What is Endo Agar?

• Endo Agar, also known as Endo’s medium, is a microbiological growth medium that was developed by Endo to differentiate gram-negative bacteria based on their ability to ferment lactose, while inhibiting the growth of gram-positive bacteria. It was originally designed for the isolation of Salmonella typhi, but it is now primarily used as a coliform medium.
• One notable feature of Endo Agar is that it inhibits gram-positive bacteria without the use of bile salts, which were traditionally employed for this purpose. Instead, Endo achieved inhibition by incorporating sodium sulfite and basic fuchsin into the medium.
• The resulting Endo Agar, also referred to as Fuchsin Sulphite and Infusion Agar, proved effective in isolating the typhoid bacilli. Over the years, modifications to the medium have been made to enhance its performance and specificity.
• Endo Agar is currently recommended as a selective medium for confirming the presumptive test for members of the coliform group in both clinical and non-clinical samples. It supports the growth of most gram-negative organisms while inhibiting the growth of gram-positive organisms.
• In the context of Endo Agar, coliform organisms refer to those that ferment lactose in the medium. These lactose-fermenting organisms, such as Escherichia coli, produce colonies with a green metallic sheen. On the other hand, non-lactose-fermenting organisms, including Salmonella species, produce clear and colorless colonies.
• The faint pink color of Endo Agar aids in differentiating lactose-fermenting and non-lactose-fermenting organisms, making it a valuable medium for identifying coliform bacteria in microbiology laboratories.

Principle of Endo Agar

The principle of Endo Agar is based on several key components and reactions that allow for the selective differentiation of bacteria based on their lactose fermentation abilities. Here are the main principles of Endo Agar:

1. Peptone: The medium contains peptone, which provides nitrogen, carbon, vitamins, and minerals necessary for bacterial growth.
2. Selectivity: Sodium sulfite and basic fuchsin are incorporated into the medium to selectively inhibit the growth of gram-positive organisms.
3. Aldehyde Reaction: Lactose-positive colonies exhibit a red color due to the aldehyde reaction with sodium sulfite and basic fuchsin. This reaction occurs when lactose-fermenting bacteria produce aldehydes during lactose fermentation.
4. Metallic Sheen: When organisms ferment lactose, the production of aldehydes results in the development of a metallic sheen. This sheen is particularly pronounced in Escherichia coli, where fuchsin crystallizes and imparts a permanent greenish metallic luster to the colonies.
5. Colorless Colonies: Lactose non-fermenting bacteria do not produce aldehydes, and as a result, they form clear and colorless colonies on the medium.
6. Pink Colonies: Coliforms, which are lactose-fermenting bacteria, produce pink colonies due to the fermentation of lactose.

By observing the color and appearance of the colonies on Endo Agar, valuable information about the lactose fermentation capabilities of the bacteria can be inferred:

• Colorless, beige, or non-pink growth indicates that lactose is not utilized, suggesting a Gram-negative non-coliform organism.
• Good growth with pink/red color indicates that lactose is utilized, suggesting a Gram-negative coliform organism.
• Good growth with a bright metallic sheen on the pink/red colonies suggests that lactose is highly utilized and points to a Gram-negative coliform organism.

It is important to note that if there is poor or no growth on the medium, it is likely that the organism is a Gram-positive organism rather than a Gram-negative one.

These principles of Endo Agar enable microbiologists to differentiate between lactose-fermenting and non-lactose-fermenting bacteria, particularly coliforms, aiding in the identification and classification of organisms in the laboratory.

Composition of Endo Agar

Ingredients	Gms/liter
Peptone	10.000
Lactose	10.000
Dipotassium phosphate	3.500
Sodium sulphite	2.500
Basic fuchsin	0.500
Agar	15.000

Final pH (at 25°C): 7.5±0.2

Preparation of Use of Endo Agar

To prepare and use Endo Agar, follow the steps outlined below:

1. Suspend 41.5 grams of Endo Agar in 1000 ml of distilled water.
2. Heat the mixture to boiling, ensuring complete dissolution of the medium.
3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Mix the medium well before pouring it into sterile Petri plates.
5. Note: If the solidified culture medium appears too red, you can remove the color by adding a few drops (maximum 1 ml per liter) of a freshly prepared 10% Sodium sulfite solution. Boil the medium again after adding the Sodium sulfite solution.
6. Streak the specimen onto the surface of the Endo Agar as soon as possible after receiving it in the laboratory. The streak plate technique is primarily used to isolate pure cultures from specimens containing mixed flora.
7. If culturing directly from a swab, roll the swab over a small area of the agar surface at the edge, and then streak from this inoculated area.
8. It is recommended to also inoculate a nonselective medium, such as Columbia Agar with 5% Sheep Blood, to provide an indication of other organisms present in the specimen.
9. After inoculation, incubate the plates at 35 ± 2°C for 18 to 24 hours. Ensure that the plates are protected from light during incubation.

Following these steps will allow for the preparation and appropriate use of Endo Agar in the laboratory setting. It enables the selective isolation and identification of lactose-fermenting coliform bacteria while inhibiting the growth of gram-positive organisms.

Result and Colony Characterisitcs of Different Organisms on Endo Agar

Interpreting the results on Endo Agar involves examining the growth and colony characteristics of different organisms. Here is the interpretation of results on Endo Agar:

1. Klebsiella aerogenes: The organism shows good-luxuriant growth with pink colonies.
2. Enterococcus faecalis: Poor growth is observed, with small pink colonies.
3. Escherichia coli: There is good-luxuriant growth of the organism, and the colonies appear pink to rose red with a metallic sheen.
4. Klebsiella pneumoniae: The organism displays good-luxuriant growth, with pink mucoid colonies.
5. Proteus vulgaris: The organism shows good-luxuriant growth, and the colonies appear colorless to pale pink.
6. Pseudomonas aeruginosa: The organism exhibits good-luxuriant growth, and the colonies appear colorless and irregular.
7. Salmonella Typhi: The organism displays good-luxuriant growth, and the colonies appear colorless to pale pink.
8. Enterobacter cloacae: There is good growth of the organism, and the colonies appear pink.
9. Salmonella Typhimurium: The organism shows good-luxuriant growth, and the colonies appear colorless.
10. Salmonella Enteritidis: The organism exhibits good-luxuriant growth, and the colonies appear colorless.
11. Shigella flexneri: The organism displays good-luxuriant growth, and the colonies appear colorless.
12. Enterobacter aerogenes: The organism shows good to excellent growth, and the colonies exhibit a green metallic sheen.

By observing the growth and colony characteristics on Endo Agar, it is possible to differentiate between different organisms based on their ability to ferment lactose and their resulting colony colors. Lactose-fermenting organisms typically produce pink or red colonies, while non-lactose-fermenting organisms form colorless colonies. The metallic sheen seen in some organisms, particularly Escherichia coli, is a distinctive feature.

These interpretations help in the identification and differentiation of various gram-negative bacteria, including members of the coliform group and Salmonella species, on Endo Agar.

Organisms	Growth
Klebsiella aerogenes	Good-luxuriant growth; pink colonies
Enterococcus faecalis	Poor growth; small pink colonies
Escherichia coli	Good-luxuriant growth; pink to rose red with a metallic sheen
Klebsiella pneumonia	Good-luxuriant; pink mucoid colonies
Proteus vulgaris	Good-luxuriant growth; Colourless to pale pink colonies
Pseudomonas aeruginosa	Good-luxuriant growth; Colourless, irregular colonies
Salmonella Typhi	Good-luxuriant growth; Colourless to pale pink
Enterobacter cloacae	Good growth; Pink colonies
Salmonella Typhimurium	Good-luxuriant growth; colorless colonies
Salmonella Enteritidis	Good-luxuriant growth; colorless colonies
Shigella flexneri	Good-luxuriant growth; colorless colonies
Enterobacter aerogenes	Good to excellent growth; Green, metallic sheen

Quality Control

Quality Control of Endo Agar involves several aspects, including the cultural response, appearance, gelling, color and clarity of the prepared medium, reaction, pH, and the growth and cultural characteristics of specific organisms.

• Cultural Response: Endo Agar is observed for its cultural characteristics after an incubation at 35-37°C for 18-24 hours. The cultural response provides valuable information about how the medium supports the growth of different microorganisms.
• Appearance: Endo Agar appears as a light pink to purple homogeneous free-flowing powder. This physical characteristic helps in identifying and distinguishing the agar from other media.
• Gelling: The gelling property of Endo Agar is described as firm and comparable to a 1.5% Agar gel. This information ensures that the agar solidifies properly and provides an appropriate consistency for microbial growth.
• Color and Clarity of Prepared Medium: When prepared, the medium exhibits an orangish-pink color and appears clear to slightly opalescent. Fine precipitates may form in Petri plates. These visual cues are essential for assessing the medium’s quality and determining if any abnormalities or contaminations are present.
• Reaction: The reaction of a 4.15% w/v aqueous solution of Endo Agar at 25°C indicates a pH of 7.5±0.2. The pH level is a crucial parameter to monitor, as it influences the growth and biochemical reactions of microorganisms.
• pH: The acceptable pH range for Endo Agar is 7.30-7.70. This specification ensures that the pH of the medium falls within the optimal range to support the growth of target organisms while inhibiting the growth of undesirable ones.

Organism Inoculum (CFU), Growth Recovery, Colour of Colony, and Cultural Response: Several specific organisms are used for quality control testing of Endo Agar. These organisms include:

• Bacillus subtilis subsp. spizizenni ATCC 6633
• Enterococcus faecalis ATCC 29212
• Escherichia coli ATCC 25922
• Klebsiella pneumoniae ATCC 13883
• Proteus vulgaris ATCC 13315
• Pseudomonas aeruginosa ATCC 27853
• Salmonella Typhi ATCC 6539
• Staphylococcus aureus subsp. aureus ATCC 25923
• Enterobacter cloacae ATCC 13047
• Salmonella Typhimurium ATCC 14028
• Salmonella Enteritidis ATCC 13076
• Shigella flexneri ATCC 12022

For each organism, the inoculum size (CFU), growth recovery, color of colony, and cultural response are evaluated. These parameters help determine if the agar supports the growth of the organisms, if there is inhibition of growth, and the characteristic appearance of the colonies.

Overall, the quality control of Endo Agar ensures that the medium meets specific criteria in terms of physical properties, pH, and its ability to support the growth of target organisms. These tests are crucial for maintaining consistency and reliability in microbiological laboratory procedures.

Uses of Endo Agar

Endo Agar has several important uses in microbiology. Here are the key applications of Endo Agar:

• Differentiation of Lactose-Fermenting and Non-Lactose-Fermenting Organisms: Endo Agar is commonly used for the differentiation of lactose fermenting and non-lactose fermenting intestinal organisms, especially during the confirmation of the presumptive test for coliforms. Lactose fermenting organisms produce pink or red colonies, while non-lactose fermenting organisms form colorless colonies on Endo Agar.
• Microbiological Examination of Water and Wastewater: Endo Agar is recommended by the American Public Health Association (APHA) as an important medium for the microbiological examination of water and wastewater. It helps in the detection and enumeration of coliform bacteria, including fecal coliforms, following the presumptive test for coliforms.
• Analysis of Dairy Products and Foods: Endo Agar is used in the microbiological examination of dairy products and various foods. It is employed for the detection and isolation of coliforms and fecal coliforms, which can indicate the presence of fecal contamination or poor hygiene during processing.
• Confirmation of Drinking Water Quality: Endo Agar plays a role in confirming the detection and enumeration of coliform bacteria in drinking water. It is used following the initial presumptive test for coliforms to provide more accurate results and ensure the safety of drinking water.
• Enumeration of Coliforms in Water: Endo Agar is used for the enumeration of coliforms in water using the membrane filtration method. This method involves filtering a known volume of water through a membrane filter, which is then placed on Endo Agar to allow the growth of coliform bacteria. The colonies that form can be counted to determine the coliform concentration in the water sample.

Endo Agar’s selectivity and differential properties make it a valuable medium for various microbiological analyses, particularly in the assessment of water quality, dairy products, and food safety.

Limitations of Endo Agar

Endo Agar, despite its usefulness, has certain limitations that need to be considered. Here are the limitations of Endo Agar:

• Growth of Other Gram-Negative Bacteria and Yeasts: While Endo Agar is primarily selective for Enterobacteriaceae, it is possible for other gram-negative bacteria and yeasts to grow on this medium. Therefore, additional biochemical tests are required for further confirmation and identification of the specific organisms.
• Sensitivity to Light: Endo Agar should be protected from exposure to light. Light exposure can cause photooxidation, which can decrease the productivity of the medium and affect the interpretation of results. It is important to store and handle the plates appropriately to maintain their integrity.
• Overheating: Overheating of the medium should be avoided as it can destroy its productivity. Care must be taken during the preparation and sterilization process to ensure that the medium is heated appropriately without causing excessive damage or alteration.
• Further Confirmation Required: Although Endo Agar is useful for differentiating lactose-fermenting and non-lactose-fermenting organisms, further biochemical tests are necessary for confirmation. These tests help in identifying specific bacterial species and distinguishing them from other similar organisms.
• Suppression of Sheen: If the inoculum used is too heavy, it may suppress the characteristic metallic sheen produced by lactose-fermenting coliforms. This can affect the interpretation of results, making it important to ensure proper inoculum density for accurate observations.
• Atypical Colony Appearance: Occasionally, non-coliform organisms may produce colonies with a typical metallic sheen, leading to potential misinterpretation. Similarly, coliform organisms may produce colonies that are atypical, including dark red or nucleated colonies without the characteristic sheen. These variations in colony appearance can complicate result interpretation and require additional testing for accurate identification.

Considering these limitations, it is crucial to use Endo Agar in conjunction with other selective and confirmatory tests to ensure accurate identification and differentiation of organisms.

FAQ

References

• http://exodocientifica.com.br/_technical-data/M029.pdf
• https://www.bd.com/resource.aspx?IDX=8974
• https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/343/460/278-endo-agar-104044.pdf
• https://www.neogen.com/categories/microbiology/m-endo-agar/
• https://www.merckmillipore.com/Web-IN-Site/en_US/-/EUR/ViewCountrySelection-RedirectToCountry?CountryCode=IN&CookieAcceptance=accepted&bd=1&ga=null&SourceDomain=Merck-US-Site&TargetURL=https%3A%2F%2Fwww.emdmillipore.com%2FUS%2Fen%2Fproduct%2FENDO-agar%2CMDA_CHEM-104044&ReferrerURL=https%3A%2F%2Fwww.google.com%2F
• http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0479&c=UK&lang=EN
• https://www.carlroth.com/com/en/media-for-coliforms-ecoli/endo-agar-(base)/p/x920.1
• https://tools.thermofisher.com/content/sfs/manuals/IFU1420.pdf
• https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU453942.pdf