The EMB agar is a selective and differential medium used for solid culture isolation of Gram negative bacilli, especially the Enterobacteriaceae and other Gram negative bacteria undemanding. It is also known by the acronym EAM, which stands for eosin-methylene blue.
This medium was created by Holt-Harris and Teague in 1916. It contains peptone, lactose, sucrose, dipotassium phosphate, agar, eosin, methylene blue, and water. It is very similar to MacConkey Agar, especially when using Levine's Modified EMB Agar, which does not contain sucrose.
Metallic gloss of Escherichia coli on EMB agar Source: Carmen Moreno González, from Wikimedia Commons
In fact, each laboratory decides whether to work with one or the other, since they fulfill the same function, although biochemically they are different.
It even has the same drawback as classic MacConkey agar in terms of swarming production by the genus Proteus. Therefore, to avoid this phenomenon, the agar concentration can be increased by up to 5%.
Basis
Selective
EMB agar is subtly selective because it contains the aniline dyes (eosin and methylene blue), which act as inhibitors, preventing the growth of most Gram positive bacteria and some fastidious Gram negative rods.
However, this agar has the drawback that some Gram positive bacteria can resist the presence of the inhibitory substances and grow as small colorless punctate colonies, such as Enterococcus faecalis and some Staphylococcus.
Certain yeasts can also grow, such as the Candida albicans complex, which will give very small pink colonies. Chlamydospores can even develop from this yeast if the sample is deep seeded.
Differential
On the other hand, EMB agar is also a differential medium, since these dyes together (eosin and methylene blue) have the property of forming a precipitate at acidic pH, therefore they serve as indicators of its production.
Thus, weakly lactose or sucrose fermenting bacteria produce purple colonies within 24 to 48 hours. For example the genera Klebsiella, Enterobacter and Serratia.
Those bacteria that strongly ferment lactose, such as Escherichia coli, or sucrose, such as Yersinia enterocolitica or Proteus penneri, form a greenish black precipitate, giving a characteristic metallic luster appearance in these species.
It should be noted that if EMB levine medium (without sucrose) is used, Yersinia enterocolitica and Proteus penneri will produce clear colonies.
Bacteria that do not ferment lactose or sucrose are nourished by the presence of peptones, which provide the amino acids and nitrogen necessary for bacterial growth, and produce clear colonies. For example, the genera Salmonella and Shigella, among others.
Likewise, it is important to note that the Acinetobacter genus can present lavender-blue colonies, even though it is not a lactose fermenter or sucrose, but has the property of fixing methylene blue on its cell wall. This can also happen with other oxidative bacteria.
Preparation
The original dehydrated medium is light beige in color.
To prepare this culture medium, 36 grams of the dehydrated medium must be weighed and suspended in a flask containing one liter of distilled water.
After letting the mixture rest for 5 minutes, take the flask to a heat source, mixing vigorously and constantly until it boils and dissolves completely.
Subsequently, the already dissolved culture medium must be sterilized using the autoclave at 121 ° C for 15 minutes.
At the end of the time, it is removed from the autoclave and left to stand briefly. Then, still warm (45-50 ° C), 15-20 ml of agar is served in each sterile Petri dish. The medium should be litmus blue.
After serving the plates are left slightly uncovered until the agar cools slightly. They are then covered and allowed to solidify completely. Subsequently, they are arranged in inverted plate holders and stored in a refrigerator (8 ° C) until use.
This procedure is preferably carried out in a laminar flow hood or in front of a Bunsen burner to avoid contamination.
It is important to bear in mind that each commercial house will indicate the amount to be weighed to prepare the culture medium.
The final pH of the medium must be 7.2 ± 0.2
Applications
This medium is used to sow urine and feces or any type of clinical specimen, especially if the presence of non-fastidious Gram-negative bacilli is suspected, such as the bacilli belonging to the Enterobacteriaceae family, which grow very well on this medium.
Enteropathogenic bacteria of the Shigella and Salmonella genera are distinguished by their colorless or slightly amber colonies.
Other non-lactose fermenting bacilli such as Aeromonas, Pseudomonas, Acinetobacter, among others, also grow.
Likewise, this medium is very useful in the microbiological analysis of food and water, as it is ideal for the complete confirmatory phase of the determination of coliforms, that is, to corroborate the presence of E. coli from turbid EC broths, from of the most probable number technique (MPN).
QA
To verify that the freshly prepared culture medium is working well, control strains can be planted to observe the characteristics of the colonies and verify that they give as expected.
For this, ATCC strains or well-identified strains of E. coli, Enterobacter aerogenes, Klebsiella sp, Salmonella typhimurium, Shigella flexneri, Pseudomonas aeruginosa and some Gram positive bacteria, such as S. aureus, can be used.
E. coli is expected to generate well developed blue-black colonies with a green metallic luster. Whereas, Enterobacter aerogenes and Klebsiella sp should give well developed bluish-black mucous colonies.
On the other hand, Salmonella typhimurium and Shigella flexneri, must develop large colonies, colorless or with a slight amber color.
Finally, the genus Pseudomonas aeruginosa grows as colorless colonies of irregular size, while Gram positive bacteria should be totally inhibited or grow sparsely with very small colonies.
Final thoughts
Sometimes sterilization causes the methylene blue to be reduced, showing a medium orange color. In order for the methylene blue to oxidize and recover the purple color, it must be mixed gently until the color is recovered.
Also, after sterilization the dye may precipitate, so it should be mixed well before serving the Petri dishes.
References
- Camacho A, Giles M, Ortegón A, Palao M, Serrano B and Velázquez O. 2009. Techniques for Microbiological Analysis of Foods. 2nd ed. Faculty of Chemistry, UNAM. Mexico.
- Carranza C, León R, Falcón N, Neumann A, Kromm C. Characterization and Distribution of Potentially Pathogenic Escherichia coli Strains Isolated from Broiler Chickens from Poultry Farms in Peru. Rev. investiga. vet. Peru 2012 23 (2): 209-219. Available at: scielo.org.
- Laboratorios Conda SA Eosin and Methylene Blue Agar. 2010. Available at: condalab.com
- Britannia Laboratories. Levine EMB (With Eosin and Methylene Blue) 2011.Available at: britanialab.com
- BD Laboratories. BD EMB Agar (Eosin Methylene Blue Agar), Modified. 2013.Available at: bd.com
- Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. (5th ed.). Argentina, Editorial Panamericana SA
- Forbes B, Sahm D, Weissfeld A. 2009. Bailey & Scott Microbiological Diagnosis. 12 ed. Argentina. Editorial Panamericana SA