Media Sterlization

Media Sterlization:

Media can be treated by variety of methods like radiations, filtration, ultrasonic treatment and heat. Practically the universal and most favourable method for sterilization is the steam.

Kinetics of Sterlization:

The destruction of microorganisms by steam or the moist heat can be described by the first order chemical reaction and can be represented by the following equation:

-                                                                                                 dN / dt = kN                               Eq no. 1

N is no. Of viable organisms present

t is the time of sterilization treatment.

k  is the reaction rate constant or the specific death rate

Upon integration of equation no.1 we get:

                                          N_t / N₀ = e ^–kt                           Eq no. 2

N₀ is no. of viable organisms present at the start of sterilization

N_t  is the no. Of viable organisms after treatment period t

Upon taking natural logarithms of the Eq. No. 2 we get:

                                           ln (N_t / N₀)  =  -kt                    Eq no.3

 

                  

                                   

 

 A plot of natural logarithm of  N_t / N₀ against time will yield a straight line ; the slope of which is equal to – k (see the fig. ABOVE)

So we can make 2 anomalous predictions from the plot:

1.  An infinite time is required to achieve sterile conditions. ( Nt  = zero)

2.   After a certain period of time there will less than one viable cell present.

But the above plots can be observed only when the sterilization of pure culture is done in one physiological form only and that too under the ideal sterilization conditions. Thus, the value of k is not only species dependent but also on the physiological form of the cell e.g. endospores of genus Bacillus will be more heat resistant than the vegetative cells.

Considering this factor Richards in 1968 has produced a series of graphs (see Graphs below) which illustrate the deviation from the theory which can be expressed in the practice.

              

      GRAPH 1

  GRAPH 2

Above 3 graphs illustrate the effect of time of heat treatment on the survival of a population of bacterial endospores.

Graph 1 shows the initial population increase resulting from heat activation of spores in early stages of sterilization; followed by the decline in viable spore number. Activation o spores is significantly more than their destruction during early stages of the process and therefore viable number of spores increases before the observation of exponential decline.

Graph2 illustrates the initial stationary period followed by the decline; owing to the fact that the death of the spores is compensated by the heat activation of spores.

Graph 3 shows the population decline at a sub maximum rate due to the death of the spores.

 GRAPH 3

Now we know that with any first order reaction, the reaction rate increases with temperature increase due to increase in the reaction rate constant which is the case of destruction of microorganisms (i.e.specific death rate constant; k).

Thus, the relationship between the temperature and reaction rate constant can be expressed by Arrhenius Equation:

                                                     dlnk/dT  = E/RT²                                                  Eq no 4:

E is the Activation energy

R is gas constant

T is absolute temperature

Upon integration of Eq. no 4 we get:

                                                   k= Ae^{- E/RT}                                                            Eq. no 5:

A is Arrhenius constant

On taking natural Logarithms Eq no. 5 becomes:

                                                     ln k = ln A  - E/Rt                               Eq. No.6:

By combining Eq. no. 3 and 5 following expression can be derived for heat sterilization of a pure culture at a constant temperature:

                                               ln (N_t / N₀)  = A.t.e ^–E/Rt                       Eq. no. 7

Deindoerfer and Humphery used the term  ln (N_t / N₀)  as a design criterion for the sterilization which has been called as DEL Factor or NABLA factor.

Del factor is a measure of fractional reduction of viable organism count produced by a certain heat and time regime. Therefore:

                                                ∇    =    ln (N_t / N₀₎

           But;         ln (N_t / N₀₎ = kt

                  &                kt = A.t. e ^–E/Rt           

               Thus,        ∇   = A.t. e ^–E/Rt                                    Eq. no. 8

On rearranging equation no. 8 becomes :

                        ln t = E/RT +  ln  ( ∇ /A)                 eq. no 9

Thus, a plot of the natural logarithm of the time required to achieve a certain ∇ value against the reciprocal of the absolute temperature will yield a straight line, the slope of which is dependent on the activation energy.( see fig below)

     

So a regime of time and temperature can be determined if we want to achieve the desired Del factor. However , as we know fermentation medium is not an inert mixture of components so it is very likely that deleterious reactions occur , resulting in loss of nutrient quality. Two types of reactions occur during sterilization as follows:

(i) Interactions between nutrient components of the medium. A common occurrence during sterilization is the Maillard-type browning reaction which results in discoloration of the medium as well as loss of nutrient quality. These reactions are normally caused by the reaction of carbonyl groups, usually from reducing sugars, with the amino groups of amino acids and proteins. Problems of this type are normally resolved by sterilizing the sugar separately from the rest of the medium and recombining the two after cooling.

(ii) Degradation of heat labile components. Certain vitamins, amino acids and proteins may be degraded during a steam sterilization regime. In extreme cases, such as the preparation of media for animal-cell culture, filtration may be used.

P.S I do not owe any of the above text.

References:

 Shuler, M.L. and Kargi, F. (2002) Bioprocess Engineering: Basic Concepts. 2 nd Edition. Prentice Hall, 

Stanbury, P. , Whittaker, A. (2004) Principles of Fermentation Technology