Mannitol Salt Agar: Principle, Uses, and Results

Mannitol salt agar (MSA) is a selective, differential, and indicator medium used to isolate and identify Staphylococcus aureus from the clinical specimen. As its name suggests, mannitol salt agar (MSA) contains 1% mannitol (sugar), 7.5% salt, and agar as a solidifying agent. Members of the genus Staphylococcus can tolerate high salt concentration (7.5%) and grow on mannitol salt agar.


Mannitol salt agar (MSA) is a selective and differential medium.

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

Selective medium

Incorporating 7.5% sodium chloride in the medium helps select only those bacteria that can tolerate high salt concentrations. Staphylococci species can tolerate this salt concentration, but other pathogenic bacteria may not. This concentration inhibits the growth of most other gram-positive and gram-negative bacteria. Thus MSA selectively isolates Staphylococcus spp i.e. selective media for Staphylococcus spp.

Differential Medium

Pathogenic staphylococci, i.e. Staphylococcus aureus is able to ferment mannitol, but coagulase-negative staphylococci (CONS) are not.  So, if that particular specimen contains S. aureus, it ferments mannitol and changes the pH of the medium to acidic. As MSA contains phenol red as a pH indicator, at pH levels below 6.9, the medium is a yellow color. But if CONS grow, they cant ferment mannitol, so the color of the media around the bacterial colony does not change to yellow; it appears pink. So, MSA is also a differential medium.

Remember that in the neutral pH (6.9 to 8.4) the color of phenol red is red; while above pH 8.4, the color of phenol red is pink. Other commonly used media that contain Phenol red as pH indicator are; TSI Agar, urea base agar, and XLD agar.

Composition of Mannitol Salt Agar

  1. Enzymatic digest of casein (source of nitrogen, vitamin, and carbon)
  2. Enzymatic digest of animal tissue (source of nitrogen, vitamin, and carbon)
  3. Beef extract (source of nitrogen, vitamin, and carbon)
  4. D-Mannitol: Only carbohydrate source present in the medium
  5. Sodium chloride 
  6. Phenol red (pH indicator)
  7. Agar

Final pH: 7.4 ± 0.2 at 25 oC

Preparation of Mannitol Salt Agar

You can purchase prepared mannitol salt agar from commercial suppliers, get the powder, and prepare the media in your laboratory. Mannitol salt agar is best prepared from ready-to-use dehydrated powder, available from most suppliers of culture media. The medium is usually used at 11.1 g in every 100 ml of distilled water (concentration may vary depending on the manufacturer).

  1. Prepare the medium as instructed by the manufacturer. Sterilize by autoclaving at 121oC for 15 minutes.
  2. When the medium has cooled to 50-55oC, mix well, and dispense it aseptically in sterile Petri dishes. Date the medium and give it a batch number.
  3. Store the plates at 2-8oC in plastic bags to prevent moisture loss.
Growth of S. aureus and CONS on MSA
Growth of S. aureus and CONS on MSA (Image source: Pearson Education)

Shelf life: Several weeks, providing there is no change in the appearance of the medium to suggest contamination, deterioration, or alteration of pH.

The medium’s pH should be within the range of pH 7.3 to 7.7 at room temperature.

Staphylococcus saprophyticus (coagulse-negative Staphylococci) may ferment mannitol, producing yellow halo around colonies in MSA thus resembling S. aureus.

Colony Characteristics in Mannitol Salt Agar

  1. Escherichia coli: Does not grow
  2. Staphylococcus epidermidis: Colorless to pink colonies
  3. Staphylococcus aureus: Yellow colonies; may have a yellow halo around colonies.

Note: Do not perform coagulase test from the colonies isolated from mannitol salt agar.


  1. When grown on mannitol salt agar, some Micrococcus (Micrococcus is a normal flora of human skin, mucosa, and oropharynx), such as M. luteus (yellow) can produce yellow colonies. M. roseus (red) produces pink colonies on MSA. Find out the difference between Micrococcus and Staphylococcus here
  2. Enterococcus faecalis and Enterococcus faecium (the most common enterococcal species that have been isolated from human infections) are salt-tolerant bacteria. They can ferment mannitol and produce lactic acid, producing yellow-colored colonies on MSA. Catalase test can help to differentiate between Enterococcus (-ve) and Staphylococcus (+ve).

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.

38 thoughts on “Mannitol Salt Agar: Principle, Uses, and Results

  1. so if i am trying to detect MRSA on msa plate and i have colonies with yellow surroundings what should i do next before i assume that they are s.aureus and culture them on mueller hinton agar with oxacillin . thanx for this amazing website

    1. Dear Mohammed,
      As you might have already know that all yellow colonies in Mannitol Salt Agar is not given only by Staphylococcus aureus but also by other salt tolerating, Mannitol fermenting bacteria. So some simple tests like Gram staining (to see typical gram positive cocci in cluster) and coagulase test are always helpful to confirm that the isolate is S. aureus. Read troubleshooting above again.

  2. how about klebisiella?
    We’ve tried to grow an unkown in MSA, does klebsiella grow on MSA?
    The result in MAC is mucoid , so we’re guessing klebsiella
    We have contaminants growing on some portions of the MSA, bigger colonies doesn’t exhibit fermentation, smaller colonies did.

    1. Dear merz cov
      We use MSA for special purpose only, to differentiate S. aureus from other CONS and/or while screening presence of S. aureus in anterior nares of health care professionals. So far, we have not isolated Klebseilla from MSA. MSA is supposed to inhibit gram negative bacilli. To identify the isolate, please follow the basics. Perform Gram staining. Culture in Blood Agar and MacConeky Agar. Choose further tests on the basis of Gram Staining and Culture reports to identify the isolate.

  3. Dear Prof Tankeshwar,
    Thank you so much for this article, very well written.
    If an MSA plate has mostly yellow colonies (4) and one pink colony can this mean that there are two types of bacteria growing? For example Staph aureus from the infection site and maybe Strep epidermidis that was normally part of the patient’s microflora and is harmless?

    1. Marianna, thank you for the comment. It may be Staphylococcus epidermidis, not Strep epidermis. Please perform Catalse Test, Coagulase Test, Gram Staining to identify the isolate. Staphylococcus epidermidis is the normal skin flora and when the sample is swab (not pus), skin contamination is likely to occur while taking the swab.

    1. Utibeabasi, thank you for the comment. Please go through the blog post, it is now included there.

      1. What two factors does the Mannitol Salt test indicate about a microbe if the test is positive for both?

        1. Hello Erika
          Sorry failed to understand your query. Can you rephrase and post the query again?

      1. Dear Casey and Anum,
        yes, as far i know clinically relevant species of Staphylococcus grows well in MSA. In clinical sample our concern is mainly with 3 species, S. aureus and CONS (S. epidermidis & S. saprophyticus), all of them grow well. If you want to know about any other particular species, please let me know, i will try to help whatever i can.

  4. Thank you for the information. What’s the principle of yellow colony and medium made by coagulase-positive staph? It’s due to mannitol fermentation as far as I understood. But some say if staph is coagulase positive, it makes yellow colony and surrounding yellow medium. Does coagulase make staph ferment mannitol? no.. Is there any relationship between these? or just is it coincidence of features of S.aureus who ferment manntol and is coagulase positive as well?

  5. I have Staphylococcus isolates that grew very well and turned the color yellow on MSA plates, but the VITEK test showed that they were S. saprophyticus !! how can this happen??
    in this case how can I differentiate S. aurues from other Staph. species on MSA ?? .

    1. Yes Micrococcus can grow on this media. It contains a high concentration (about 7.5%-10%) of salt (NaCl), making it selective for Gram-positive bacteria (Staphylococcus and Micrococcaceae).

  6. Can a Micrococcus ferment mannitol? and why is there gas in a durham tube but no color change? Can this be the result of Micrococcus? lastly can Micrococcus ferment any sugars at all?

  7. thank you for this article, it is very informative for me and my colleagues. I want to ask if Gram negative bacilli can grow on MSA ? we had cultured an unknown wound swab on MSA the colonies are very faint. when we performed Gram stain it was Gram negative bacilli. it really made me confused . please answer me.
    thank you

  8. Hello sir
    It’s very informative post.thank you so much for it.I have a question. I’m a research student and my work is isolation of staphylococcus from human skin, for this purpose I use MSA to culture staphylococcus species but I m very confused to identifying then. I mostly find yellow, mucoid, sticky, shining, colonies and after gram staining it shows small team positive cocci.some in culstures and some diplococci.kindly guide me what should I consider them ? What test should I perform to differentiate them, because after reading this artical I’m confused these are sthaplocooci or micrococci?
    Plz sir reply me asap.

  9. This artical is very informative, but I do have a question. What is the purpose of the Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue? I need to know this for school. 🙂

  10. hello sir
    thank you for the article. my question is what is the difference between S saprophyticus and S aureus colonies on MSA?

  11. Thanks a lot …
    I need to ask u on different subject I’m trying to understand …It’s about MIC & how can I change for example 16 mcg which is sensitive on plate to a minimum dose of that drug in human body, cause I ask many physicians & even pharmacists about their benefit of MIC to decrease a dose, Unfortunately no body knows … Please can u help me?

    1. SKK please read this article. It explains this concept well: (The information you have provided is not sufficient to give any recommendations).

  12. Hello. I am hoping you can answer a question for me. I have grown an unknown bacteria on a mannitol salt agar plate and have both white and yellow colonies, suggesting I have S. epi and S. aureus. However, all of the agar is still pinkish red in color. I know that some species of S. aureus will not ferment mannitol. The question I have is, does M. luteus form yellow colonies without the agar color change? And, if so, would the colonies be large? I am used to very small colonies of Micrococcus. Also, if it is not M. luteus or S. aureus (in these yellow colonies) what could it be?

    Thank you in advance.

    1. Daniella, thanks for your query. We should not be confused with pigment production and the change in the color of the medium because of the changes in pH. In the case of Micrococcus, it’s the pigment production, not the pH changes in the medium. So, the pigment will appear even if you sub-culture the isolate in other commonly used culture media such as Chocolate Agar, Blood Agar etc. So the yellow-colored colonies in MSA may be that of Micrococcus. If it’s Micrococcus, you wont see typical gram like clusters in Gram staining and the size of coccus is slightly bigger than those of Staphylococcus. Did you perform the coagulase test from that isolate to confirm/rule out it as Staphylococcus aureus?

  13. I apologize for asking this separately, but I just thought about it.
    Would exposure to an antibiotic cause S. aureus to grow yellow colonies without mannitol fermentation in MSA?

    1. Pigment production and color change due to alternation in media pH are two different features of an isolate. Staphylococcus aureus produces golden pigment (find more about its basis here) but I do not think, just an exposure to a particular antibiotics in our laboratory during culture-sensitivity testing can change the pigment-producing capabilities (If you come across any literature/research-article that is sharing about such phenomenon, please let me know).

  14. Hi, i want to ask, can acid sample change the color in media without any colonies grow in MSA

    1. This is possible. The formation of yellow color in MSA is due to the fermentation of Mannitol and formation of acid. Color of the media will be changed if acid is formed or added. The intensity of the color depends on the amount/concentration of the acid formed/added.

  15. Thank you Im very interested in Microbiology I hope Im going to gain many things from you

    1. Can you please elaborate about this? If you are asking about ‘antimicrobial susceptibility testing’, it’s already covered. Please check and let me know.

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