Endo Agar- Composition, Principle, Preparation, Results, Uses

Endo Agar is a microbiological growth medium that was originally developed by Endo for the isolation of Salmonella typhi, the causative agent of typhoid fever. It is now mostly used as a coliform medium to differentiate gram-negative bacteria on the basis of lactose fermentation while inhibiting gram-positive bacteria. Coliforms are a group of bacteria that are commonly found in the intestinal tract of humans and animals, and are used as indicators of fecal contamination and water quality.

Endo Agar is a selective and differential medium for the isolation and differentiation of gram-negative enteric bacteria, especially coliforms. The composition of Endo Agar per liter of distilled water is as follows:

• Peptone: 10 g
• Proteose peptone: 10 g
• Lactose: 5 g
• Dipotassium phosphate: 3.5 g
• Sodium sulfite: 0.5 g
• Basic fuchsin: 0.075 g
• Agar: 12.5 g

The final pH of the medium is 7.5 ± 0.2 at 25°C.

The peptone and proteose peptone provide nitrogen, carbon, vitamins, and minerals required for bacterial growth. Lactose is the fermentable carbohydrate that allows the differentiation of lactose fermenters and non-fermenters. Dipotassium phosphate acts as a buffer to maintain the pH of the medium. Sodium sulfite and basic fuchsin make this medium selective by suppressing gram-positive organisms and imparting a pink-red color to lactose fermenting colonies. Agar is the solidifying agent that provides a firm surface for bacterial growth.

Endo Agar can be modified by adding other ingredients to enhance its performance or specificity. For example, sodium chloride can be added to increase the osmotic pressure and inhibit the growth of non-halophilic bacteria. Sodium lauryl sulfate can be added to inhibit the growth of gram-positive cocci and spore-forming bacilli. Bromocresol purple can be added to replace basic fuchsin as an indicator of lactose fermentation.

Endo Agar should be stored in a cool and dry place, away from light and moisture. It should be used before the expiry date indicated on the label. It should be prepared according to the manufacturer`s instructions or standard laboratory procedures. It should be checked for sterility, pH, color, and clarity before use. It should be discarded if there are any signs of contamination, deterioration, or alteration.

Endo Agar is a selective and differential medium that allows the growth of gram-negative bacteria while inhibiting the growth of gram-positive bacteria. The selective property of the medium is due to the presence of sodium sulfite and basic fuchsin, which act as inhibitors of gram-positive organisms. The differential property of the medium is based on the ability of bacteria to ferment lactose, which is the only carbohydrate source in the medium.

Lactose-fermenting bacteria produce acid from lactose, which lowers the pH of the medium and triggers a chemical reaction between sodium sulfite and basic fuchsin. This reaction results in the formation of red-colored compounds that impart a pink or red color to the colonies of lactose-fermenting bacteria. Some lactose-fermenting bacteria, such as Escherichia coli, produce aldehydes as a by-product of lactose fermentation. These aldehydes react with basic fuchsin and form crystalline precipitates that give a metallic sheen to the colonies.

Lactose-nonfermenting bacteria do not produce acid or aldehydes from lactose, and therefore do not change the color or appearance of the medium. They form clear or colorless colonies that are easily distinguished from the lactose-fermenting ones.

Endo Agar is mainly used for the isolation and differentiation of coliforms, which are gram-negative, lactose-fermenting bacteria that are indicators of fecal contamination. Coliforms produce pink or red colonies with or without a metallic sheen on Endo Agar. Non-coliforms, such as Salmonella and Shigella, produce colorless colonies on Endo Agar.

Endo Agar is a ready-to-use solid medium that can be purchased from commercial suppliers or prepared in the laboratory. To prepare Endo Agar in the laboratory, follow these steps:

1. Suspend 41.5 grams of Endo Agar powder in 1000 ml of distilled water.
2. Heat the mixture to boiling until the powder dissolves completely.
3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Mix well before pouring into sterile Petri plates.

Note: If the solidified culture medium is somewhat too red, then to remove the color add a few drops (max. 1 ml/liter) of a freshly prepared 10% Sodium sulfite solution and boil.

To use Endo Agar for the isolation and differentiation of coliforms and other intestinal bacteria, follow these steps:

1. Streak the specimen as soon as possible after it is received in the laboratory. The streak plate method is used primarily to isolate pure cultures from specimens containing mixed flora.
2. Alternatively, if the material is being cultured directly from a swab, roll the swab over a small area of the surface at the edge; then streak from this inoculated area.
3. A nonselective medium such as Columbia Agar with 5% Sheep Blood must also be inoculated to provide an indication of other organisms present in the specimen.
4. Incubate plates, protected from light, at 35 ± 2°C for 18 to 24 hours.

Endo Agar is a differential medium that allows the distinction of lactose-fermenting and non-lactose-fermenting gram-negative bacteria based on the color and appearance of the colonies. The following are some general guidelines for interpreting the results on Endo Agar:

• Lactose-fermenting bacteria produce pink to red colonies with or without a metallic sheen. The sheen is more pronounced with Escherichia coli, which produces a greenish metallic luster. The pink color is due to the formation of aldehydes from lactose fermentation, which react with sodium sulfite and basic fuchsin in the medium. The sheen is caused by the crystallization of fuchsin on the surface of the colonies.
• Non-lactose-fermenting bacteria produce colorless to beige colonies that are transparent or opaque. These bacteria do not produce aldehydes from lactose fermentation, and thus do not react with the medium components. Some non-lactose-fermenters may produce hydrogen sulfide, which results in blackening of the medium around the colonies.
• Gram-positive bacteria are generally inhibited by the medium and do not grow well or at all. If they do grow, they may produce small, pinpoint colonies that are difficult to see.

The following table summarizes some common examples of bacteria and their typical appearance on Endo Agar:

It is important to note that Endo Agar is not a definitive test for identifying bacteria, and that further biochemical tests are required for confirmation. Also, some exceptions and variations may occur in the colony morphology of different strains or species of bacteria. Therefore, Endo Agar should be used in conjunction with other selective and differential media, such as Eosin Methylene Blue (EMB) Agar or MacConkey Agar, for a more comprehensive analysis of gram-negative intestinal bacteria.

Endo Agar is a selective and differential medium that is widely used for the detection and enumeration of coliform bacteria in water, food, dairy products, and other environmental samples. Coliform bacteria are gram-negative, lactose-fermenting rods that are indicators of fecal contamination and potential pathogens. Endo Agar allows the differentiation of coliforms based on their ability to ferment lactose and produce aldehydes, which react with the sulfite-fuchsin reagent in the medium and produce a characteristic red color with or without a metallic sheen. Non-coliform bacteria usually produce colorless or pale pink colonies on Endo Agar.

Some of the specific uses of Endo Agar are:

• Endo Agar is recommended by the American Public Health Association (APHA) as an important medium in the microbiological examination of water and wastewater, dairy products, and foods. It is used to confirm the presence of coliforms after a presumptive test using a lactose broth or a lauryl tryptose broth. A positive presumptive test is indicated by gas production in the broth, which suggests the presence of lactose-fermenting bacteria. A confirmatory test is then performed by inoculating Endo Agar plates with the broth culture and observing for typical coliform colonies after incubation. A positive confirmatory test is indicated by red colonies with or without a metallic sheen on Endo Agar.
• Endo Agar is also used for the detection and isolation of fecal coliforms from milk, dairy products, and food. Fecal coliforms are a subset of coliforms that are able to grow at 44.5°C and are more specific indicators of fecal contamination than total coliforms. Fecal coliforms include Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter freundii. To isolate fecal coliforms, Endo Agar plates are inoculated with the sample and incubated at 44.5°C for 24 hours. Fecal coliforms produce red colonies with or without a metallic sheen on Endo Agar at this temperature, while other coliforms and non-coliforms do not.
• Endo Agar is used for the enumeration of coliforms in water by the membrane filtration method in a laboratory setting. The membrane filtration method involves passing a known volume of water through a sterile membrane filter that traps the bacteria present in the water. The filter is then placed on an Endo Agar plate and incubated at 35°C for 18 to 24 hours. The number of coliform colonies on the plate is counted and multiplied by the dilution factor to obtain the coliform count per 100 ml of water. The membrane filtration method is more accurate and sensitive than the multiple tube fermentation method for counting coliforms in water.

Endo Agar is thus a useful medium for detecting and enumerating coliform bacteria in various samples and assessing their sanitary quality. It is also helpful for isolating pure cultures of coliforms for further identification and characterization by biochemical tests.

• Endo Agar is not a highly selective medium, as it allows the growth of other gram-negative bacteria and yeasts besides Enterobacteriaceae. Therefore, it is important to use a nonselective medium such as Columbia Agar with 5% Sheep Blood to provide an indication of other organisms present in the specimen.
• Endo Agar is sensitive to light exposure, which may lead to photooxidation and decrease the productivity of the medium. Therefore, it is recommended to protect the plates from light during incubation.
• Endo Agar should not be overheated, as it may destroy the productivity of the medium. The medium should be boiled only until dissolved and sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
• Endo Agar may not produce reliable results if the inoculum is too heavy, as it may suppress the sheen effect. Therefore, it is advisable to streak the specimen as soon as possible after it is received in the laboratory and use a small amount of inoculum.
• Endo Agar may not differentiate coliforms and non-coliforms accurately, as some non-coliform organisms may produce typical sheen colonies and some coliform organisms may produce atypical colonies, including dark red or nucleated colonies without sheen. Therefore, further biochemical tests must be carried out for further confirmation.

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