introduction to microbiological assay

8/11/2019 Introduction to Microbiological Assay

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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

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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

introduction to microbiological assay

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