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