I'm not familiar with CaCO3 inclusion method.

I wonder what would be pH of the medium after solidification.

In my experience, higher pH than 7.5 would not favor lactic acid bacteria growth.

However, I suggest some other methods I've frequently used.

First of all, colonies in the picture seems to be quite a lot. There might be a chance of picking more than two colonies with such agar plates.

It would be better spread in serial dilutions from 10^-1 to 10^-6 range.

If the sample is a fermented food, ~10^8 CFU/mL is expected depending on the state of fermentation. Therefore, you would get two digit to three digit colonies (30-300 colonies in classical saying) in 10^-5 or 10^-6 diluted and spread samples.

When you get clearly separated colonies, pick a little portion of a colony with clean tips (10 uL or 100 uL tips work fine), then mix well into a droplet of 3% H2O2 (3~10% pick one, works fine). Give it 1~3 minutes to react, then see if oxygen bubbles are formed.

As most lactic acid bacteria are catalase negative, colonies with no bubble are likely to be a lactic acid bacteria.

For pH indication method, addition of phenol red to MRS media might work, but due to yellow color of MRS media and phenol red at low pH, it will be hard to identify. Bromo Cresol Blue (BCP) might be another option which is purple at pH 7ish and turns yellow at low pH. PCA agar with BCP is another option but will not be favorable for lactic acid bacteria.

Adding BCP to MRS may work fine. Please see the following link for how much BCP to add.

http://www.himedialabs.com/TD/M1351.pdf

Again, if too many colonies cover the agar plate, most of media will turn yellow, so serial dilution will help a lot.

I hope this answer help you out.

Best regards,

Bo Ram Beck

It is expected to have a bigger size of colonies after 48 hours of incubation. Better dilute your sample one more time and try to pick colonies after that. You may wait for a few more hours to have larger sizes of colonies that will help you to clearly understand the morphological characteristics of lactic colonies.

Bo Ram Beck Thank you so much for the reply, your suggestions will really help me a lot. But I have a confusion on how much % of H2O2 should be used for catalase test as some literature have suggested 15% H2O2 for identifying anaerobes?

Mohammad Afzal Hossain Thank you so much for your suggestions.

Soibam Ngasotter 3 to 30% range works fine. You can use 10% for your convenience. I tried 3, 5, 10, and 30% and every concentration worked well.

Concentration will affect time to produce oxygen bubble, but more than 3% will have enough reactant (H2O2) for bacterial catalase. Thus, it is more like about saving reagents.

Iam suggested to make serial dilution of your sample and adjusted PH value, will be help you to see clear zone

The above suggestions are absolutely fine. But try one robust approach also. Use a 10-2 or 10-3 dilution sample.

Do the quadrant streaking from these dilutions and incubate.

observe the clear zone after incubation and pick all the colonies from the clear zone and by repetitive quadrant streaking purify it.

As I said earlier, it's a robust approach, but it will work for sure. For your reference, I am providing you one of my plates

Bo Ram Beck Nihan Beyatli Thank you for your response and suggestions.

Kumar Pranaw Thank you for sharing your plate as it gave me more idea of how the colonies should look like.

While adding Caco3 to obtain clear background on MRS plate to easily differentiate Lactic acid bacteria from other floras.

Lactic acid bacteria produces lactic acid as an end product of fermentation/growth in any suitable medium. When you add CaCO3 in media, lactate react with it and form Ca-lactate and so, you get clear area/zone surrounded the colonies of lactic acid bacteria.

Ref below paper and differentiate LAB from other bacteria in MRS agar supplemented calcium carbonate

0.5% calcium carbonate is enafe for differentiating LAB to other bacterias.

In the above-attached plate, I have used 1% CaCO3

Dear sir,

If using more percentage of Caco3, difficult to identify of LABs. 0.5 or 1 doesnot shows any deviations of LAB. I also checked more than 5% and observed high lactic acid producing LAB. This only for screening for plate of LAB. I checked LAB cultivated in MRS broth at Caco3 for production of my metabolites EPS, GABA and selenium. Caco3 interfer spectroscopic results.

serial dilution with 10-4 or 10-5 will be helpful in isolation of colonies. Also 1%Caco3 for MRS solid medium is useful in determination of enzyme production and the activity is determined by estimation of clear zone.

Jabastin Jayamanohar Salha G Desouky Thank you very much for your valuable suggestions.