Fields related to Microbiology. • Industrial Microbiology. • Analytical Microbiology.
• Medical Microbiology. • Environmental Microbiology ...
MICROBIOLOGICAL ASSAY IN PHARMACY Marlia Singgih Wibowo School of Pharmacy ITB
Fields related to Microbiology • • • •
Industrial Microbiology Analytical Microbiology Medical Microbiology Environmental Microbiology
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
How we use Microbes • Biomass • Metabolites (primary and secondary) • Enzymes • Biotransformation • Genetic material
Analytical Microbiology • Microbes as test microbes : test result based on microbial response to samples • Microbes as contaminant (in pharmaceutical products, food or cosmetics)
Medical Microbiology • • • • •
Study pathogenicity of patogenic microbes New drug design thru patogenic invasion Study of antigen structure and function Designing vaccine etc
Environmental Microbiology • • • • •
Bioremediation Waste Treatment Biopesticide designing Fertilizer etc
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
Stages in microbiological analysis Preparation of method, instruments and reagents ↓ Identification of sample ↓ Sampling ↓ Qualitative Analysis ↓ Quantitative Analysis ↓ Report
Category of assays in microbiology • • • • •
Direct method Culture techniques Enumerasi Method Alternative Method Rapid Method
Direct Method •
• • •
Direct observation by naked eyes (macroscopic observation) Microscopic observation (using microscope) Dye Method / Staining method DEFT (Direct Epifluorescent Filter Technique)
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
Morphology of Aspergillus niger under optical microscope
DEFT (Direct Epifluorescent Filter Technique) • Polycarbonate membrane Filter • Fluorocromatic dye will fluoresence after conjugation . Example : Acridine orange : Conjugation with dsDNA : Green ssRNA : Orange Life cells : Yellow Dead cells : Bright Green
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 .
Culture on agar media for : • • • • • •
Isolation Determination Storage Growing Production Analysis
Enumeration Method • • •
Plate Count MPN Count (Most probable Number) : based on turbidity Physicochemical Analysis : based on metabolites or component of cells
Alternative Methods • • • •
Biochemical method Dye-reduction Test Electrical Methods ATP Determination
Biochemical Methods: Enzymes
An APIZYM strip Enzymes tested for include: acid/alkaline phophatase, trypsin, chymotrypsin, galactosidase, glucosidase, glucuronidase, proteases, fucosidase.
©M. Ryan
Biochemical Methods: Metabolites
Thin Layer Chromatography of Secondary Metabolites (Example of Patulin production by Penicillium spp.)
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
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
ATP Determination • ATP will be found in live cell : product of metabolism • Measured by physicochemical method : activity of enzimatic luciferase and substrate luciferin Î chemiluminesence
ATP assay Luciferin + Luciferace + ATP Luciferin-Luciferace-AMP + PP + O2 Oxyluciferin-Luciferase-AMP + H2O Oxyluciferin + Luciferase + AMP + hµ (560 nm)
Rapid Method • •
Immunochemical method Microbiological method : molecular
Immunochemical Method • Principle of analysis : based on Antigen-Antibody reaction • Antigen react with specific Ab Î complex of AgAb • Ag-Ab complex can be visualised by several ways (spectrophotometric, spectrofluorometric methods, etc.)
Enzyme Linked-Immunosorbent Assay
Antigen Complex AgAb
Conjugate enzyme with Ag-Ab Substrate
Antibody enzimatic Reaction
Product
Immunochemical Method • • • •
EIA / ELISA (Enzyme Immuno Assay) RIA (Radio Immuno Assay) IFA (Immuno Fluoresence Assay) LIA (Luminescence Immuno Assay)
Molecular Method • Hybridization • Amplification
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
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
PCR
3’ 5’
5’ 3’ Denaturation 94 °C
3’
5’
5’
3’ Annealing primer 72°C
3’
5’
5’
3’
3’
5’
5’
3’
Polimerisation 72 °C
3’
5’
dsDNA new
5’
3’
dsDNA new
Repeating process
Denaturation Annealing Polymerisation
Process after PCR • Detection process of PCR product by DNA electrophoresis (agarose gel) • DNA bands compared to DNA marker • Sequencing
DNA Electrophoresis
DNA marker
Product of PCR
PCR Fingerprints of Replicates of an isolate of Metarhizium anisopliae, After 2 Years of Preservation with Mr Primer
L to R:1,18 100 bp ladder,2-6 lyophilised ,7-11 mycelial plugs in water , 12-16 cryopreserved, 17 control ©M. Ryan
