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Microbial Limit Tests • They are applied to all raw materials and preparations that do NOT require to be sterile. • They cover the total microbial viable aerobic count (TC) and the absence of specified pathogens. • They are carried out according to the method mentioned in different Pharmacopeias. • • • • Microbial limit test includes: 1. Preparation of sample, 2. Total aerobic microbial viable count, 3.Test for absence of pathogens. • Preparation of sample: • ENSURE that the sample does not inhibit the multiplication of microorganisms that may be present, under the test conditions. 1.Oral Syrup A. Total aerobic microbial count Materials 1. Bottle of oral syrup (sample). 2. Three test tubes each containing 9 ml sterile phosphate buffered saline (PBS) pH 7. 3. Six test tubes of soya bean casein digest agar containing 0.5% soy lecithin & 4% polysorbate 20. 4. Six sterile Petri dishes. 5. Four sterile 1 ml pipette. (10-1) (10-2) (10-3) Sample Pour agar then mix 8wise Colony growth after incubation Plate (1) colonies 10-1 Plate (2) colonies 10-2 Plate (3) colonies 10-3 Procedure A. Total aerobic microbial viable count Pour plate method • 1. The sample is subjected to 10-fold serial dilution • 2. Thoroughly mix each dilution (by means of a new 1 ml pipette) 3. With a sterile 1 ml pipette, aseptically transfer 1 ml of each dilution into each of 2 sterile Plates. 4. Mark each plate with the dilution present. 5. Pour the molten agar medium, into Petri dishes, and mix the bacterial suspension with the medium. 6. Incubate at 30-35oC for 48 to 72 hours. • What to do to have a successful experiment? • 1) Mix WELL your m.o suspension in: a) The 9-ml PBS tubes b) The Petri dishes together with molten agar. Example of a plate with bad mixing Example of good mixing (homogenouslydistributed colonies) 2) Pour your Molten agar at 50oC (Not too HOT nor too COLD) Example of a plate with NO colonies, condensed water vapor can be observed on the inner surface of the cover 3) Invert your plates immediately after solidification of poured agar medium to prevent condensed water vapor droplets on the inner side of the plate lid from falling-down over the agar surface. Example of a plate with uncountable patch over agar surface Results • Count the number of colonies • Choose the plates with 30 - 300 colonies • Express the average of the two plates in terms of the number of microorganisms per gram or per ml of sample V.C.= No. of c.f.u./dilution factor Pharmacopeal limit is < 10,000 CFU/ml or g of the sample. Results • Case 1: - If no microbial colonies are recovered from the plates of the initial 1:10 dilution (10-1): Express the results as “less than 10 CFU per g or per ml of sample .” i.e. Sample has a microbial count within the limit (<10000 CFU/ml) and the batch is accepted. Results • Case 2: • If plates of one of the dilutions (for example 10-2) contain 30 - 300 colonies (for example 80 CFU), calculate the viable count as follows: No. of CFU 80 • V.C = Dilution factor = 10-2 = 8000 CFU/ml Then the sample has a microbial count within the limit (<10000 CFU/ml) and the batch is accepted. Results - Case 3: - If plates of more than one dilution show colony count in the range of 30- 300, calculate the viable count for each dilution, then calculate the average CFU/ml. 10-1 10-2 10-3 - For example Dilution No. of CFU V.C1 = V.C2 = No. of CFU Dilution factor No. of CFU Dilution factor = = C.V1 + C.V2 Total V.C= 2 >300 290 10-2 31 10-3 290 31 = 29000 CFU/ml = 31000 CFU/ml • Case 3 (cont.): • Then the total V.C = 29000 + 31000 = 2 30000 CFU/ml • Then the sample has a microbial count above the acceptable limit (>10000 CFU/ml) and the batch is rejected. Results • Case 4: If plates of all three dilutions (10-1, 10-2 and 10-3) contain colonies more than 300 then: • Then the sample needs further dilution in order to calculate the viable count.