Principle, Uses, Preparation, And Interpreting The Results Of Simmons Citrate Agar

Scientific tests and processes are the proverbial bread and butter of laboratory work.

Principle, Uses, Preparation, And Interpreting The Results Of Simmons Citrate Agar

Without them, not only would our understanding of the sciences be incredibly different, and potentially worse off as well, but we would not have a way to reliably test for things in the presence of living tissues, such as infectious pathogens and other foreign micro-organic bodies.

However, if you are new to the field, or are just looking at these processes for research purposes, it can often feel like these tests are difficult to wrap your heads around.

In some cases, they can almost feel deliberately obtuse with the scientific phrasing and terms that are used.

But as frustrating as they can be, these tests are vital to the continued research of the field of microbiology, so it is important to try and grapple with them.

This is why we have tried to reach a middle ground with this guide. In it, we will try and explain what the purpose of the Simmons citrate agar is, as well as its composition, and the sort of results you can expect it to produce, in the simplest ways possible, so that anyone can understand it.

What Is Simmons Citrate Agar?

Before we get into the nitty-gritty of what this agar is used for, we should probably explain what it is in the first place!

Like many other types of tools used in the field of microbiology, Simmons Citrate agar is like many other types of gelatinous agar, as it is used as a substance that can allow for the growth and propagation of cultures and colonies of bacteria to grow in it.

Allowing them to be observed and researched by scientists and other experts that wish to study them, without any risk to them.

However, unlike many other types of agar that are used in research, Simmons citrate agar is what is known as a selective and differential medium for bacteria to be grown in.

What Is A Selective Medium?

A selective medium in microbiological research is a substance that will only promote the growth of very specific organisms, usually only those that can live in the conditions presented in the medium of choice.

In the case of Simmons Citrate agar, the test effectively creates an environment where bacteria that are successfully living and thriving in it use citrate as their sole source of carbon and ammonia ions.

Other organisms that either do not primarily use this method of obtaining the necessary carbon and ions for cell functions will not thrive in these living conditions, and those that use no citrate will not survive at all.

What Is A Differential Medium?

We also mentioned that Simmons Citrate agar is a differential medium, which is a substance that can allow an observer to tell apart and distinguish between different bacteria cultures that are growing in the medium.

The biochemical properties of the bacteria that are being grown usually result in different effects on the medium, especially when a nutrient or chemical indicator is added to it.

Such as neutral or phenol red, making these chemical results of the different cultures even more visible to human observation.

What Is The Composition Of Simmons Citrate Agar?

So, what is the composition of Simmons Citrate agar substance that makes it able to carry out its functions?

Well, the following list of ingredients that goes into Simmons Citrate agar are measured in amounts that relate to their presence in a liter of deionized water:

  • 15 gm of Agar material
  • 5 gm of Sodium Chloride
  • 2 gm of Sodium Citrate
  • 1 gm of Ammonium Dihydrogen Phosphate
  • 1 gm of Dipotassium Phosphate
  • 0.2 gm of Magnesium Sulfate
  • 0.08 gm of Bromothymol Blue

Research Uses For Simmons Citrate

As we have already established, Simmons Citrate agar mediums are an excellent way for the researcher to establish which microorganisms use citrates as their primary source of carbon and other vital nutrients.

This makes the test very useful in the identification of a variety of bacteria that researchers may wish to research or detect the presence of in a given sample.

The bacteria that can be found are considered gram-negative, meaning that they will retain a stain after gram staining of a sample has happened, which includes many species such as Salmonella enteritidis and other Salmonella species, as well as Escherichia coli.

This makes the Simmons citrate agar test a very useful method to have on hand for detecting these species.

The Principles Of Using Simmons Citrate Agar

The Principles Of Using Simmons Citrate Agar

So, with citrate being the main testing factor that is being recorded and measured in a Simmons Citrate agar test, what are the principles that underpin the test itself.

Well, generally speaking, in this test, the ammonium dihydrogen phosphate and the sodium citrate found in the composition act as the sole source of both nitrogen and carbon for the bacteria in the test, with dipotassium phosphate acting as a buffer for them.

The salt, or sodium chloride, acts as a balance for osmosis to continue taking place in the medium as its chemical composition of it changes.

Bacteria that can use the ammonium dihydrogen phosphate will grow unimpeded in the medium, spreading rapidly and easily, especially when compared to other bacteria in the sample.

Bacteria that can grow in these conditions will also often build an excess of alkaline or basic byproducts as a result of the experience and will convert the citrate in the medium into pyruvate, a substance that is incredibly useful in organic metabolic cycles that take place in virtually all living organisms.

Preparing Simmons Citrate Agar For Use

So, with everything else out of the way, we can now discuss what you need to do to prepare a Simmons citrate agar for testing yourself.

The following steps should be followed as close to the letter as possible.

  • Suspend the accumulated ingredients for the agar, which should amount to roughly 24.28 grams, into 1000 milliliters of distilled or deionized water.
  • Heat should then be applied until the medium has completely dissolved into the water.
  • The water should then be mixed well before it is distributed into flasks or tubes. If the tubes are around 16- millimeters, you should add around 5 to 6 milliliters to them.
  • The tubes should then be sterilized by autoclaving them at around 15 pounds of pressure for 15 minutes, at 121 degrees Celsius
  • After that, cool them in a slanted position.
  • When stored in a refrigerator correctly, the tubes will have a shelf life of around 6 to 8 weeks.
  • The tubes should be a deep forest green

Interpreting The Results Of Simmons Citrate Agar

A positive test from the experiment will change the color of the tube from a deep green to bright blue color, thanks to the presence of the alkaline byproduct that will occur in citrate-dependent bacteria. Otherwise, the solution will stay green.


As you can see, the test, whilst seeming pretty difficult to understand at first, is pretty simple once it is broken into its separate steps.

Jennifer Dawkins

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