1.4. Indian Institute of Technology - Madras

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.

In this chunk, we discuss the standards used to measure microorganisms in water. The Central Pollution Control Board (CPCB) in India has set a standard that allows only 5 microorganisms per 100 milliliters of water, indicating that this is a safe limit for drinking water. The text specifies that most microorganisms of concern are pathogens, primarily bacteria, which can cause diseases. It's important to note that, while there are measures for water, standards for bacteria found in air are still being developed.

Think of drinking water as a clear glass of lemonade. Just like you'd want your lemonade to be refreshing and free of any unwanted fruits or items, we want our water to be free from harmful microorganisms. The standard of 5 per 100 ml is similar to saying you can have at most 5 floating pieces of fruit in your lemonade – any more than that, and it's no longer considered safe to drink.

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.

Counting microorganisms in water samples can be very challenging because of their tiny size (1 to 10 microns) and the volume of the sample. The method described involves filtering 100 ml of water to collect microorganisms on a filter paper and then searching for small spots that may contain the bacteria. This is difficult because the number is small and locating them on a relatively large filter paper is comparable to finding a few tiny seeds scattered on a large table.

Imagine trying to find a few grains of sand on a large beach. You have to sift through a huge area to find just a handful of grains. This is similar to the challenge scientists face when searching for tiny bacteria in a large water sample using filter paper.

One of the old standard methods is that people use what is called 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... So, when it becomes big you can see it. So, you have formation of a colony, one bacterial cell will multiply 2, 4, 6, 8 it multiplies in some fashion and whatever was this one single dot, you cannot see now has become a colony.

The culturing method involves taking a small amount of water, introducing it to a nutrient-rich medium, and letting the bacteria grow. This method exploits the ability of bacteria to multiply rapidly. After incubation, the once invisible bacteria form visible colonies, which can be counted. Each colony comes from a single bacterial cell, representing how many bacteria were in the original sample. By using this method, scientists can get a count of the bacteria present even when they were too tiny to see initially.

Think of it like planting seeds in a garden. Initially, you cannot see the seeds, but with enough time and the right conditions (soil, water, sunlight), they grow into visible plants. Similarly, the bacteria you cannot see in the water sample multiply into visible colonies after being given the right nutrients.

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 very high concentration is you already have a lot of dots and at the end of one day, you may get a 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.

When the concentration of bacteria in a sample is extremely high, counting them becomes problematic because the colonies can merge into a mass, making it difficult to distinguish between the different colonies. To overcome this, scientists dilute the sample before culturing. For instance, if there's an estimated concentration of 100 colonies, diluting the sample helps ensure that when cultured, distinct colonies form, allowing for accurate counting.

Imagine trying to pick out individual beads from a bowl filled with thousands of colorful beads closely packed together. It would be quite confusing! However, if you poured the beads onto a larger surface with more space, it would be much easier to pick out and count specific colors. Similarly, diluting bacterial samples helps in counting them accurately.

So, microbes are treated like particles, so you can also look at it like a particle and look at it in a microscope... So, flow cytometry is used in diagnostic analysis in blood. When you do blood analysis, you will see them laying there using flow cytometry will count the number of red blood cells, white blood cells, and all the cells which are about the same order of magnitude.

In addition to traditional culturing methods, advanced techniques like flow cytometry can be used for counting bacteria. This technique forces a sample of liquid, containing microorganisms, through a narrow passage where they pass one by one past a detector, which counts them. This is similar to how blood cells are analyzed, offering a different approach to microbial analysis.

Consider a busy highway during rush hour. A sensor at the beginning of the highway counts each car as it passes by, even though they are moving quickly and closely together. Flow cytometry works in a similar way, counting the individual bacteria as they flow through the pathway, providing a rapid and efficient count.

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