<51> Uji Batas Mikroba Farmakope Indonesia ed.4 (<61>Microbial Limit Test) and Identification method. Marlia Singgih Wibowo School of Pharmacy ITB

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1 <51> Uji Batas Mikroba Farmakope Indonesia ed.4 (<61>Microbial Limit Test) and Identification method Marlia Singgih Wibowo School of Pharmacy ITB

2 Why we need Microbial Limit Test? To predict number of viable aerobic microbes in all pharmaceutical products (from raw material to finished product) and to state that the products are free from certain contaminant To guarantee that pharmaceutical products are safe, qualified and benefit

3 General Statement Incubation : place a container in thermostatic controlled room with temperature range between 0 o and 5 o C for 24 to 48 hours Growth: presence of viable microbes or presumed proliferation of viable microorganisms

4 Preliminary Test Validation is conducted based on the fact that the samples did not inhibit microbial growth Peliminary test is conducted against : Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Salmonella sp

5 General Procedure 1 ml of culture (not less than10 - CFU of microbial cells from 24 hours old culture) is added into samples with : Phosphate Buffer ph 7.2 Media Fluid Soybean-Casein Digest (FSCD) Media Fluid Lactose Medium (FLM) If there is no growth observed, the test is not valid, and need some modification in procedure

6 MODIFICATION OF PROCEDURE 1. Increase dilution fluid volume with same volume of sample 2. Add inactivator agent in dilution fluid. Combination of modification 1 and 2 until the growth appears

7 INHIBITOR Some agents can be added into the media to neutralize inhibitor : Lecitin from soyabean : 0.5 % w/v Polysorbate 20 : 0.4 % w/v Alternative : repeat the test using Media Fluid Casein Digest-Soy Lecithin-Polysorbat 20

8 If the addition of in activator and increasing volume cannot induce the growth of viable microbes, or samples cannot be tested by using membrane filter, it is concluded that there is bacterice action from the sample Therefore the sample cannot be used for MLT The test should be conducted on determination of inhibitory effect and bactericide effect

9 Preparartion of Sample Prepare 10 ml or 10 gram of sample Specimen is treated according to its physicochemical properties and will not change the volume and naturally bioburden microbes in the sample.

10 Solid samples which is not soluble in dilution fluid Decrease the size of particles, suspend in specific media with following tests: Total Aerobic Microbial test Test on Staphylococcus aureus and Pseudomonas aeruginosa Test on Salmonella sp and Escherichia coli

11 For liquid samples, suspend the samples in the water or hydroalcoholic (contain ethanol < 0%), For solid samples which is easily dissoled into 90 ml of Phosphate buffer ph 7,2 or other media Test conducted for : Total Microbial limit test Test for Staphylococcus aureus Pseudomonas aeruginosa Test for Salmonella sp and Escherichia coli

12 Samples are not soluble in water, ointment, cream Suspend it with emulgator i.e. polysorbate Use mechanical blender, if necessar up to 45 o C. Test included : Aerob TotalMicrobial Limit Test Staphylococcus aureus and Pseudomonas aeruginosa Test for Salmonella sp and Escherichia coli

13 Aerosol sample Cooling the container in ice bath for 1 hour, open the container and leave the sample until reach room temperature. Let the propellant free, take the sample 10 g or 10 ml from 10 containers, place into culture media Test for : Total Aerobic Microbial Limit Test Test for Staphylococcus aureus and Pseudomonas aeruginosa Test for Salmonella sp and Escherichia coli If the result cannot be concluded, repeat the test with 20 samples

14 Microbial Limit Test (Total Aerob Count) 10.0 gram or 10.0 ml of specimen (sample) dissolve in : Phosphate Buffer ph 7.2, Media FSCD or Media FCDSLP Volume adjusted Up to 100 ml. Plate Count Method Multiple Tube Method

15 Plate Count Method Dilution should be made to get the counting between 0-00 colonies Pipette 1 ml of dilute sample into 2 sterile plates Volume of media : ml Media SCDA (45 o C) Close the plate with its lid, let the agar solidified Up side down the plate, and then incubate for hours Count the growth. Average the result If there is no growth observed, report as less than 10 microbes per g or ml sample.

16 Multiple Tube Method

17 Most Probable Number (Nilai Duga Terdekat) in Multiple Tube method Observed Combinations of Number of Tubes Showing Growth in Each Set No.of mg (or ml) of specimen per tube 100 mg (0,1 ml) 10 mg (0,01 ml) 1 mg (0,001 ml) Most probable Number (MPN) of microbes per g or ml >

18 Staphylococcus aureus and Pseudomonas aeruginosa test On specimen add media FSCD to 100 ml, mix, and incubate If any growth observed, inoculate it into Media VJA (or BPA or MSA) and Media CETA Close the lid, turn it up side down, incubate See table 2 and for confirmation of Staphylococcus aureus dan Pseudomonas aeruginosa

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20 Morphology of Staphylococcus aureus on Selective Media Agar Selective Media Vogel Johnson Agar (VJA) Medium Mannitol Salt Agar (MSA) Medium Specific Morphology/ colonies Black colonies surround by yellow zone Yellow colonies with yellow zone Gram Staining Positive Positive Baird Parker Agar (BPA) Medium Black, shiny colonies, with clear zone 2 to 5 mm Positive

21 Staphylococcus aureus on VJA

22 Staphylococcus on mannitol agar

23 Staphylococcus aureus on Baird-Parker Agar

24 Coagulase Test for Staphylococcus aureus Colonies from Media VJA (BPA,MSA) into tubes containing 0.5 ml of mamalian plasma (rabbit, and/or horse) with or without inhibitor substances. Incubate in waterbath 7 o C Control positive and negative Observe the samples after and 24 hour. If no coagulase : Staphylococcus aureus Free

25 Coagulase test for Staphylococcus aureus

26 Deoxyribonuclease for S.aureus

27 Specific morphology of Pseudomonas aeruginosa on Selective Agar Media and Diagnostic tools Media Cetrimide Agar CETA (Medium) Specific morphology Generally greenish Fluorescence colony oxydase under uv light Gram Staining Greenish Positive Negative, rods Pseudomonas Agar (PAF) Medium for Fluoresin deteksi Pseudomonas Agar (PAP) Medium for pyocyanin detection Generally it is not showing any color or yellowish Generally greenish Yellowish Positive Negatif, rods Blue Positive Negatif, rods

28 Detection of Pseudomonas aeruginosa Cetrimide agar Containing ammonium Quaternary agent as antiseptic agent, not for Pseudomonas PAF and PAP 90 % of Pseudomonas producing pigments: Fluorescein (yellow) Pyocyanin (Blue/green) Oxyidase test Pseudomonas do not ferment Kovac s oxydase reagent

29 Oxydase test and Pigment (Pseudomonas aeruginosa) Inoculate a colony of suspect on Media CETA to media PAF and PAP on petri dish Close the lid, turn it up side down, incubate at 5 o ± 2 o C for a period of days. Observe the colony under UV light, see table for confirmation

30 Pseudomonas aeruginosa test on Cetrimide Agar

31 Pseudomonas aeruginosa on Media PAF dan PAP

32 oxydase test for P.aureginosa

33 Salmonella sp and Escherichia coli Into specimen in a container, add Media (Fluid Lactose Medium) = FLM to 100 ml and incubate Observe the growth, shake slowly. Pipette 1 ml into Media (Fluid Selenite-Cystein Medium) FSCM and (Fluid Tetrathionate Medium) FTM Mix and incubate for 12 hours to 24 hours

34 Specific morphology of Salmonella sp on Selective media Agar Media Brilliant Green Agar Medium Xylose-Lysine- Desoxycholate Agar Medium Bishmut Sulfite Agar Medium Pemerian koloni Small, transparent, colorless or pink to white opaque (frequently surrounded by pink to red zone) Red, with or without black centres Black or green

35 Brilliant Green Agar Medium for Salmonella

36 Xylose-Lysine-Desoxycholate (XLD)Agar Medium

37 Desoxycholate-Citrate medium

38 Bishmut Sulfite Agar Medium

39 Triple Sugar Iron Agar

40 Specific morphology of Escherichia coli on MacConkey Agar Medium Colony Brick red, may have surrounding zone Gram staining Negative rods (cocco bacilli)

41 Detection of Escherichia coli MCA Bile salt inhibit bacteria not from intestine origin Crystal violet inhibit cocci LEMBA Coli ferment lactose into pyruvate acid, become acidic, then eosin metylene blue is presipitated, produce metal shine

42 E.Coli pada MacConkey s Agar

43 Test Indol untuk E.coli

44 E.Coli on eosin-methylene blue agar