So, here if we look at the standards that people use for analysis of microorganisms, for example CPCB standards for microorganisms will be like say 5 per 100 ml or 5 microorganisms for 100 ml. So, the microorganisms we are talking about are predominantly pathogens. Many of the pathogens are bacteria, there are a few viruses and all that but mainly bacteria and for water quality, people count the number of bacteria in it. Here we focus on water only. There are bacteria in air also, fungal spores and all but there are no standards for it yet. It is still going on people are trying to develop it but it will take some time for it to happen.

So, how do you count say 5 per 100 ml? This bacteria size is around 1 to 10 microns, it means if I take 100 ml sample, I have to see it, it is difficult to count, so you need a microscope. So how do you do this? You take 100 ml of sample you filter it put it on a filter paper and observe the filter paper if somewhere in the filter paper there are 5-micron spots. Filter papers are big 2.5 centimeters in size, you have to search for 5 microns somewhere, so, it is not very easy.

So, this is a big challenge, counting microbial populations is a big challenge. One of the old standard methods is that people use what is called as a culturing method. A lot of people work on these various ways of doing it, but one of the simplest methods is to take a water sample and you culture the bacteria on a nutrient medium. Typically, what you do is, you take a plate filled with some nutrients. There are some standard nutrients. Nutrient is something in which the bacteria will grow, uses that as a substrate and it will multiply.

So, what people do is they take say 1 ml of water sample and put it on a nutrient medium. And in the sample say there are 5 bacteria and you cannot see it because it is micron size. And then you incubate it for a day or 24 hours at some temperatures 30 degrees centigrade or 25 degrees centigrade. Then what happens is, they allow the bacteria to multiply. So, the bacteria multiply and it becomes a small cluster, multiply and grow around here. So, when it becomes big you can see it.

So, what if it has 100 already? If you have 100, you have no problem because there are other methods of doing it. But if you do culturing when you have a very high concentration, you already have a lot of dots and this at the end of one day, you may get the big jumble, a big mass, and you cannot differentiate how many originally were there. So, usually the analysis prefers that, if you have a very large number, you dilute it so that you can get distinct masses of colony forms.

So, microbes are treated like particles, so you can also look at it like a particle and look at it in a microscope and there are a lot of instruments now available, which use microscopy in order to count bacterial cells and these are not standard methods, but people use what is called as flow cytometry.

So, people use other ways of detecting bacteria also which includes putting a dye something called a staining. They put a dye, this dye will go and absorb on different organisms in order to distinguish between which bacteria which fungus it is and then you can use what is called as a fluorescence microscope in order to detect and count.

In general, if the concentration of microorganisms is very high, it will show up as turbidity. So water is not clear, which means it could be because a lot of bacteria is there so it is a suspended particulate matter; and that’s why there is turbidity. The turbidity is one measure of microorganisms but not always, you cannot be sure. So the only way to make sure that it is a microorganism is to put it on and see if it is growing.