E. Coli Under The Microscope – Types, Techniques, Gram Stain, Hanging Drop Method

Of all the bacteria on the planet, E. coli is perhaps one of the best known, even to those working outside the fields of microbiology and the wider scientific community. 

E. Coli Under The Microscope - Types, Techniques, Gram Stain, Hanging Drop Method

Keen to learn more about the types of E. coli, the techniques used to detect it, and the methods involved? Then read on for all you need to know.

What Is E. Coli?

Escherichia coli, also known as E. coli, is a type of bacteria found in a variety of environments, including soil and the intestines of animals, as well as a number of foods.

E. coli is a genus of bacteria that belongs to the genus Escherichia.

The majority of strains of E. coli are harmless – and indeed, many have benefits to the intestinal tract of humans.

Others, however, prove to be far more dangerous, and some can have serious health consequences – and these are the types that the majority of people will tend to associate with the term “E. coli”.

Perhaps the best-known example of a dangerous strain is E. coli 0157:H7.

The more harmful and dangerous strains of E. coli can be of serious risk to humans and can be responsible for a number of health issues, including pneumonia, respiratory illnesses, and urinary tract infections.

In some cases, these conditions can be extremely serious.

E Coli is important in the intestinal tract because it aids digestion and helps the body to absorb the essential vitamins available in food more easily and quickly.

In addition, some strains of E. coli are also useful in helping to prevent and inhibit the growth and breeding of harmful bacteria, which can also cause serious health issues for humans.

Types Of E.Coli

There are a number of types of E. coli, and some are pathogenic. This means that they are prone to causing illnesses both inside and outside the intestinal tract of humans.

Most pathogenic types of E. coli cause diarrhea in affected individuals and are usually the result of contaminated or dirty food, eaters, or animals.

There are six main types of E. coli deemed to be pathogenic, and these are:

  • STEC – Shiga toxin-producing E. coli. Most commonly associated with food poisoning and food-based outbreaks, this may also be referred to as VTEC – verocytotoxin-producing E. coli, or EHEC – enterohemorrhagic E. Coli.
  • (ETEC) – Enterotoxigenic E. coli 
  • (EPEC) – Enteropathogenic E. coli 
  • (EAEC) – Enteroaggregative E. coli 
  • (EIEC) – Enteroinvasive E. coli
  • (DAEC) – Diffusely adherent E. coli 

What Is Gram Staining?

In order to understand the way in which we can detect and understand E. coli, we first need to establish an understanding of gram staining.

The Gram stain is a differentiating technology that is commonly used to classify bacteria. By imbuing the cells with color, the staining technique distinguishes between two types of bacteria (gram-positive and gram-negative). 

E. coli is considered to be a gram-negative bacteria – they stain negative on a gram stain.

This is largely due to the properties of E. coli; the bacteria comes with an additional outer membrane, and this is made from lipopolysaccharides as well as phospholipids, both of which create a negative overall charge to the cell wall, creating a negative gram stain result.

What Are The Requirements For A Gram Stain Of E. Coli?

There are a number of things you will need to perform a Gram Stain of E. coli, and these include:

  • A compound light microscope
  • Bunsen flame or heater
  • Wire loop
  • Slides that are clean and free from grease
  • A marker pen
  • Basic dye – crystal violet
  • Gram’s iodine
  • Decolorizing agent – 95% ethanol
  • 1% safranin or dilute carbol fuchsin or neutral red
  • A positive control strain of E. coli
  • A negative control strain of E. coli

How To Take A Bacterial Smear

How To Take A Bacterial Smear

Once you have collected all of the necessary equipment, the process for taking a bacterial smear is follows:

  • Take a slide, and make sure that this is clean and free from grease.
  • Use the wire loop to take 0.85% saline, and place this right in the center of the slide.
  • Use the wire to touch the surface of a colony, and place this in the saline drop to create a thin film.
  • Allow the smear to air dry completely.
  • Hold the slide at one end, and pass the slide over the flame of the burner twice or three times to heat fix the bacterial smear in place.

The Procedure For A Gram Stain

Once the smear has been sealed, you are ready to proceed with the gram stain, and this is as follows:

  • Cover the smear you have made with crystal violet. Allow this to stand for a minute, and then rinse the smear with tap water.
  • Once rinsed, you can cover the smear using the Gram’s iodine. Allow this to stand for one minute, then rinse again under the tap.
  • Use 95% alcohol to decolorize the smear, then run this under the tap water.
  • Cover the smear with safranin, and leave this to stand for one minute.
  • Rinse the smear under the tap water, and allow it to air dry.
  • You can then observe the smear. This should be done first under low power, or 10x objective, and then once again under oil immersion (100x) objective.

Once the procedure is complete, you will be able to observe the results and determine whether E. coli is present in the specimen, and so in the wider sample.

Hanging Drop Technique

Another option for the analysis of E. coli is the hanging drop technique, and this offers the advantage of allowing scientists and biologists to analyze living microorganisms.

You will need:

  • A slide of glass
  • Paraffin wax
  • A wire loop
  • Cover slip
  • Heater or Bunsen burner
  • A broth culture of motile bacteria, preferably harvested within the last 24 hours.

To conduct a hanging drop analysis on your E. coli sample, you will need to carry out the following steps:

  • Add a paraffin ring to your glass slide, and then use a lab scoop or scraper to add paraffin or Vaseline to the corners of your cover slip.
  • Use your wire loop to add the culture to the center of the cover slip
  • Turn the slide over the sample, so that the sample is between the cover slip and the slide.
  • Place the slide on the microscope with the cover slip on top
  • Switch to the lowest power, and view the specimen through the microscope. You will need to establish whether the bacteria are moving in a range of directions and that it is changing position regularly.

Why Do We Need To Test For E. Coli?

One of the main reasons for testing for E. coli is to reduce the risk of contamination and illness in humans; as we have mentioned, there are various strains that can pose a real risk of human health and wellbeing. 

It is important, therefore, to ensure that the presence of E. coli can be eliminated and managed as much as possible, particularly in public areas and spaces such as pools and spas.

These areas tend to start by treating pool water with bacteriological and chemical testing, and this is often required as part of the protocol to keep the space open. 

This means that if E. coli is present, it can be picked up and tackled as soon as possible before there is a serious risk to human help.

Most facilities and public spaces of this kind will have a rigorous E. coli testing procedure in place to help prevent outbreaks.

In these situations, testing is generally performed using a sample of water, and this could be for, a river, lake, the sea, a hot tub, or a spa facility.

One of the methods described and detailed above is typically used, and this means that fast, decisive action can be taken in the event that a positive strain of potentially pathogenic bacteria is present.

It is also worth noting that as with a number of contaminants that are predominantly waterborne, temperature consistencies can be a major factor in keeping E. coli at bay.

So this is a key consideration for those who are in a position where E. coli could be a major issue or risk.

Final Thoughts

E. coli is a bacteria that has a bad reputation, but it is important to note that there can be a number of advantages to it, particularly with regard to the human gut and intestinal system.

The pathogenic strains, however, can cause serious risks to human health, so it is imperative that these are detected and resolved as quickly as possible.

By using the techniques outlined above, scientists can locate and detect potentially dangerous strains of E. coli quickly and effectively.

This can play a major role in ensuring that public health is protected as much as possible.

By learning how to detect and eliminate E. coli, scientists are playing a key role in this – and research continues to grow and improve.

Jennifer Dawkins