 In this module, we will talk about microbial growth kinetics. But in this module, we will talk about different methods which are used for the estimation of the growth. As we discussed in previous modules that growth is divided into four different phases lag, log, stationary and decline phase. But these four phases can only be determined if we actually know the growth. How we can estimate the growth of a cell of a microorganism? So in this module, we can see that there are different approaches to estimate the growth. Here you can see there are six different approaches by which we can estimate the microbial growth. The first method is turbidity method and the second is total count, third is the viable count, fourth one is biochemical analysis, fifth that is the physical method which called as graduated capillary tube and the last that is dry or a wet cell mass estimation. So as concerned that turbidity methods, in this method when the growth happen the number of cells increase due to the increase in the number of the cells, cell culture become turbid. So due to the turbidity when we check on the spectrophotometer, basically that measure the cloudiness and spectrophotometers act on the beer-lamored law. As you can see in this slide, this is beer-lamored law which actually measure the amount of light that pass through the suspension of the cell. If there is a more turbidity, then there will be the less light is allowed to pass through the culture. So spectrophotometer give the reading in two terms. One is absorbance and other is the transmittance. In this equation, you can see the relationship between the absorbance as well as the transmittance. So in this picture, you can see that how the spectrophotometer act. In this slide, you can see that this tube is blank, there is no growth and the light is directly pass and then there are 100% transmittance and we can say that absorbance is 0. So on other hand, in the second, you can see here the growth is and the number of cells are in the capillary and then there is a few lights. One beam is only allowed to cross through. So we show that the transmittance decreases as well as the absorbance increases. So this one is the method by which we can estimate the growth in term of number of cells as the cell increase turbidity increase which can be easily measured by the spectrophotometer. But there are some different limitations in this method because sometimes when the medium color is dark by the utilization of the substrate, turbidity also increase. Sometimes that can affect on the estimation of the growth. So we need an alternative method for the microbial growth. So the second method that is the total count. So the total count is basically you can say that cell growth measurement in which we can also count the total cells that include the viable cells as well as the dead cells. So the total count is mostly done with the help of a slide which we called as hemacytometer so this hemacytometer if the culture is very diluted we can directly use that culture. But when the culture is very turbid then we have to first dilute that sample. So in this picture you can see that what is hemacytometer. So here you can see that hemacytometer is basically a glass slide in which there is a standard cavities are formed. You can see in this slide here there are different boxes small box, medium box and large box. And the large box has the diameter you can say the diameter that is the width, length and the depth. Most of the hemacytometers as concerned the large box that is 1 millimeter long 1 millimeter in width and then 0.1 millimeter in its depth. So by counting the cells in different compartment we can estimate that what is the total count in the culture medium. As concerned the viable count viable count is mostly done in the petri plate having some solid media which mostly called as agar medium along with other supplements. So the viable cell is defined at the cell which is able to divide and decrease in the cell number or sorry increase in the cell number. So the viable count the normal way to perform the viable count is to determine the number of cells in the sample which is capable of forming colonies on a suitable medium. So here we can assume those cells which have the ability to grow on the cells then we can count on the plate that is basically the colony count. So as there is the count of the cells we can call as colony forming unit CFU that is mostly done by a simple counting of those all the colonies. So sometimes when we do the viable counting we have to dilute the samples gradually which normally known as the dilution method. In this picture you can see that first of all if we have a sample grown in a flask you take one ml of the sample and we dilute into a specific amount of the volume and then gradually we diluted and we form a different types of dilution and then we take the sample out of each dilution and we culture on the plate and then you can see here the number of colonies decreasing as per the dilution is increased. So by this method we can easily estimate the viable cells present in any culture but as concerned the biochemical analysis it is the direct biochemical measurement of some cellular components such as nitrogen content, total protein, DNA material or any special other compounds which is not the part of the medium. So as concerned the indirect measurement of the chemical activity such as the rate of oxygen consumption or the rate of carbon dioxide production or vice versa. In short the we can use the biochemical analysis by measuring the rate of respiration. So other fifth and sixth method that is the graduated capillary tube that is mostly used in a yeast culture when there is a culture we centrifuge the yeast culture or a bacterial culture in a graduated capillary tube. So when there is a specific volume and then after the centrifugation we estimate the height of the pallet then we can estimate the growth of the culture. And the same way the dry and the wet mass we just centrifuge and then weigh the pallet as a wet mass or then we dry in an oven and then we can estimate the dry weight of the cell. So by these six different methods we can estimate the growth in any fermentation vessel.