Culture Media: PH Indicators, Color of Colonies

Colony color or pigmentation is mostly exploited characteristics of bacteria which aids in the differentiation and identification of the isolates. Except when there is a growth of pigmented bacteria. The characteristics pigmentation observed in the culture media is because of the changes in the pH of the medium (i.e. development of the characteristics color based on the use of pH indicator).

pH indicators give characteristics color in different pH e.g., phenol red gives yellow color in acidic pH. So whenever fermenters grow in sugar-containing media, they produce acid and give yellow-colored colonies. For e.g. mannitol fermenter S. aureus in mannitol salt agar, sugar fermenter in XLD agar, etc. In this post, we are grouping some of the bacteriological media on the basis of pH indicator used.

pH indicators and their range pH indicators with their color range

pH indicators used in different culture media

Bromothymol Blue pH range: 6.0 (yellow)- 8.0 (blue) Phenol Red

pH range: 6.8 (yellow)- 8.4 (red)

Neutral Red

pH range: 6.8 (red)- 8 (yellow)

Cystine Lactose Electrolyte Deficient Agar (CLED) Mannitol Salt Agar MacConkey Agar
Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar Triple Sugar Iron (TSI) Agar Deoxycholate Citrate Agar (DCA)
OF Medium Urease Test Medium Salmonella-Shigella Agar
Simmons Citrate Agar Xylose Lysine Deoxycholate Agar (XLD)  

pH indicator: Bromothymol blue

It is a color indicator that turns yellow at acidic pH. At a neutral pH, bromthymol blue is green. At pH 7.5 or above, bromthymol blue turns royal blue.

Lactose fermenting (Yellow colonies) and Lactose Non fermenting colonies in CLED
Lactose fermenting (Yellow colonies) and Lactose Non-fermenting colonies in CLED
  1. Cystine Lactose Electrolyte Deficient Agar (CLED): In  CLED media, lactose fermenting colonies of Escherichia coli give pale yellow colonies whereas non-lactose fermenting colonies of Proteus, Salmonella, etc gives blue colored colony.
  2. Simmons Citrate Agar: Simmons citrate agar tests the ability of organisms to utilize citrate as a sole carbon source and ammonium dihydrogen phosphate as the sole source of nitrogen. Organisms that can utilize citrate as their sole carbon source also utilize ammonium dihydrogen phosphate creating an alkaline environment in the medium, turning the medium blue.
  3. Hugh and Leifsons Medium (OF Medium): OF medium (oxidative-fermentative test) is used to determine if gram-negative bacteria metabolize carbohydrates oxidatively (producing weak acid), by fermentation (producing mixed acid), or are nonsaccharolytic and therefore have no ability to use the carbohydrate in the media (no acid production). The high concentration of acid produced during fermentation will turn the bromthymol blue indicator from green to yellow in the presence or absence of oxygen.
  4. Thiosulfate Citrate bile salts sucrose (TCBS) agar is used for the selective isolation of Vibrio spp. TCBS has a very high pH (8.5 to 9.5) to suppress other intestinal flora. pH indicator bromothymol blue is used in this media, so if the organism utilizes the sucrose, it lowers the pH of the media and yellow-colored colonies are seen in TCBS.

pH indicator: Neutral Red

Red at pH 6.8 and below, yellow at pH 8 and above.

Mixed growth of mucoid Lactose fermenting colonies and NLF colonies in MacConkey Agar
Mixed growth of mucoid Lactose fermenting colonies and NLF colonies in MacConkey Agar
  1. MacConkey Agar: It is a selective and differential medium. The pH indicator helps to differentiate between lactose fermenting and lactose non-fermenter. E coli and other lactose fermenting bacteria give pink-colored colonies in MacConkey agar. Whereas non-lactose fermenter gram-negative bacilli produce pale yellow colonies.
  2. Deoxycholate Citrate Agar (DCA): Lactose non-fermenter produces colorless colonies. Coliform bacteria if present form pink colonies.
  3. Salmonella-Shigella Agar: It is a selective media used to isolate Salmonella and Shigella. Lactose fermenter produces red-pigmented colonies. Whereas non-lactose fermenter (e.g., Salmonella) grows as translucent colonies (colorless) with or without black centers (neutral red does not have any role in black color formation, it’s because of action of sodium thiosulphate and ferric citrate which are also other constituents of SS Agar).

pH indicator: Methylene Blue and Eosin dyes

  1. Eosin Methylene Blue (EMB) Agar: EMB agar is useful for the isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli.
    • Coliform bacteria: Purplish black colonies
    • Noncoliform bacteria: Colorless colonies

Malachite green as pH indicator

  1. Lowenstein Jensen Medium: Lowenstein Jensen medium is used to isolate Mycobacterium tuberculosis.

pH indicator: Phenol red

It is yellow at pH 6.8 and red at pH 8.4 and above.

Yellow colonies of S. aureus in Mannitol Salt Agar (MSA). Image source: ASM
Yellow colonies of S. aureus in Mannitol Salt Agar (MSA).
Image source: ASM
  1. Mannitol Salt Agar (MSA): Staphylococcus aureus grows in mannitol salt agar, ferments mannitol, and produces (acid) yellow colonies with yellow zones. Whereas most coagulase-negative staphylococci (CONS) and micrococci do not ferment mannitol and grow as small red colonies surrounded by red or purple zones.
  2. Triple Sugar Iron Agar (TSI) Test: TSI contains three sugar (glucose, sucrose, and lactose) along with an iron source. It helps in the identification of members of the family Enterobacteriaceae on the basis of their fermentative capability, gas production, and H2S gas production.
    1. Lactose fermenters: yellow/yellow
    2. Non-lactose fermenter: red/yellow
    3. Non-fermenter: red/red
  3. Urea Agar base: Urease-producing organisms give pink-red color to the media as they utilize urea with the formation of ammonia the pH of the media changes to an alkaline condition.
  4. Xylose Lysine Deoxycholate (XLD) Agar: It is useful for the isolation and differentiation of Salmonella and Shigella spp which appear as pink-colored colonies (as they do not ferment carbohydrate). Whereas the other nonpathogenic gram-negative enteric bacilli which produce yellow colonies (as they ferment one or more of the sugars present in the media).


  1. Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.
  2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

6 thoughts on “Culture Media: PH Indicators, Color of Colonies

  1. Fastidious bacteria growing on NA (Nutrient agar) do not need pH indicator. It is their characteristic off course there is change in media pH. Morphological colony character and pigment is one of the criteria for basic identification. We should not consider polymorphic character at this juncture. Any way this is basic characteristic we may go for further identification after screwing for the objective for which isolation is under taken.

  2. I have isolated a colony from cow mouth flora but I can’t understand which organism it is? One gives yellow pale orange gram positive motile, second gives white sticky gram positive motile, and third is gram negative non motile pale white colony. These three organism isolated,
    how can I determine it ?

  3. thank you for the information, may you please help me give the preliminary identification of the colonies that i obtained from the cultures on nutrient agar from the different food substrates(rice, samosa, meat soup bean soup and wheat bread). their colours were; cream, pure white, yellow and golden yellow. waiting forward for your response.

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