BRIGHT GREEN AGAR: RATIONALE, PREPARATION, AND USES - BIOLOGY - 2024

The brilliant green agar is a solid culture medium with high degree of selectivity. It is used exclusively for the isolation of strains of the genus Salmonella, however there are some exceptions, such as the typhi and paratyphi species that do not grow on this medium.

The search for the genus Salmonella is frequent in stool, water or food samples. In this sense, this medium can be very useful. This agar was created in 1925 by Kristensen, Lester and Jurgens, later it was modified by Kauffmann.

Bright green agar. Fermenting and non-fermenting colonies respectively. Source: MJ Richardson / Invisible Worlds exhibition - 2012geograph.org.uk/photo/2815842 Creative Commons License.

It is composed of pluripeptones from peptic digest of animal tissue and pancreatic digest of casein, it also contains yeast extract, sodium chloride, lactose, sucrose, phenol red, bright green and agar-agar.

It is characterized by being a rather inhospitable environment for most bacteria, favoring the growth of Salmonella, however some coliforms are capable of subsisting in it, developing weakly.

It is important to note that the Shigella genus does not grow on this medium, nor does Salmonella typhimurium or Salmonella paratyphi. Therefore, if you want to isolate these microorganisms, other media should be used, such as XLD agar, among others.

Basis

Bright green agar

Each of the components that make up the medium performs a specific function that determines the characteristics and properties of the agar.

Pluripeptones and yeast extract are the source of nutrients from which microorganisms take the nitrogen and minerals necessary for their development. Lactose and sucrose are sources of energy for microorganisms that are capable of fermenting them.

Bright green is the inhibitory substance that prevents the growth of Gram positive bacteria and a large number of Gram negative microorganisms.

Sodium chloride provides osmotic stability to the medium. While phenol red is the pH indicator, it turns color when detecting acid production from carbohydrate fermentation.

Non-fermenting colonies of lactose and sucrose grow on this medium in a pinkish or transparent white color, on a red background. For example, bacteria of the genus Salmonella.

While lactose or sucrose fermenting bacteria capable of growing on this medium develop yellow-green or yellow-green colonies on a greenish-yellow background. For example, Escherichia coli and Klebsiella pneumoniae.

Bright green agar (BGA) variants

There are other variants of bright green agar; Novobiocin Brilliant Green Glucose (NBG) Agar and Novobiocin Brilliant Green Glycerol Lactose (NBGL) Agar.

Novobiocin Bright Green Glucose Agar

Contains Trypticase Soy Agar, Ferric Ammonium Citrate, Sodium Thiosulfate Pentahydrate, Phenol Red, Glucose, Bright Green, Novobiocin, and Distilled Water.

It is used for the isolation of Salmonella colonies from stool samples.

In this case, bright green and novobiocin are the inhibitory substances that prevent the growth of Gram positive bacteria and some Gram negative microorganisms.

Sodium thiosulfate is the source of sulfide and ferric citrate is the source of iron, both necessary to reveal hydrogen sulfide production through the formation of a black ferric sulfide precipitate.

Glucose is the fermentable carbohydrate and phenol red is the pH indicator.

In this medium, Salmonella colonies develop large with a black center surrounded by a reddish halo and followed by a clear visible area. Some strains of Citrobacter freundii produce colonies identical to that of Salmonella.

Novobiocin Brilliant Green Glycerol Lactose Agar (NBGL)

This medium contains trypticase soy agar, ferric ammonium citrate, sodium thiosulfate, lactose, glycerol, bright green, novobiocin, and distilled water.

The difference between this medium and the previous one is that glucose is substituted by lactose and glycerol and phenol red is not used.

The medium is also used to isolate Salmonella species, the colonies develop black, due to the production of hydrogen sulfide.

Only colonies that do not produce acid from glycerol or lactose achieve sufficient H ₂ S production, because low pH interferes with H ₂ S formation. This results in colorless colonies for most species of Proteus and Citrobacter.

Preparation

-Weigh 58 grams of the commercially obtained dehydrated medium. Add it to a liter of redistilled water. Mix, let stand a few minutes, and place the mixture on a heat source until completely dissolved.

-Autoclave at 121 ° C for 15 minutes, do not exceed the sterilization time.

-Let it rest and serve while hot in sterile Petri dishes. The final pH should be 6.9 ± 0.2.

-Let solidify and store in the fridge until use. Before seeding the plates should take room temperature.

-The powder medium is green in color and prepared takes an orange-brown or reddish-green color, depending on the pH and the commercial company. A very brown color is indicative that the agar was overheated.

-Once the agar has solidified, it is not recommended to recast, as the medium deteriorates.

Uses / applications

This medium is used to search for strains of the genus Salmonella from stool samples and dairy foods, among others.

Because it is a rather inhospitable environment, it is advisable to sow an abundant inoculum if the direct sample is used. Otherwise, a pre-enrichment and enrichment of the specimens should be done before sowing in this medium.

As some strains of Salmonella are inhibited or grow with difficulty, it is advisable to accompany this medium with other selective agar for Salmonella.

Every colony with a typical Salmonella characteristic must be submitted to biochemical tests for its definitive identification.

QA

To test the good performance of the bright green agar medium, ATCC strains can be used to observe their development on it.

The most frequent strains used for quality control are: Salmonella enteritidis ATCC 13076, Salmonella typhimurium ATCC 14028, Proteus mirabilis ATCC 43071, Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922, Shigella flexneri ATCC 12022, Staphylococcus aureus ATCC 6538.

The first 3 should give pinkish or transparent white colonies on a red background. Salmonella with good development and Proteus with little or regular growth.

For Klebsiella and Escherichia, yellow-green colonies with a yellow background are expected and in the case of Shigella and Staphylococcus they should be inhibited.

The dehydrated medium must be stored at room temperature, in a dry place, because the medium is very hygroscopic.

References

1. Laboratorio Difco Francisco Soria Melguizo SA Brilliant Green Agar. 2009
2. Britannia Laboratory. Bright Green Agar. 2015.
3. BD Laboratory. BD Brilliant Green Agar. 2013.
4. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. (5th ed.). Argentina, Editorial Panamericana SA
5. Forbes B, Sahm D, Weissfeld A. 2009. Bailey & Scott Microbiological Diagnosis. 12 ed. Argentina. Editorial Panamericana SA