Iam working with lactic acid bacteria from yogur. For the growth of the lactic bacteria of the yogurt I use medium MRS oxoid agar but never grows any microorganism. I sow in bulk and incubate at 37ºC for 3 days and any microorganism grows. I also use double layer of agar.
Could someone give me some advice to get the growth of lactic bacteria ?.
Hola Raul,
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Article PRODUCCIÓN BIOTECNOLÓGICA DE ACIDO LÁCTICO: ESTADO DEL ARTE ...
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Dear Raul
LAB need anaerobic conditions so please incubate it in anaerobic jars or incubator.
by using MRS, the growth of LAB is 99.9% especially from yogurt as it contain high numbers of LAB mixed cells of two main strains dominant in yogurt.
please do not hesitate to communicate with me for any help
Regards
Mr. Rahul,
As per my experience u can simply streak diluted sample on Sterile MRS agar plate of pH 6.5 and incubate it at 30 to 35oC for 48 hrs aerobically. Because LAB forms an immersed growth in agar as submerged colony if it is anaerobic. Or use a closed jar with candle, that will create microaerophilic conditions. U may get creamy white, pale, white colored colonies of LAB. Confirm it by catalase test. It is negative for LAB. U may get similar colonies of yeasts also, so better to check it microscopically. Go for KOH test for Gram nature determination.
Dear Raul
By using two layer method you provide anaerobic condition, and MRS Agar is the best medium for Lactobacillus, therefore you must work on your method and incubator degree (from 27 to 37 c).Also Sometimes the second layer medium that ready for cover the first layer is hot and it can kill all bacteria on the first layer.
Please do not hesitate to contact me for more help
Sincerely
culture lactic acid bacteria shoud be at anaerobic condition, not only the MRS medium.
To isolate lactic acid bacteria from yogurt ... you must cultured in MRS broth first and incubate at 28- 30C for 3 days under anaerobic condition (using anaerobic jar) , then cultured in MRS agar and incubate anaerobicaly for 3 days at 28-30C.
MRS is a nice media for LAB. So good choice. My points regarding your problems:
1. If the yogurt sample is too old it will turn too acidic and the LAB flora might deteriorate
2. If the samples are collected form market, check whether or they are free of antibiotics (I now some use it to prevent post acidification)
3. Some genera might take up to a week to show up so keep the temperature to 30oC and wait patiently.
4. If your samples are full of yogurt curds (solid types) you should increase the vortex time and make sure you can pipette evenly.
5. If you employed serial dilution and took the higher figures, due to one of the reasons I mentioned above the count might be too low for you to count. So try to include lower dilutions or increase the initial volume (1:10= 5ml:50ml, 10ml:100ml)
6. Some fermented products are dominated by a specific group of LAB demanding you to include extra additives like amino acids in MRS, if you have priori knowledge of the product then you can identify what to include in it.
For the isolation of lactic acid bacterial strains you must added n gramme of your sample to 5ml of (MRS) broth and incubated at 30°C under anaerobic condition (For this you can added 2-3ml of parafin oil). After 72h of incubation, each mixture must be diluted in 9ml of sterile peptone water (0.2% w/v) up to the dilution 10-10. 100µl of each dilution were spread onto MRS agar and incubated at 30°C for 72 hours.
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