Characterization and

Screening of Microorganisms

Elin Julianti
Marlia Singgih Wibowo
School of Pharmacy ITB
 What is Microbiology?
 Microbiology is a specialized area of
biology that deals with living things
ordinarily too small to be seen with the
naked eyes (microorganisms).

 Microorganisms : bacteria, fungi,

viruses, protozoa and some parasitic
worms.
The Importance of Microorganisms
 Agriculture
 Food
 Diseases
 Energy/Environment
 Biotechnology
Products from Microorganism
 Microorganism and Disease
 Many types of microorganisms are
potentially pathogenic

 Number of new diseases increase : SARS,

AIDS, encephalitis
 Microbiologists use 5 basic techniques to grow,
examine and characterize microorganisms in
the lab.

 5 basic techniques (5‘I’s): inoculation,

incubation, isolation, inspection and
identification.
 Why we need to characterise and
screen microbial isolates?

1. To ensure the correct taxonomy to allow selection of

the correct preservation procedure (anatomy,
morphology, locomotion)

2. To asses the suitability of the preservation applied and

if necessary, to modify and improve preservation
procedure

3. To facilitate the monitoring of long-term stability of the

microorganisms
 Purpose of Screening:

Detection of novel products (metabolites, vitamins,

enzymes) and the potential for other economic
uses :
biological control
biodegradation
bioremediation
chemical processes (transformation,
bioaccumulation)
food or food processes
for assaying and testing.
  Characterization Methods

 Qualitative methods (Observations, microscopy)

 Quantitative methods (growth rate, sporulation
capacity)
 Biochemical methods (chromatography/ enzyme
assays, protein analysis)
 Molecular methods (PCR, Sequencing,
functional genomics)
 Qualitative methods

methods of visual characterisation

and analysis = phenotypic methods)
 CULTURE MORPHOLOGY & ANATOMY

1. ANATOMICAL STUDIES (shape and size of spores,

chloroplasts, intracellular organisation, etc.)

2. CULTURE MORPHOLOGY (pigmentation, colony radius,

hyphal form and aggregation, gross appearance, etc.)

3. SPORULATION (abundance, type)

4. LOCOMOTION (Nematodes and flagellate organisms)

 Cultural morphology of Fusarium sp.

Cultural and morphological characteristics of Fusarium oxysporum f.

sp. passiflorae isolate Fus-01. A, B, C: colony aspect on PDA, CMA
and MALT, respectively.
 Microscopy

• Analysis of morphology, anatomy and

intracellular organisation.
• Detection of biochemicals and macrostructures
using staining techniques
• Identification using oligonucleotides of organism
with specific stains
• Assessment of viability
 Types of microscopy
 Light microscopy
 Scanning Electron Microscopy
 Transmission Electron Microscopy
 Phase contrast microscopy
 Fluorescence
Chaetomium globosum under light microscope
Diatoms (SEM images)

http://www.bgsu.edu/departments/biology/algae/SEM/
Transmission Electron Micrographs

http://www.kochi-u.ac.jp/~mine/en/spermen.html
Phase-contrast microscopy

https://en.wikipedia.org/wiki/Phase-contrast_microscopy
 Fluorescence Microscopy

Griffithsia japonica
Liberated spermatia
Cell surface is stained with
FITC-Con A.
Scale bar = 10 micrometre.
photo by Michiko Ohnishi

http://www.kochi-u.ac.jp/~mine/en/spermen.html
Quantitative methods

(methods of measurement)
 Physiology

1. GROWTH RATES (Bacteria, Fungi, Algae)

2. THIN LAYER CHROMATOGRAPHY : Metabolites (fungi)
, Chlorophylls (algae)
3. RATE OF PHOTOSYNTHESIS: Algae and
photosynthetic bacteria
4. RATE OF RESPIRATION
5. ENZYME PRODUCTION / SUBSTRATE UTILISATION:
Bacteria and Fungi (using commercially available kits
APIZYM, BIOLOG etc.)
 Growth rates
Fungal growth rate can be determined by measuring the
mean hyphal radial extension over a set period of time
using defined media and growth conditions. Results can
be plotted on graphs and analysed, statistically, to allow
comparisons.

The growth of unicellular organisms (e.g. bacteria,

yeasts, protozoa and micro algae) or cells, can be
determined by counting the number of (viable) cells
over a set period of time. Growth curves can then be
plotted (e.g. time ‘v’ log number of cells) to allow
comparisons to be made.
 Biochemical methods

(analysis of the intra- and

extracellular chemicals produced by
living organisms)
 Chromatography

A method for separating secondary metabolites

and other biochemicals (e.g. chlorophylls). Once
separated, metabolites can be collected and
characterised if required.

Thin Layer Chromatography (TLC):

a simple, inexpensive method, samples to be
loaded onto a stationary phase (e.g membranes
attached to a suitable support). A solvent (liquid)
phase then ascends by capillary action carrying/
separating components as it progresses.
 Thin layer chromatography of secondary metabolite standards
 Detection of Patulin using
Thin Layer Chromatography (TLC)

Image courtesy of R. Paterson

TLC of Ubiquinones

Ubiquinones are a class

of terpenoid lipids that
are involved in electron
transport.
There unique structure
and profiles, which are
often unique to
individual species, have
made them a powerful
tool for use in microbial
chemotaxonomy
 Chromatography: Other techniques
High Performance Liquid Chomatography (HPLC)
A highly automated system for separating the chemical
components of complex mixtures, that gives rapid reproducible
results. Samples are extracted into solvents which are then
passed through a column which separates the components.

Gas Chromatography (GC) often used in microbiology for the

detection and analysis of membrane lipids. It is used for the
identification and characterisation of bacteria and yeasts. The
method involves vaporising the sample in a carrier gas (e.g
helium), the substance/gas is carried along a heated tube at a
characteristic rate (Partition coefficient) and then detected as it
exits the tube. The output is then compared against a library of
profiles and a best match selected.
 Protein analysis
Proteins can be detected & separated by
electrophoresis, ultracentrifugation and
chromatography methods. It can be detected using
antibody techniques (such as ELISA), or Protein
structure can be elucidated using x-ray
crystallography, mass spectrometry and peptide
sequencing.

Cell breakage (in an appropriate buffer) is required to extract

proteins for analysis. For example : physical, chemical or
sonification.
Pectinase gel
ELISA (enzyme linked immuosorbent assay)

1. An immunological method

2. Utilises computer aided technology

3. Very sensitive (like all immunological methods)

4. Utilises antibodies specific to the antigens of a

particular biomolecule (e.g. proteins,
hormones)

5. Highly Reproducible
 Plate tests

 Biochemical investigations carried out in vitro

in Petri dishes on designated nutrient media ,
under pre-specified experimental conditions.
Mainly for bacteria, filamentous fungi and
yeasts.

 Many diverse applications including nutrient

utilisation studies, sexual compatibility,
enzyme activity assessments,
antagonism/synergism experiments and
antibiotic/ antiseptic assays.
 Carbon source utilisation

Pink colour
represents
utilisation of
the sole carbon
source
Shows
differentiation
between two
species of
Colletotrichum
Nitrogen source utilisation

Left to right:
Urea
Hypoxanthine
Nitrite
Nitrate
Protein hydrolysis
Vegetative Compatibility testing: Colletotrichum

•The ability to form a

stable heterokaryon by
anastomoses
•Hyphae differing at one
or more loci are unable
to form a stable
heterokaryon
•Used to determine the
genetic structure of
asexual fungal
populations
Ligninolytic activity
1 2 3

Plates 1 and 2 exhibit ligninolytic activity

(presumptive test for ability to metabolise lignin)
 Enzyme assays and carbohydrate utilisation

UTILISATION: The ability of an organism to directly utilise a

specific nutrient source.
ENZYME ASSAY/ TESTS: The ability of an organism to produce
intra- or extracellular enzymes capable of breaking down a
specific substrate.

Many commercially available kits available including APIZYM,

API OCH, BIOLOG.
Researchers can devise their own utilisation/ assay tests using
substrates that are bound to chromo or fluorogenic chemicals. For
example, 4MU (4-methylumbelliferyl) compounds available from
Sigma
Carbohydrate utilisation: Biolog GN Plate

Image courtesy of N. Mapuranga

 APIZYM strip

Enzymes tested for include: acid/alkaline phophatase, trypsin,

chymotrypsin, galactosidase, glucosidase, glucuronidase,
proteases, ficosidase.
 API 20NE strip (utilisation / identification of bacteria)

Assimilation of malate, caprate, mannitol, arabinose; reduction of

nitrates, hydrolyis of esculin and gelatn; inodole production,
acidification of glucose.
MOLECULAR METHODS

(analysis of genetics and

functional genomics)
 PCR Techniques

Since its conception in the mid 1980’s, the

Polymerase Chain Reaction (PCR) has
revolutionised the study of biology at the molecular
level.

PCR has allowed:

Study of microbial population biology at the molecular level
An understanding of genes and gene expression
Characterisation (taxonomy, species definition)
  PCR Fingerprinting

RAPD-PCR (Random Amplified Polymorphic DNA)

SSR-PCR (Simple Sequence Repeat)
VNTR-PCR(Variable Number Tandem Repeat)
AFLP-PCR (Amplified Fragment Length Polymorphism)

Uses :
 Population Biology

 Host Pathogen Interactions

 Genetic Stability / Variability

 Molecular Characterisation
Example PCR Fingerprints of fungal
isolates, with GACA4 Primer

SHOWING INTERSPECIFIC VARIATION

A+T-rich DNA digest showing mtDNA bands for
characterisation of Colletotrichum spp.

SHOWING INTRASPECIFIC VARIATION

 Points to be considered
 Where possible, use validated (i.e. properly
characterised) strains for study
 Optimise preservation method for the
microorganism
 When undertaking comparisons, ensure
control of variables
 Select appropriate methodology for tests
 Investigate “strange” results