Uji Batas Mikroba Farmakope Indonesia ed.4 (
Transcript of Uji Batas Mikroba Farmakope Indonesia ed.4 (
<51> Uji Batas MikrobaFarmakope Indonesia ed.4 (<61>Microbial Limit Test) and Identification method
Marlia Singgih WibowoSchool of Pharmacy ITB
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
General Statement
Incubation : place a container in thermostatic controlled room with temperature range between 30o and 35o C for 24 to 48 hoursGrowth: presence of viable microbes or presumed proliferation of viable microorganisms
Preliminary Test
Validation is conducted based on the fact that the samples did not inhibit microbial growth Peliminary test is conducted against :
Staphylococcus aureusEscherichia coliPseudomonas aeruginosaSalmonella sp
General Procedure
1 mL of culture (not less than10-3 CFU of microbial cells from 24 hours old culture) is added into samples with :
Phosphate Buffer pH 7.2Media 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
MODIFICATION OF PROCEDURE
1. Increase dilution fluid volume with same volume of sample
2. Add inactivator agent in dilution fluid3. Combination of modification 1 and 2 until the
growth appears
INHIBITOR
Some agents can be added into the media to neutralize inhibitor :
Lecitin from soyabean : 0.5 % w/vPolysorbate 20 : 0.4 % w/v
Alternative : repeat the test using “Media Fluid Casein Digest-Soy Lecithin-Polysorbat 20”
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 sampleTherefore the sample cannot be used for MLTThe test should be conducted on determination of inhibitory effect and bactericide effect
Preparartion of Sample
Prepare 10 mL or 10 gram of sampleSpecimen is treated according to its
physicochemical properties and will not change the volume and naturally bioburden microbes in the sample.
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 andPseudomonas aeruginosaTest on Salmonella sp and Escherichia coli
For liquid samples, suspend the samples in the water or hydroalcoholic (contain ethanol < 30%), For solid samples which is easily dissoled into 90 mLof Phosphate buffer pH 7,2 or other media Test conducted for :
Total Microbial limit testTest for Staphylococcus aureusPseudomonas aeruginosaTest for Salmonella sp and Escherichia coli
Suspend it with emulgator i.e. polysorbateUse mechanical blender, if necessar up to 45o C.Test included :
Aerob TotalMicrobial LimitTest Staphylococcus aureus andPseudomonas aeruginosaTest for Salmonella sp and Escherichia coli
Samples are not soluble in water, ointment, cream
Aerosol sampleCooling 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 TestTest for Staphylococcus aureus and Pseudomonas aeruginosaTest for Salmonella sp and Escherichia coli
If the result cannot be concluded, repeat the test with 20 samples
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
Plate Count MethodDilution should be made to get the counting between 30-300 coloniesPipette 1 mL of dilute sample into 2 sterile platesVolume of media : 15-20 mL Media SCDA (45oC) Close the plate with its lid, let the agar solidifiedUp side down the plate , and then incubate for 48-72 hoursCount the growth . Average the resultIf there is no growth observed, report as “less than 10 microbes per g or ml sample”.
Multiple Tube Method
Most Probable Number (Nilai Duga Terdekat) in Multiple Tube methodObserved Combinations of Number of Tubes Showing Growth
in Each SetNo.of mg (or mL) of specimen per tube
100 mg (0,1 ml)
10 mg(0,01 ml)
1 mg(0,001 ml)
3333
3333
3210
>11001100500200
3333
2222
3210
29021015090
3333
1111
3210
1601207040
3333
0000
3210
95604023
Most probable Number (MPN) of
microbes per g or ml
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 CETAClose the lid, turn it up side down, incubateSee table 2 and 3 for confirmation of Staphylococcus aureus dan Pseudomonas aeruginosa
Morphology of Staphylococcus aureuson Selective Media Agar
SelectiveMedia
Specific Morphology/ colonies
Gram Staining
Vogel Johnson Agar (VJA)Medium
Black colonies surround by yellow zone
Positive
Mannitol Salt Agar (MSA) Medium
Yellow colonies with yellow zone
Positive
Baird Parker Agar (BPA) Medium
Black, shiny colonies, with clear zone 2 to 5 mm
Positive
Staphylococcus aureus on VJA
Staphylococcus on mannitol agar
Staphylococcus aureus on Baird-Parker Agar
Coagulase Test forStaphylococcus 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 37o CControl positive and negativeObserve the samples after 3 and 24 hour.If no coagulase : Staphylococcus aureus Free
Coagulase test for Staphylococcus aureus
Deoxyribonuclease for S.aureus
Specific morphology of Pseudomonas aeruginosaon Selective Agar Media and Diagnostic tools
Media Specific morphology
Fluorescence colony
oxydaseunder uv
light
GramStaining
Cetrimide Agar CETA (Medium)
Generally greenish
Greenish Positive Negative, rods
Pseudomonas Agar (PAF) Medium for Fluoresindeteksi
Generally it is not showing any color or yellowish
Yellowish Positive Negatif, rods
Pseudomonas Agar (PAP) Medium for pyocyanindetection
Generally greenish
Blue Positive Negatif, rods
Detection of Pseudomonas aeruginosa
Cetrimide agarContaining ammonium Quaternary agent as antiseptic agent, not for Pseudomonas
PAF and PAP90 % of Pseudomonas producing pigments:
Fluorescein (yellow) Pyocyanin (Blue/green)
Oxyidase testPseudomonas do not ferment Kovac’s oxydase reagent
Oxydase test and Pigment (Pseudomonas aeruginosa)
Inoculate a colony of suspect on Media CETA to media PAF and PAP on petri dishClose the lid, turn it up side down, incubate at 35o ± 2oC for a period of days.Observe the colony under UV light, see table 3 for confirmation
Pseudomonas aeruginosa test on Cetrimide Agar
Pseudomonas aeruginosa on Media PAF dan PAP
oxydase test for P.aureginosa
Salmonella sp and Escherichia coli
Into specimen in a container, add Media (Fluid Lactose Medium) = FLM to 100 ml and incubateObserve the growth , shake slowly. Pipette 1 ml into Media (Fluid Selenite-Cystein Medium) FSCM and (Fluid Tetrathionate Medium) FTMMix and incubate for 12 hours to 24 hours
Specific morphology of Salmonella spon Selective media Agar
Media Pemerian koloni
Brilliant Green Agar Medium
Small, transparent, colorless or pink to white opaque (frequently surrounded by pink to red zone)
Xylose-Lysine-DesoxycholateAgar Medium
Red, with or without black centres
Bishmut Sulfite Agar Medium
Black or green
Brilliant Green Agar Medium for Salmonella
Xylose-Lysine-Desoxycholate (XLD)AgarMedium
Desoxycholate-Citrate medium
Bishmut Sulfite Agar Medium
Triple Sugar Iron Agar
Specific morphology of Escherichia coli on MacConkey Agar Medium
Colony Brick red, may have surrounding zone
Gram staining Negative rods (cocco bacilli)
Detection of Escherichia coli
MCABile salt inhibit bacteria not from intestine origin Crystal violet inhibit cocci
LEMBAColi ferment lactose into pyruvate acid, become acidic, then eosin metylene blue is presipitated, produce metal shine
E.Coli pada MacConkey’s Agar
Test Indol untuk E.coli
E.Coli on eosin-methylene blue agar