The agar Hektoen or Hektoen enteric agar solid medium is a selective and differential culture. It was created at the Hektoen Institute by King and Metzger for the isolation of enteropathogenic bacteria of the Shigella and Salmonella genera.
The medium is composed of proteose peptone, yeast extract, bile salts, lactose, sucrose, salicin, sodium thiosulfate, sodium chloride, iron citrate, ammonium citrate, bromothymol blue, acid fuchsin and agar. This formulation makes it possible to differentiate the Shigella and Salmonella genera from the rest of the bacteria capable of growing in this medium.
A. Virgin Hektoen agar B. Seeded Hektoen agar. Source: Flickr.com/The original uploader was Philippinjl at French Wikipedia.
Although there are other media with the same function as Hektoen agar, it has a greater advantage compared to other media, especially when you want to recover Shigella species.
The species of both genders cause severe gastrointestinal problems in humans due to the consumption of contaminated food; therefore transmission is fecal - oral. That is why the use of Hektoen agar is mainly indicated in the microbiological analysis of stool and food samples.
Basis
Hektoen Agar contains peptones and yeast extract as a source of nutrients, providing the essential elements for microbial development.
However, it also has bile salts that act by inhibiting the growth of some bacteria, especially Gram positive and some Gram negative. It is for this reason that it is considered a selective medium.
On the other hand, Hektoen agar is a differential medium. This property is conferred by the presence of fermentable carbohydrates such as lactose, sucrose and salicin, together with the pH indicator system, represented by bromothymol blue and acid fuchsin.
All bacteria capable of growing on this medium that do not belong to the genus Salmonella and Shigella will develop salmon or orange colonies with the exception of the genus Proteus. This is due to the fermentation of one or more of the carbohydrates present, which acidifies the medium, which makes the pH indicator turn.
For its part, the genus Shigella and Salmonella are not capable of fermenting any of the carbohydrates present, using only peptones as an energy source, which alkalizes the medium and therefore their colonies are blue-green.
Bacteria capable of forming hydrogen sulfide (colorless gas) can also be distinguished in this medium. Sodium thiosulfate acts as the source of sulfur while iron citrate is the developer. Both compounds make possible the formation of a black precipitate of iron sulfide that shows the reaction.
The black precipitate in the center of the colony with a transparent halo around it gives a fish-eye appearance. This characteristic suggests the presence of the genus Salmonella.
Finally, the sodium chloride maintains the osmotic balance and the agar provides the solid consistency to the medium.
Preparation
Weigh 76 g of the dehydrated medium and dissolve in a liter of distilled water. Shake the mixture vigorously and then let it rest for 10 to 15 minutes. It can be heated and boiled, giving rotary movements until its total dissolution. This medium is not autoclaved.
When the medium reaches a temperature of approximately 45 ° C, a volume of 20 ml is poured directly into sterile Petri dishes.
The agar is allowed to solidify. At that time they are ready for use. It is recommended to use them immediately. If this is not possible, they are kept in the refrigerator until use.
Plates should be removed from the refrigerator in advance before seeding to bring them to room temperature.
The pH of the medium should be 7.5 ± 0.2. The color of the dehydrated medium is purple and the prepared medium is brownish-green.
Use
The use of Hektoen agar is recommended for the search for bacteria of the Shigella and Salmonella genus in stool and food samples.
The possibility of isolating these bacteria is considerably increased if the sample is previously enriched in special broths, such as selenite broth, cystine selenite broth, tetrathionate broth, etc.
The inoculum must be strong and the sowing is done by streaking. The plates are incubated at 37 ° C for 24 to 48 hours in aerobiosis.
Incubation for 48 hours is recommended because the characteristics of the colonies are clearer for their interpretation and differentiation at this time.
QA
To perform quality control on this medium, certified bacterial strains are used, such as: Salmonella typhimurium ATCC 14028, Salmonella enteritidis ATCC 13076, Shigella flexneri ATCC 12022 and Shigella sonnei ATCC 25931.
The expected results are as follows: Salmonella typhimurium and Salmonella enteritidis should develop blue-green colonies with or without a black center. Whereas the Shigella species will grow as blue-green colonies.
Strains of Escherichia coli ATCC 29212, Proteus mirabilis, Klebsiella pneumoniae ATCC 700603, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 can also be included.
In these cases, the observed characteristics are the following: E. coli and K. pneumoniae will develop salmon-orange colonies in this medium, with a precipitate of the same color around them. Whereas, Proteus will develop blue-green colonies with or without a black center.
While S. aureus and E. faecalis must be inhibited, E. faecalis occasionally manages to grow as very small, yellow colonies.
On the other hand, since this medium is not autoclaved, it is important to evaluate the sterility of the medium. Therefore, from each batch prepared, one to two uninoculated plates should be incubated at 37 ° C for 24 hours in aerobiosis.
Obviously no growth of any kind is expected on the plate.
Limitations
-Proteus species can develop in this medium and the characteristics of their colonies can be confused with Salmonella or Shigella species. For this reason, any suspicious colony must be confirmed with biochemical tests.
-It is necessary to accompany the use of this medium with other less selective agars, because if the microorganism sought is found in low concentrations it may not develop in this medium.
-Do not overheat during preparation, as excessive heat alters the composition of the medium.
-Unusually, colonies of lactose fermenting Salmonella may appear that may go unnoticed.
References
- Wikipedia contributors. Hektoen enteric agar. Wikipedia, The Free Encyclopedia. March 13, 2019, 23:38 UTC. Available at:.wikipedia.org / Accessed March 16, 2019.
- BD Laboratories. BD Hektoen Enteric Agar (HE Agar). 2013.Available at: bd.com
- Britannia Laboratories. Hektoen Enteric Agar. 2015.Available at: britanialab.com
- Difco Francisco Soria Melguizo Laboratories. Hektoen agar. Available at: f-soria.es
- Difco & BBL Manual, Hektoen Enteric Agar. 2nd Edition. Available in: bd.com/europe