction, the reaction rate increases with
increase in temperature due to an increase in the reaction rate
constant, which in sterilization of media is k
• Relationship between temperature and the reaction rate constant was
demonstrated by Arrhenius and is represented by
In k = In A - E /RT
E = activation energy
R = gas constant The constant factor in the equation of state for perfect gases
T = absolute temperature .
A = Arrhenius constant
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• From this equation a plot of ln k against a the reciprocal of the
absolute temperature gives a straight line
• Plot is called an Arrhenius plot
• Enables a calculation of the activation energy and prediction of the
reaction rate for any temperature
• Thus a plot of the natural logarithm of the time required to achieve a
certain Del factor, against the reciprocal of the absolute temperature
will yield a straight line, the slope of which is dependent on the
activation energy.
• Same degree of sterilization may be obtained over a wide range of
time and temperature regimes
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Del Factor
• Del factor ( V ) = In (No/Nt),
where No is the number of organisms at the start of sterilization

and Nt is the number remaining after time t.
• Therefore, the Del factor is a measure of the fractional reduction in viable organism
count produced by a certain heat and time regime.
• The larger the Del factor, the greater the sterilization regime required.
• Term commonly used in the fermentation industry
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Del Factor
The DEL FACTOR achieved at the different temperatures during the

treatment cycle must be calculated
Thus the DEL FACTOR of the whole process is equal to the sum of the del
factors of each of its constituents;
t = h + m + c
t = del factor whole process
h = " " heating up period
m = " " holding period (maintained) period
c= " " cooling-down period
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Batch Sterilization Profile Example
Temperature verse time in a batch sterilization process
Simplified calculations for deactivation of spores and medium components 27

Containment
• Refers to preventing materials from leaving a
process
i.e. keeping micro-organisms within a system
• Whilst sterilization is primarily focused on
destroying all bacteria present
• Types of containment:
1. Primary containment
2. Secondary containment 28
Primary Containment
• Protection of personnel and the immediate lab environment from
exposure to infectious agents
• Requires use of proper storage containers, good microbial techniques
and biological safety
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Secondary Containment
• Deals with protection of the environment external to the lab
• This is provided by facility design and operational practices
30
Containment Design
• Concerned with laboratory bio-safety
• Physical containment of micro-organisms required
• Usually by isolation to environment, biologically

secure cabinets or rooms
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Importance of Containment Design
• Allows no leakage of harmful micro-organisms
• Reaction may not go into completion, yields become

poor if not contained
• Failure to contain may alter environmental

parameters i.e. pH, temp, chemical composition
• Entry of other micro-organisms or chemical

composition may negatively affect the process
reaction
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Types of Containment
• Primary Containment
Protection of personnel and the immediate
laboratory environment e.g. biological safety cabinets
• Secondary Containment
Protection of the environment’s external to the
laboratory from exposure to infectious materials
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Topic Outline

1. Definition of sterilization and containment

•To avoid loss of productivity - contaminated product will overweigh

• Use pure inoculum

• Microorganisms are not killed instantly on exposure to a lethal agent

• Population decreases exponentially by a constant fraction at constant

• Several factors influence the effectiveness of any antimicrobial

Represented by the following equation -dN / dt = k .N

where N = number of viable organisms present

On taking natural logs, equation is reduced to

Plots of the proportion of survivors and the natural logarithm of the

• Thus a value of Nt of less than one organism remaining is considered in

where No is the number of organisms at the start of sterilization

• Term commonly used in the fermentation industry

The DEL FACTOR achieved at the different temperatures during the

Temperature verse time in a batch sterilization process

Simplified calculations for deactivation of spores and medium components 27

• Physical containment of micro-organisms required

• Usually by isolation to environment, biologically

• Reaction may not go into completion, yields become

• Failure to contain may alter environmental

• Entry of other micro-organisms or chemical

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