Endo created Endo Agar to distinguish gram-negative bacteria based on lactose fermentation and inhibit gram-positive bacteria. The latter were not inhibited by bile salts, as was traditional. Endo was able to inhibit gram-positive bacteria using sodium sulfite, basic fuchsin.
Endo Agar (also known as Fuchsin Sulphite or Infusion Agar) was used to isolate the typhoid bacteria. Over the years, many modifications have been made to this media. Endo Agar, a select medium that can confirm the presumptive test of members of the coliform family from clinical and nonclinical samples, is recommended today.
Principle of Endo Agar
Peptone is a component of the medium that provides nutrients, vitamins, and minerals for bacterial growth. This medium is selective because it suppresses gram-positive organisms. The aldehyde reaction of Basic Fuchsin and Sodium Sulfite with Lactose-positive colonies results in a reddish color. When the organism ferments Lactose, it produces aldehydes that give off a metallic sheen. Clear, colorless colonies of Lactose-non-fermenting bacteria are formed.
Coliforms produce pink colonies when lactose is fermented, while non-fermenters of lactose produce colorless colonies. This reaction occurs with Escherichia coli. The fuchsin crystallizes and gives the colonies a permanent greenish metallic luster. Lactose should not be used if the growth is pink or colorless.
Lactose can be used to stimulate pink/red growth. This bacteria is likely a Gram-negative. Lactose can be used to stimulate good growth in pink/red with a bright metallic sheen. This bacteria is likely a Gram-negative. If it has poor growth or none, it may be a Gram-positive organism.
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 and Method of Use of Endo Agar
- Take 41.5 grams and mix it with 1000 ml of distilled water.
- To dissolve the medium completely, heat to boiling
- For 15 minutes, sterilize by using an autoclave at 15 lbs pressure (121 degC).
- Before pouring into sterilized Petri dishes, mix well.
- After receiving the specimen in the laboratory, immediately begin to treat it.
- The streak plate is used to separate pure cultures from mixed flora specimens.
- Alternativly, culture the material directly with a swab. Roll the swab across a small area at the edge of the surface and then streak from that inoculated area.
- Columbia Agar containing 5% Sheep blood must be used as a non-selective medium to inoculate the specimen. This will provide information about other organisms.
- For 18-24 hours, incubate plates at 35 +-2degC.
Note: If the solidified medium is too red, add a few drops of Sodium sulfite solution (max. 1 ml/liter of freshly prepared 10% Sodium sulfite solutions and boil.
Result Interpretation 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 |
Uses of Endo Agar
- This medium is used to differentiate lactose-fermenting from non-lactose fermentation intestinal organisms, especially during confirmation of the presumptive coliform test.
- APHA recommends Endo Agar as a key medium for microbiological analysis of water, wastewater, dairy products, and food.
- Endo Agar can be used to confirm the detection and enumeration coliform bacteria after presumptive testing of drinking water.
- It can also be used to detect and isolate coliforms, fecalcoliforms, and other coliforms in milk, dairy products, or food.
- Endo Agar can be used to count coliforms in water using the membrane filter method in a laboratory.
Limitations of Endo Agar
- Other than Enterobacteriaceae and gram-negative bacteria, yeasts and other gram-negative bacteria may also be grown.
- Avoid exposing the medium to sunlight as it can cause photooxidation and reduce the medium’s productivity.
- Avoid overheating the medium as it can cause irreparable damage to its productivity.
- Additional biochemical tests are required to confirm the results.
- The sheen can be reduced if the inoculum becomes too thick.
- Non-coliform organisms can sometimes produce typical sheen colonies. Sometimes, coliform organisms can produce unusual colonies. These include dark red colonies or nucleated colonies that lack sheen.
