Post on 18-Mar-2019
MICROBIOLOGICAL ASSAY IN PHARMACYMarlia Singgih WibowoSchool 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 analysisPreparation 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 nigerunder optical microscope
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
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
©M. Ryan
Enzymes tested for include: acid/alkaline phophatase, trypsin, chymotrypsin, galactosidase, glucosidase, glucuronidase, proteases, fucosidase.
An APIZYM strip
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 luciferinchemiluminesence
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 complex of Ag-
Ab• Ag-Ab complex can be visualised by several ways
(spectrophotometric, spectrofluorometricmethods, etc.)
Enzyme Linked-Immunosorbent Assay
Antigen
Antibody
Complex Ag-Ab
Conjugate enzyme with Ag-Ab
Substrate
ProductenzimaticReaction
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’ 5’3’5’
Denaturation 94 °C
3’5’
3’ 5’
3’ 5’
3’5’
Annealing primer 72°C
3’ 5’
3’5’Polimerisation72 °C
3’5’
3’ 5’ dsDNA new
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
©M. Ryan
L to R:1,18 100 bp ladder,2-6 lyophilised ,7-11 mycelial plugs in water , 12-16 cryopreserved, 17 control