Sterilization

1
Sterilization
Sterility on a practical basis means the absence
of any detectable viable organism. Pure culture
only the desired organism is detectably
present. Sterilization methods Medium
equipment Thermal deactivation
(steam). Heat-sensitive materials in medium
filtration (microfiltrator pore
sizeslt0.2µm). Heat-sensitive equipment UV
radiation (energy intensive) or chemical agents
(toxic to the desired culture. e.g.70
ethanol-water, and ethylene oxide (gas). Gas
(air) sterilized through adiabatic compression
process for supplying oxygen (2200C) followed by
filter (e.g. glass wool filter).
2
Sterilization

• Thermal deactivation
• The probability of an unsuccessful sterilization
  
• 1- P0(t)
• The probability of extinction (successful
  sterilization)
• P0(t)1-p(t)N0
• where p(t) is the probability that an individual
  will still be viable at time t. N0 is the number
  of individuals (cells or spores) initially
  present.
• Assume first order death rate
• p(t)e-kdt
• 1- P0(t)1-1-p(t)N0 1-1-e-kdtN0
• where the specific death rate kd
  ae-E0d/RT(1/time), constant at specified T. Eod
  is the activation energy for the death of the
  organism.

3
Sterilization

• Decimal reduction time (D) time for the number
  of viable cells to decrease tenfold.
• EN(t) N0 p(t)
• EN(t) N0 e-kdt
• 0.1 e-kdD
• D2.303/kd
• EN(t) is the expected value of the number of
  individuals present at time t.

4
Sterilization

• 1- P0(t) 1-1-e-kdtN0
• kd ae-E0d/RT
• From the above equation
• Known N0, T, t, determine Kd, the probability of
  an unsuccessful sterilization is determined.
• Given N0, T, acceptable probability of failure
  e.g. 10-3, required time can be determined
• Higher Kd tends to achieve low probability of
  sterilization failure. Normally at 121oC.
• Kd of vegetative cells gt 1010 min-1, spores
  0.5-5 min-1. The major concern is spores.

5
Sterilization Chart
e.g. N0108, Kd1min-1 (1210C),
t26min Kdt26 Probability of failure 1-P0.001
e.g. N0108, Kd1min-1 (1210C), 1-P0.001
Kdt26 Required sterilization time tkdt/kd26
min
(M. Shular, Textbook, p.319)
6
Batch Sterilization
(Sterilization)
Temperature verse time in a batch sterilization
process
Simplified calculations for deactivation of
spores and medium components
7
Sterilization
Degradation of components in the medium in
sterilization process. vitamin and growth
factor The inactivation of viability is much
more sensitive to temperature changes than the
degradation of important growth factors in the
medium It is important to assure complete
killing of foreign organisms without the
destruction of important components in the
medium.
8
Sterilization
Degradation of components in medium Assume the
degradation rate of such components is first
order. dC/dt-kdC The degradation rate
constant kd can also be related to temperature by
Arrhenius equation. Integrating the above
equation, ln C/C0-kdt or CC0e-kdt where C0
is the initial concentration of the component. To
determine the components remaining active the
temperature T ? determine kd ? with known t,
determine C.
9
Sterilization

• Degradation of components in the medium.

Kd,2
C2
C1
?t2
C0
C1
?t1
Kd,3
C2
C3
?t3
Kd,1
kd ae-E0d/RT