microbiological assay in pharmacy - download.fa.itb.ac.iddownload.fa.itb.ac.id/filenya/handout...
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MICROBIOLOGICAL ASSAY IN PHARMACYMarlia Singgih WibowoSchool of Pharmacy ITB
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Fields related to Microbiology• Industrial Microbiology• Analytical Microbiology• Medical Microbiology• Environmental Microbiology
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Industrial Microbiology• Using microbes to produce useful products (thru
fermentation process) :– Drugs substances : antibiotic, hormones, etc.– Biomedical Products : vaccine, antisera, monoclonal
antibodies, etc.– Excipients : sweeteners, flavour additives, etc.
• Producing analytical reagents
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How we use Microbes
• Biomass• Metabolites (primary
and secondary)• Enzymes• Biotransformation• Genetic material
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Analytical Microbiology • Microbes as test microbes
: test result based on microbial response to samples
• Microbes as contaminant (in pharmaceutical products, food or cosmetics)
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Medical Microbiology• Study pathogenicity of patogenic microbes• New drug design thru patogenic invasion• Study of antigen structure and function • Designing vaccine • etc
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Environmental Microbiology • Bioremediation• Waste Treatment• Biopesticide designing• Fertilizer • etc
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Why microbiological quality is needed to be tested in pharmaceutical product ?
• Pharmaceutical Products : Medicines, cosmetics, medical devices, Households, Food supplements
• Products should be safe and useful • Pathogenic Microorganisms → How
microorganisms contaminating product• How pathogenic microorganisms can be detected
and how many is the amount of the contaminant? • Qualitative and Quantitative Analysis
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Stages in microbiological analysisPreparation of method, instruments and reagents
↓Identification of sample
↓Sampling
↓Qualitative Analysis
↓Quantitative Analysis
↓Report
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Category of assays in microbiology
• Direct method• Culture techniques • Enumerasi Method • Alternative Method• Rapid Method
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Direct Method• Direct observation by
naked eyes (macroscopic observation)
• Microscopic observation (using microscope)
• Dye Method / Staining method
• DEFT (Direct Epifluorescent Filter Technique)
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DIRECT METHOD
• Direct observation : morphology, spores color, colony color and shape
• Microscopic obsv : shape and motility, shape and color of hyphae, using optical or inverted microscope
• Staining Method: Gram staining for bacteria• DEFT (Direct Epifluorescent Filter Technique) : Samples
are filtered, and then staining, observation using epifluoresence microscope
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Morphology of Aspergillus nigerunder optical microscope
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DEFT (Direct Epifluorescent Filter Technique)• Polycarbonate membrane Filter• Fluorocromatic dye will fluoresence after
conjugation . Example :Acridine orange : Conjugation with dsDNA : Green
ssRNA : OrangeLife cells : YellowDead cells : Bright Green
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CultureTechnique
Eventhough many advance method have been applied for identification and characterization of microorganisms, culturing techniques /inoculation onto microbiology medium still needed, especially for confirmation of identity. Commonly, they use universal Media, selective media, etc .
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Culture on agar media for :• Isolation• Determination• Storage • Growing• Production• Analysis
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Enumeration Method
• Plate Count• MPN Count (Most probable Number) : based
on turbidity• Physicochemical Analysis : based on
metabolites or component of cells
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Alternative Methods
• Biochemical method• Dye-reduction Test• Electrical Methods• ATP Determination
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Biochemical Methods: Enzymes
©M. Ryan
Enzymes tested for include: acid/alkaline phophatase, trypsin, chymotrypsin, galactosidase, glucosidase, glucuronidase, proteases, fucosidase.
An APIZYM strip
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Biochemical Methods: Metabolites
Thin Layer Chromatography of Secondary Metabolites
(Example of Patulin production by Penicillium spp.)
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Dye-reduction Test• Based on redox reaction of a pigment (dye)• Dye will take an electrone from an aktive biological system
and will produce color• In General : oxsidized form will produce color, and reduced
form will be colorless • Dyes: methylene blue, resazurin, triphenyltetrazolium
chloride
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Electrical Methods
• When microorganisms growing, the activity of cells will change the composition of the medium, and will change the properties of its electricity.
• The measurement of : conductance, capacity, and impedance
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ATP Determination
• ATP will be found in live cell : product of metabolism
• Measured by physicochemical method : activity of enzimatic luciferase and substrate luciferinchemiluminesence
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ATP assay
Luciferin + Luciferace + ATP
Luciferin-Luciferace-AMP + PP + O2
Oxyluciferin-Luciferase-AMP + H2O
Oxyluciferin + Luciferase + AMP + hµ (560 nm)
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Rapid Method
• Immunochemical method • Microbiological method : molecular
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Immunochemical Method• Principle of analysis : based on Antigen-Antibody
reaction• Antigen react with specific Ab complex of Ag-
Ab• Ag-Ab complex can be visualised by several ways
(spectrophotometric, spectrofluorometricmethods, etc.)
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Enzyme Linked-Immunosorbent Assay
Antigen
Antibody
Complex Ag-Ab
Conjugate enzyme with Ag-Ab
Substrate
ProductenzimaticReaction
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Immunochemical Method
• EIA / ELISA (Enzyme Immuno Assay)• RIA (Radio Immuno Assay)• IFA (Immuno Fluoresence Assay)• LIA (Luminescence Immuno Assay)
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Molecular Method
• Hybridization • Amplification
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Hybridization
• Process of denaturation of dsDNA to ssDNA by heat
• Recombination of the ssDNA with an ssDNA that has been probed (label)
• The Hybrid DNA will be labelled and can be detected by instruments
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Amplification Technique• PCR (polymerase chain reaction) • Target DNA is denaturated using heating process in
thermocycler• Primer (a short oligonucleotide ) complement to each end
of the ssDNA• Synthesis of the DNA starts from the primer• Cycling Process : 20 – 50 x
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PCR
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3’ 5’3’5’
Denaturation 94 °C
3’5’
3’ 5’
3’ 5’
3’5’
Annealing primer 72°C
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3’ 5’
3’5’Polimerisation72 °C
3’5’
3’ 5’ dsDNA new
dsDNA new
Repeating process
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Denaturation
Annealing
Polymerisation
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Process after PCR
• Detection process of PCR product by DNA electrophoresis (agarose gel)
• DNA bands compared to DNA marker• Sequencing
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DNA Electrophoresis
DNA marker Product of
PCR
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PCR Fingerprints of Replicates of an isolate of Metarhizium anisopliae, After 2 Years of
Preservation with Mr Primer
©M. Ryan
L to R:1,18 100 bp ladder,2-6 lyophilised ,7-11 mycelial plugs in water , 12-16 cryopreserved, 17 control