1.6. Monitoring and Measurement of Microorganisms

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.

In this section, we learn about the standards set for counting microorganisms, particularly in water. The Central Pollution Control Board (CPCB) has established guidelines stating that for water quality analysis, there should be at most 5 pathogenic microorganisms per 100 milliliters of water. Pathogens are harmful microorganisms, primarily bacteria, which can cause diseases. Therefore, monitoring these levels is crucial for ensuring safe water quality.

Imagine checking your drinking water for safety like you would check the amount of salt when cooking. Just as you wouldn't want too much salt in your food, you want to ensure that the water has a safe level of harmful microorganisms.

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.

Counting microorganisms is challenging because they are very small, typically between 1 to 10 microns in size. To count them, a 100 ml water sample is needed, but direct counting with the naked eye is impossible. Therefore, a microscope is necessary to observe and count these tiny organisms accurately.

Think of trying to count grains of sand on a beach. It's nearly impossible to do it by eye because they are so tiny. Just as a magnifying glass can help you see the sand better, a microscope helps scientists see and count microorganisms.

One of the old standard methods is that people use what is called a culturing method. ...you take a plate filled with some nutrients. ...you take say 1 ml of water sample and put it on a nutrient medium.

An established method for counting microorganisms is the culturing method. In this process, a water sample (1 ml) is placed on a nutrient-rich medium in a petri dish. The nutrients allow bacteria to grow and multiply. After incubating for 24 hours at a specific temperature, bacterial colonies form, making it easier to count them.

It's similar to baking bread. You mix ingredients to create the right environment for yeast to grow and rise. Just as the yeast multiplies in the dough, bacteria multiply in the nutrient medium, forming visible colonies.

So, this is called as CFU or a colony forming unit. ...So therefore, you cannot get an immediate value of this thing...

The results of culturing are expressed as Colony Forming Units (CFUs). This refers to the number of visible colonies that grow from one original bacterial cell. Since incubation takes time, it is impossible to get instant results. After waiting, scientists can count the colonies formed and relate this back to the volume of the original sample.

Imagine planting seeds in a garden. You can't see how many plants will grow right away; you must wait for them to sprout and develop. In the same way, the bacteria must be given time to grow into colonies before counting them.

...if you have a very large number, you dilute it so that you can get distinct masses of colony forms.

When there is a high concentration of microorganisms in a sample, researchers dilute the sample to obtain manageable colony counts. For instance, if a water sample contains 100 bacteria, diluting it by tenfold allows for easier counting of colonies without overlapping.

Think of trying to drink a very strong juice concentrate. If it's too strong, you add water to make it more palatable. In microbiology, diluting a sample helps make the microorganism concentration easier to manage and count.

So, there are a lot of instruments now available, which use microscopy in order to count bacterial cells ...this is a big challenge, microbial analysis is a very big challenge.

Flow cytometry is an advanced technique used to count microorganisms in a sample. The sample flows through a narrow channel, allowing individual bacteria to be counted as they pass by a laser. Although this method is powerful, it’s not yet a standard practice due to concerns about accuracy and sample representation.

It's like counting cars on a busy road using a camera. If you stand at one point, you may not see all the cars—some may be blocked from view. Just like that, flow cytometry can miss counting some bacteria if the sample isn't representative.

People use other ways of detecting bacteria also which includes putting a dye something called a staining. ...you can look at the bacteria look at morphology you can look at DNA analysis...

To accurately identify microorganisms, researchers often use staining techniques. A special dye is added to the sample, allowing different types of bacteria or fungi to be distinguished under a fluorescence microscope. More sophisticated techniques also involve DNA analysis to identify specific bacteria in detail.

It's akin to using food coloring to differentiate between different types of candy in a jar. By adding color, you can more easily identify what’s inside. In microbiology, staining helps researchers see and classify bacteria under a microscope.

In general, if the concentration of microorganisms is very high, it will show up as turbidity.

One visual indicator of high microorganism concentration in water is turbidity, which causes the water to appear cloudy. While turbidity can suggest the presence of microorganisms, it does not guarantee their presence, emphasizing the need for further testing to confirm actual microbial growth.

Consider muddy water after a heavy rainfall; the cloudiness is caused by particulates. Just like turbidity signals that something is mixed in the water, it indicates potential microbial contamination, but you should test to find out for sure.

The term viable is living and non-viable means it is a dead cell which will not grow. ...it will not cause probably much harm.

In microbiology, microorganisms can be categorized as viable (living and capable of growth) or non-viable (dead and unable to grow). Viable pathogens are a concern because they can cause infections, while non-viable cells, although present, do not pose the same level of danger as they cannot proliferate.

Imagine a light bulb that is on (viable) compared to one that is burnt out (non-viable). The working bulb can light up a room just as viable microorganisms can cause infections; the burnt-out bulb cannot do anything, similar to how non-viable microorganisms pose less risk.

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