- Endo Agar was developed by Endo to differentiate gram-negative bacteria on the basis of lactose fermentation while inhibiting gram-positive bacteria.
- Inhibition of the later was achieved without the use of bile salts as was traditionally used.
- Endo was successful in inhibiting gram-positive bacteria on his medium by the incorporation of sodium sulfite and basic fuchsin.
- The resulting Endo Agar, also known as Fuchsin Sulphite and Infusion Agar, was used to isolate the typhoid bacilli.
- Many modifications of this media have been done over the years.
- Endo Agar is today a selective medium recommended for confirmation of the presumptive test for members of the coliform group from clinical and non-clinical samples.
Composition of Endo Agar
Final pH (at 25°C): 7.5±0.2
Principle of Endo Agar
- The medium contains peptone which provides nitrogen, carbon, vitamins, and minerals required for bacterial growth.
- Sodium sulfite and basic fuchsin make this medium selective by suppressing gram-positive organisms.
- Lactose positive colonies exhibit a red color caused by the aldehyde reaction with the Sodium Sulfite and Basic Fuchsin.
- The development of a metallic sheen occurs when the organism produces aldehydes with the fermentation of Lactose. Lactose non-fermenting bacteria form clear, colorless colonies.
- Coliforms thus produce pink colonies on fermentation of lactose while lactose non-fermenters produce colorless colonies on the medium.
- With Escherichia coli, this reaction is very pronounced as the fuchsin crystallizes, exhibiting a permanent greenish metallic luster (fuchsin luster) to the colonies.
- If growth is colorless, beige or not pink, lactose is not utilized and it is probably a Gram-negative non-coliform.
- If good growth that is pink/red, lactose is utilized and the bacteria is probably a Gram-negative coliform.
- If good pink/red growth that has a bright metallic sheen, lactose is highly utilized and the bacteria is probably a Gram-negative coliform.
- (If poor or no growth, recall it is probably a Gram-positive organism.)
Preparation and Method of Use of Endo Agar
- Suspend 41.5 grams in 1000 ml distilled water.
- Heat to boiling to dissolve the medium completely.
- Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
- 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.
- Streak the specimen as soon as possible after it is received in the laboratory.
- The streak plate is used primarily to isolate pure cultures from specimens containing mixed flora.
- 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.
- 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.
- Incubate plates, protected from light, at 35 ± 2°C for 18 to 24 h.
Result Interpretation on Endo Agar
|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|
Uses of Endo Agar
- The medium is now used for the differentiation of lactose fermenting and non-lactose fermenting intestinal organisms, particularly during confirmation of the presumptive test for coliforms.
- Endo Agar is recommended by APHA as an important medium in the microbiological examination of water and wastewater, dairy products and foods.
- Endo Agar is used to confirming the detection and enumeration of coliform bacteria following presumptive test of drinking water.
- It is also used for the detection and isolation of coliforms and fecal coliforms from milk, dairy products, and food.
- Endo Agar is used for the enumeration of coliforms in water by the membrane filtration method in a laboratory setting.
Limitations of Endo Agar
- Besides Enterobacteriaceae, other gram-negative bacteria and yeasts may also grow.
- Avoid exposure of the medium to light, as it may lead to photooxidation and decrease the productivity of the medium.
- Overheating of the medium must be avoided, as it may destroy the productivity of the medium.
- Further biochemical tests must be carried out for further confirmation.
- If the inoculum is too heavy, the sheen may be suppressed.
- Occasionally, non-coliform organisms may produce typical sheen colonies. Coliform organisms may also occasionally produce atypical colonies, including dark red or nucleated colonies without sheen.