Sterilization on a practical basis means the absence of any detectable viable organism. : only the desired organism is detectably present. Sterilization methods: Medium & equipment:. Heat-sensitive materials in medium: (microfiltrator pore sizes<0.2μm). 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 (~220 0 C) followed by (e.g. glass wool filter).

Sterilization Thermal deactivation The probability of an unsuccessful sterilization: 1- P 0 (t) The probability of extinction (successful sterilization): P 0 (t)=[1-p(t)] N 0 where p(t) is the probability that an individual will still be viable at time t. N 0 is the number of individuals (cells or spores) initially present. Assume first order death rate: p(t)=e -k d t 1- P 0 (t)=1-[1-p(t)] N 0 = 1-[1-e -k d t ] N 0 where the specific death rate k d = αe -E 0d /RT (1/time), constant at specified T. E od is the activation energy for the death of the organism.

Sterilization (D): time for the number of viable cells to decrease tenfold. E[N(t)] = N 0 p(t) E[N(t)] = N 0 e -k d t 0.1= e -k d D D=2.303/k d E[N(t)] is the expected value of the number of individuals present at time t.

Sterilization 1- P 0 (t)= 1-[1-e -k d t ] N 0 k d = αe -E0d/RT From the above equation: -Known N 0, T, t, determine K d, the probability of an unsuccessful sterilization is determined. -Given N 0, T, acceptable probability of failure e.g , required time can be determined -Higher K d tends to achieve low probability of sterilization failure. Normally at 121 o C. K d of vegetative cells > min -1, spores min -1. The major concern is spores.

Sterilization Chart e.g. N 0 =10 8, Kd=1min -1 (121 0 C), t=26min Kdt=26 Probability of failure: 1-P=0.001 e.g. N 0 =10 8, Kd=1min -1 (121 0 C), 1-P=0.001 Kdt=26 Required sterilization time: t=kdt/kd=26 min (M. Shular, Textbook, p.319)

Batch Sterilization Temperature verse time in a batch sterilization process T (K) t time heating holding cooling Simplified calculations for deactivation of spores and medium components T (K) t time Kd1 Kd2 Kd3

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.

Sterilization Degradation of components in medium Assume the degradation rate of such components is first order. dC/dt=-k d C The degradation rate constant k d can also be related to temperature by Arrhenius equation. Integrating the above equation, ln C/C 0 =-k d t or C=C 0 e -kdt where C 0 is the initial concentration of the component. To determine the components remaining active: the temperature T → determine kd → with known t, determine C.

T (K) t time K d,1 K d,2 K d,3 Sterilization Degradation of components in the medium. C0C0 C1C1 C2C2 C1C1 C2C2 C3C3 k d = αe -E0d/RT Δt1Δt1 Δt2Δt2 Δt3Δt3