Hi,

The problem in your culture media for Lactobacilli growth.

I have question for you , Is probiotic powder to have mix culture or single culture?

The MRS with antibiotic is causes Stress for bacterial growth.

The growth needs more incubated time.

Thanks

Dear Alba

May be you have more than one strain of lactobacillus in probiotic powder, Please check it first.

For preparing serial dilution, its better using phosphate buffer instead of physiologic serum

Use vortex for shaking instead of hand shaking

Change you sampler tip each time

Good luck

Hi Alba,

You can streak the tiny colonies on separate plate and perform the 16S rRNA gene sequencing to find out whether this is a contamination or not. Make sure you use different tips for mixing each dilution and it is also important to use separate spreaders for plating. Freshly autoclaved buffer should be used for stock preparation and for making dilutions. I hope this will help.

Hi Alba,

Firstly, check the purity of the culture, in order to eliminate the mixed cultures. For example API 50 CH strips, for carbohydrate metabolism (API web)

Secondly, You can try MRS agar without cycloheximide and bromocresol purple. It is possible that the supplements (cycloheximide and bromocresol purple) in MRS, have an inhibitory effect on Lactobacillus growth, so the inhibition decreases with increasing dilution.

Generally, there is no need dilution higher than 10-10.

Hi Gordana,

I need to confirm the probiotics colonies. Three consecutive decimal dilutions were inoculated in MRS agar and incubated at 30 ºC under anaerobiosis (5% CO2) for 4 days. After the period of incubation I did the count of the colonies but I dont know this colonies . Which colonies should I count as lactic acid bacteria? Please kindly share your experience with these colonies of possible lactic acid bacteria or yeast ?

Best regards ..

Hi,

Firstly, You should first check a composition of the probiotic product whether mono culture-single strain, or strains (mixed cultures). For exemple, if probiotic product contain only Lactobacillus sp. you can do preliminary identification at the genus level (Gram staining, catalasa test and anaerobic growth). Colony of bacteria belong to Lactobacillus on MRS agar are white, round, small, with the smell of lactic acid products (yogurt). Gram positive, non sporogenic and catalase negative. In addition, you can check the microbiological purity of the product by spreading on TSA (for bacteria) and SDA (for yeast and fungi). The inoculated plates should be incubated in aerobic conditions.

Dear,

Firstly, thank you for your reply . My probiotic product is natural fermented pickles. Thanks for your help. I sucked pickled samples from mixed biota into MRS Agar medium, incubated at 30 ºC under anaerobic jar for 4 days and observed different colonies and I cannot count lactic acid bacteria. You are right, colony of bacteria belong to Lactobacillus on MRS agar are white, round, small. Can lactobacilli make different colonies on MRS Agar medium? Or are these colonies yeast?

Best regards ..

Dear,

Yeast colonies, if you mean Saccharomyces sp. are clearly different. Take the microbiological loop and pick up individual colonies, and transfer to a microscopic slide, and cover with 3% hydrogen peroxide. If there is no bubble in the air, it is most likely Lactobacillus, while Saccharomyces is bubble. Be sure to make a microscopic preparation from the same colonies and stain with Gram. Yeasts will be large, oval, with budding, while is Lactobacillus Gram positive rod-shaped.

Best regards,

Gordana

Thank you very much for all your answers. I will apply the experiment you mentioned as soon as possible.

Best regards..