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