Ttributyrin agar has been dissolved 20 g in 1 litre distilled water and sterilized by autoclaving at 121°C for 15 minutes. It has been cooled to 80°C and added 10 g neutral tributyrin. But we couldn't see a clear zone, even by Rhodococcus spp. and Pseudomonas spp.
Unlike in the case of gelatinase and lipase production detection using plate assays, lipase production is bit difficult to differentiate as the clear zones around positive colonies are not very distinct as in the case of gelatinase or amylase. A change in the opacity of the medium around the colony is considered positive for lipase production. Since the tributyrin agar as such is a kind of translucent medium it needs a keen observation to find out the difference. If you take a close look you can see a light grainy kind of appearance around the colonies that are producing lipase. If you want me to have a look please send the image of the plate to [email protected].
In spite of using tributyrene agar, you can also use nutrient agar medium and after autoclaving add sterile 1% tributyrene oil. You will get better contrast after lipase production by the studied isolates.
Suspend 23 grams (Tributyrin Agar Base) in 990 ml distilled water. Add 10 ml of Tributyrin (FD081, himedia). Mix and heat to boiling to dissolve the medium
completely. Sterilize by autoclaving at 15lbs pressure (121°C) for 15 minutes. Shake the flask and individual plate so as to maintain uniform turbidity.
Note : For proper lipase activity, it is recommended to use glass plates instead of disposable plates. Hence USE ONLY GLASS PLATES. DO NOT USE PLASTIC PLATES.
OR ELSE you can use another method.
Suspend 32.15 grams (Spirit Blue Agar) in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving
at 15 lbs pressure (121°C) for 15 minutes. Cool to 50°C and add 30 ml lipase substrate Tributyrin (FD081, himedia) slowly while agitating to obtain an
even distribution.
USE ONLY GLASS PLATES. DO NOT USE PLASTIC PLATES.
For detail composition and principle see the PDFs. All d Best.
-AKM
As mentioned above, Spirit Blue agar is an excellent medium to use.
The method mentioned by Abhas KUMAR Maharana is correct. I have followed the same and i got the results. While pouring the plates you should make sure that you mixing the tributyrin properly.
You can also try rhodamine B agar which gives you orange fluorescenece under UV light around the colonies producing lipase. I am attaching a preparation of rhodamine B agar.
Thank you so much, for your useful suggestions and answers. Thank you for everyone :)
Yes you are right Viraj Yele. Now days researchers try rhodamine B agar plate method also. I got good results from that. ALL D BEST...
We tried phenol red plates and got surprisingly good results. Zones were visible.
What will be the lipase substrate as mentioned in the protocol M445.pdf?
I am currently experiencing the same problem. I used the same method as Himani and i can't see any clear zones around the bacterial colonies. I will try the suggested methods and i will also report back. Thanks for the information shared.
Any alternative of tributyrin substrate for lipase activiity?
I have previously used Tributyrin and I got a very good result. You can search for this article: Griebeler et al., 2011. Isolation and Screening of Lipase-Producing Fungi with Hydrolytic Activity. DOI 10.1007/s11947-008-0176-5.
Screening of Lipolytic Microorganisms Using Tributyrin in the article is as follows: The screening of microorganisms that produce lipases with hydrolytic activity was carried out in Petri dishes using a medium constituted by (% m/v in distilled water): peptone 0.5%; yeast extract 0.3%; tributyrin 0.1%; agar 2%, pH 6.0. BEST OF LUCK.
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