Microbial Limit Test (MLT)
What is MLT?
Why to do MLT?
How to do?
MLT- microbial limit test.
This procedure is used to analyse the pharmaceutical samples for microbial limit . In this test we used to check the microbial availability like E.coli , Salmonella ,Pseudomonas , yeasts and molds in sample. This test is very necessary in pharmaceutical industries. So now lets see how to do MLT-
MLT for 1) water to be used in preparation of product
2) Final product or raw material –To check the microbial load in sample/product and also presence or absence of pathogenic microbes. So first , lets see which medias are required –
- coli– MacConkeys broth (MB) , MacConkeys agar (MA)
- Pseudomonas – Citrimide agar (CA)
- S . aureus –Manitol salt agar (MSA)
- Salmonella – RVSB ( Rapppaport Vassiliadis Salmonella enrichment broth ) , XLDA (Xylose lysine deoxycholate)
The selective medias ae used for isolation of Pathogenic Organisms.
- Soyabean casein digest agar (SCDA) , Sabourods dextrose agar (SDA) R-2A are used to colony count of bacteria and yeast and mold.
Total yeast and mold (TYMC) and total aerobic microbial count (TAMC) is equal to the TVC i.e total viable count)
TYMC + TAMC = TVC
There is limit for TAMC and TYMC
TAMC limit – 1000 cfu/ ml
TYMC limit – 100 cfu / ml
Pathogenic Organisms should be absent.
- MLT of water used for product – water which used for product preparation should be sterile and does not contain any bacterial contamination. So to assure that MLT should be done. It contains 2 parts – first to colony count and 2nd for specific organisms presence like Salmonella, coli , Pseudomonas.
- For total colony count – Take water sample 2ml and add it into two petriplates ( per plate 1ml). Then add R -2A media in that petri plate . Incubate for 5 days at 30-35°C. After 5 days take a colony count of that two plates and take a mean and its your CFU/ ml.
Now this is done about colony count now to check specific organism-
- For specific organism- Take 100 ml water sample and into double strain SCDM and incubate it 18-24 hrs at 30-35°C. then from that SCDM first take 0.1 ml on RVSB incubate at 30-35° C for 48-72 hrs , after incubation from RVSB take a loopful l and streak on XLDA media and incubate at 30-35°C for 18hrs and after incubation observe red colony with black spot. This test is for Salmonella. Now 2nd take 1ml from SCDM and into MB and incubate at 42-44°C for 24-48 hrs, after incubation take a loopful from MB and streak on the MA and incubate for 30-35°C for 24-72 hrs and observe brick red colony. This test is for E.coli. now 3rd from that SCDM take a loopful and streak on CA and MSA and incubate at 30-35°C for 48-72 hrs and after incubation observe green colony on CA which is of Pseudomonas and metallic sheen colony which is of S. aureus.
This is about the MLT for water.
Note – Double strain SCDM means if we make 100ml SCDM the we add 3gm of SCDM powder into 100ml water generally but in double strain we have to add SCDM powder in a double quantity means in 100 ml water 6gms add SCDM powder. That is mean double stain SCDM.
MLT for Product – Here we have to 1st prepare SCDM with polysorbate -80 (4%) and soyalecithin (0.5% ) (Note: polysorbate-80 and soyalecithin is added in SCDM to neutralize the Antimicrobial activity of Product/sample to get the actual microbial flora) pH should be adjusted as standard. Then heat it till it boils and then transfer it into 3 bumper tubes (broth tubes). Per tube 90ml. then autoclave it as standard (121° C at 15PSI for 15-20min ). After autoclave – we have 3 tubes name as A ,B , C. Then take 20gm product and in tube A and tube C per tube 10gm. Now our tube A and C contains 90 ml SCDM + 10gm product. Then from A tube take 10ml and transfer to tube B . and then these tubes should incubate at 30-35°C for 18-24 hrs.
In next step – first see what to do with tube A- from tube add 1ml to two Petri plates (duplicate ) and then pour SCDA to that plates. Incubate it at 30-35° C for 5 days, same procedure should with SDA for observation of yeast and mold. After incubation take a colony count of duplicate plates and take a mean of that two plates and its your cfu/ml.
This is for colony count .
for specific organisms– Now we have tube B and tube C
- Tube B – after incubation of tube B take a 1ml from it and transfer it to MB 100ml. Then incubate it for 24-48 hrs at 42-44C. then from MB take a loopful and streak on MA . Incubate it 30-35° C for 48-72hrs. After incubation observe red brick colony of E . coli .
- From tube B- take a loopful and streak on CA and incubate it for 48-72hrs at 30-35°C. after incubation observe colony . yellow / green colour coloby of Pseudomonas .
- From tube B -take a loopful and streak on MSA and incubate it at 30-35° C for 48-72hrs. after incubation observe yellow metallic sheen colony of aureus
Now from C tube – After incubation take 0.1ml from it and add into RVSB media and incubate it 30-35°C for 18-24 hrs. after incubation of RVSB take a loopful and streak on XLDA and incubate it 30-35°C for 18-24 hrs. observe red colour colony with black spot of Salmonella.