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MICROBIOLOGICAL ASSAYIN PHARMACY
Marlia Singgih Wibowo
School 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, monoclonalantibodies, etc.
Excipients : sweeteners, flavour additives, etc.
Producing analytical reagents
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How we use Microbes
Biomass
Metabolites (primaryand secondary)
Enzymes Biotransformation
Genetic material
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Analytical Microbiology
Microbes as test microbes
: test result based onmicrobial 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 Howmicroorganisms 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 analysis
Preparation 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 (macroscopicobservation)
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 ofhyphae, 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 ofAspergillus niger
under optical microscope
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DEFT (Direct Epifluorescent Filter Technique)
Polycarbonate membrane Filter
Fluorocromatic dye will fluoresence afterconjugation . Example :
Acridine orange :Conjugation with dsDNA : Green
ssRNA : OrangeLife cells : Yellow
Dead 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 microbiologymedium 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 systemand 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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ATP assay
Luciferin + Luciferace + ATP
Luciferin-Luciferace-AMP + PP + O2
Oxyluciferin-Luciferase-AMP + H2O
Oxyluciferin + Luciferase + AMP +h (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, spectrofluorometric
methods, etc.)
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Enzyme Linked-Immunosorbent Assay
Antigen
Antibody
Complex Ag-
Ab
Conjugate
enzyme with
Ag-Ab
Substrate
Productenzimatic
Reaction
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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 byheat
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 inthermocycler
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
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Denaturation 94 C
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3 5
3 5
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Annealing primer 72C
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3 5
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Polimerisation
72 C
35
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
markerProduct of
PCR
PCR Fingerprints of Replicates of an isolate
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g p pof 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 inwater , 12-16 cryopreserved, 17 control
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