The agar Endo or Endo medium is a solid medium, differential and with some degree of selectivity culture. The original formula was created by Endo in 1904 to differentiate lactose-fermenting from non-fermenting bacteria. Initially it was designed for the isolation of Salmonella typhi, but later the objective of the medium turned to the search for coliforms.
The principle of Endo Agar has remained, but its formulation has undergone countless changes over the years. Currently, the medium is composed of peptic digest of animal tissue, lactose, dipotassium hydrogen phosphate, sodium sulfite, basic fuchsin, and agar.
A: Virgin Endo Agar B: Seeded Endo Agar, in the middle of the agar you can notice the metallic shine of an E. coli strain. Source: A: Strolch1983 / B: LenkaM
The main use of the medium has been linked to the isolation and differentiation of Gram negative bacilli belonging to the Enterobacteriaceae family and to other close families.
For a long time it was used in the detection of coliforms in water, dairy and food samples, but today the use of this medium has been displaced by others with similar functions. However, some microbiology laboratories use this agar for the isolation of Enterobacteriaceae from samples of clinical origin.
Basis
Endo agar contains peptones that serve as a source of amino acids, nitrogen, carbon and energy, necessary for the growth of undemanding microorganisms.
On the other hand, the slightly selective character of the agar is provided by the addition of sodium sulfite and basic fuchsin; both components partially or totally inhibit the growth of most Gram positive bacteria.
The differential character is given by the presence of the fermentable carbohydrate, which in this case is lactose and basic fuchsin, which also serves as a pH indicator.
Gram-negative bacteria that grow on this agar and are capable of fermenting lactose will form strong pink colonies; being pathognomonic of Escherichia coli the formation of dark red colonies with an iridescent greenish metallic luster. This is due to the high production of acids from carbohydrate fermentation.
It should be noted that the medium around the colonies also turns a strong pink color. Whereas non-lactose fermenting Gram negative rods form pale pinkish colonies similar to medium or colorless.
Dipotassium hydrogen phosphate balances the pH of the medium and agar is the component that provides the solid consistency.
Preparation
Endo agar
Weigh out 41.5 g of the dehydrated medium and dissolve in 1 liter of distilled water. Heat the mixture with frequent stirring until there is complete dissolution of the medium. Sterilize in an autoclave at 121 ° C, at 15 lb pressure, for 15 minutes.
When removing from the autoclave, allow to cool to a temperature of approximately 45-50 ° C, shake the mixture to homogenize before serving. Pour 20 ml into sterile Petri dishes.
Allow the plates to solidify, invert and store in a plaquero or wrap with dark paper before storing in the refrigerator. It is very important to protect the prepared medium from direct light. A best practice is to prepare the exact amount that you will need.
If stored in a refrigerator, the plates should be allowed to warm up before use.
The pH of the medium should be between 7.2 to 7.6 and the color of the prepared medium is pale pink.
M-Endo agar variant
There is another version of Endo agar (m-Endo) that follows the formula of McCarthy, Delaney and Grasso, which contains more compounds and varies in the form of preparation.
This variant contains: lactose, tryptose, enzymatic digest of casein, enzymatic digest of animal tissue, sodium chloride, dibasic potassium phosphate, sodium sulfite, yeast extract, monobasic potassium phosphate, basic fuchsin, sodium deoxycholate, lauryl sulfate sodium and agar.
In this case, 51 g of the dehydrated medium is weighed and suspended in 1 liter of distilled water containing 20 ml of ethanol.
Heat slightly while stirring until the medium dissolves completely. It should not be overheated and should not be autoclaved. Once the mixture is homogeneous, serve in sterile Petri dishes and allow to solidify.
Use
In some countries, it is still used to count total and fecal coliforms in food and water samples, especially the presence of Escherichia coli as the main indicator of fecal contamination.
M-Endo Agar is recommended by the American Public Health Association (APHA) for the monitoring and control of disinfection and wastewater treatment programs, as well as in the evaluation of the quality of drinking water.
The most widely used method is membrane filtration, after enriching the sample with Lauryl sulfate broth for 2 to 4 hours.
It can also be used as a substitute for EMB agar in the microbiological analysis of food and water by the most probable number technique (MPN), specifically in the complete confirmatory phase to corroborate the presence of E. coli from cloudy EC broths.
QA
Known or certified control strains are sown to assess the quality of the prepared Endo agar batch.
Among the strains that can be used for this purpose are: Escherichia coli ATCC 25922, Escherichia coli ATCC 11775, Enterobacter cloacae ATCC 13047, Klebsiella pneumoniae ATCC 13883, Salmonella typhimurium ATCC 14028, Shigella flexneri ATCC 12022, Proteus miracaliscus ATCC 11700 and EnteroCC 14153CC1415300.
The strains are seeded by exhaustion and are incubated at 37 ° C for 24 hours in aerobiosis.
The expected results are:
- For Escherichia coli: strong red colonies, with metallic luster.
- For E. cloacae and K. pneumoniae the colonies should be pink mucoid.
- In the case of S. typhimurium, S. flexneri and P. mirabilis, the colonies are usually pale pink or colorless.
- Finally, E. faecalis is expected to be partially inhibited, therefore its growth must be poor with very small, strong pink colonies.
Limitations
-Endo medium has low selective power, therefore, it is possible that some Gram positive microorganisms such as Staphylococcus, Enterococcus and even yeasts can grow.
-Other bacilli not belonging to the Enterobacteriaceae Family can develop in this medium, such as Pseudomonas sp and Aeromonas sp. The characteristics of these strains are colorless irregular colonies.
-This prepared medium is very sensitive to light, therefore, prolonged exposure to it deteriorates the indicator system, irreversibly damaging the medium.
-The components of the medium are considered carcinogenic, therefore direct contact should be avoided.
-The dehydrated medium is very hygroscopic and must be kept in its original container at room temperature, tightly closed and in a dry environment.
References
- BD Laboratories. Endo Agar. 2013.Available at: bd.com
- Neogen Laboratories. M Endo Agar. Available at: foodsafety.neogen.com
- Endo Agar. Wikipedia, The Free Encyclopedia. 7 Sep 2017, 08:27 UTC. 28 Feb 2019, 22:55. Available at: en.wikipedia.
- MercK Laboratory. Endo agar. 2019.Available at: merckmillipore.com
- Technical Sheet Laboratories. M –Endo Agar LES. 2015.Available at: liofilchem.net