BMED2801 – Lecture 5 – Microbial identification & classification
Learning objectives
Students should be able to describe:
• Relationship between prokaryotes and eukaryotes
• Challenges, principles, methods of classifying microbes
• Methods of microbial identification:
– Microscopy
– Biochemical and physiological tests
– Chemical composition
– DNA based methods
Features distinguishing major microbial groups
• Subcellular entities: (Not cells- are smaller than cells – have DNA/genes and/or proteins) Depend on a
host cell for replication and metabolism, not truly ‘alive’ (?)
– Prion: self-replicating protein (extremely unusually. always need DNA/RNA to replicate – prions can
replicate without it), no nucleic acids  causative agent of a variety of Spongiform Encephalopathies –
brain disease causing spongy degeneration of the brain e.g. mad cow’s disease in cattle, scrapie in
sheep/goat and Creutzfeldt-Jakob disease in humans.
– Virus: nucleic acids (RNA or DNA) with protein coat e.g. bacteriophage
– Plasmid: made of DNA, like virus but no protein coat
• Prokaryotes (Bacteria):
The simplest free-living organism, independent in replication and metabolism.
• Mostly unicellular, but can show differentiation (e.g. spores) and morphological complexity (e.g.
filaments)
• Two prokaryotic Domains exist:
– Eubacteria: peptidoglycan wall, ester-lipid membrane (fatty acid + glycerol linked together by esters)
– Archaea: glycoprotein wall, ether-linked membrane (gives rigidity – able to withstand v. hot
environments) Some Archaea are halophiles (salt-loving), e.g. Halobacterium gives red colour to salt
lakes
• Eukaryotes:
More complex (in terms of structure, function and life cycle) and larger than prokaryotes, membrane
delimited true nucleus, contain membrane-bound organelles, can be multicellular
• The Domain Eukarya contains animals & plants, and three microbial groups:
– Algae: contain chlorophyll; perform photosynthesis  volvox, Cyanobacteria – some produce toxins
that form red tides that infect fish and the toxin moves through the food chain. Medical significance
related to the transmissible nature of toxins e.g. via water supply.
– Fungi: heterotrophic (use organic matter as food source), non-motile, extracellular digestion (secrete
digestive enzymes outside of cell to digest – usu. large insoluble macromolecules e.g. cellulose/starch)
e.g. Aspergillus- medically impt. - can infect lung tissue
– Protozoa: heterotrophic, motile, intracellular digestion  Amoeba is motile by pseudopodia
Distinction of prokaryotes and eukaryotes (EXAM!!)
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Some prokaryotes sometimes have structures within their cytoplasm –e.g. granules + inclusion
bodies  but these are not really organelles – are not membrane bound.
eukaryotic cell wall differs – cellulose in plants, and chitin in fungi
Prokaryotes – usu. only have one circular chromosome which is haploid – never have paired
chromosomes – therefore never reproduce sexually. Eukaryotes alternate between haploid and
diploid life cycles–> somatic cells are diploid, gametes are haploid.
advantageous trait in eukaryotes – the ability to have sex and recombine genetic material
DIFFERENCE IN RIBISOMES  Antibiotics e.g. streptomycin - that are designed to target
ribosomes to kill the bacteria – why don’t they affect our eukaryotic ribosomes? – Because
eukaryotic and prokaryotic ribosomes are diff. in size and structure (eukaryotic are bigger) – diff.
are enough to produce diff. target effects.
Also ciprofloxacin – target nucleoid DNA replication – do not affect eukaryotic DNA gyrase
because it is diff. to that of prokaryotes.
Evolution of eukaryotes from prokaryotes
• First eukaryotic cell likely arose from fusion of 2 prokaryote ancestors (one formed the nucleus the other
formed the cytoplasm) to give cell with membrane-bounded nucleus.
Subsequent steps:
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mitochondrion added  formed when prokaryote engulfed by first eukaryote cell  led to animals
and fungi
Then Chloroplast added  led to algal cell or plant.
Plant cell – highly evolved in terns of its number of organelles = actually 4 diff. prokaryotes joined together
– one is nucleus, one is cytoplasm, one is the mitochondrion and the other the chloroplast.  No longer
free living can not separate back into prokaryotes.
• Organelles in eukaryote cells (the mitochondrion and chloroplast) are closely related to bacteria - their
likely origins are two endosymbiosis events (a hypothetical evolutionary process by which some cellular
structures may have developed as a result of the incorporation of free-living prokaryotes into the
cytoplasm of eukaryotes)
Life diversifies: unicellular forms
• Prokaryotes were not replaced by eukaryotes, but evolved in parallel, diversifying to colonise new
niches e.g. purple sulfur bacteria exhibit a diversity of morphologies.
• Some eukaryotes remained unicellular/primitive state (e.g. the protozoa), some Protist - either with
mitochondrion (aerobes) e.g. plasmodium or without* mitochondria (anaerobes) e.g. giardia
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Degenerate organelle – with some function of the mitochondria – but since they grow
anaerobically – they don’t really need a mitochondrion, so it is lost.
• Other eukaryotes developed into multicellular forms
• Euk’s with mitochondria  fungi, animals
• Euk’s with mitochondria & chloroplasts  algae, plants
How to make sense of life’s diversity?
Taxonomy: the science of classifying plants, animals, and microorganisms into increasingly broader
categories based on shared features.
• Why is taxonomy important for microbiology?
– provides meaningful organisation of diverse species
– Allows predictions and hypotheses to be made – e.g. predict the behaviour of the unknown members of
these groups.
– allows accurate communication between scientists
– Essential for identification of unknown microbes
• Taxon (one taxonomic entity – a genus, a unit) (pleural - taxa): taxonomic unit, a grouping of organisms
Microbial classification presents unique challenges
• Simple morphology  lack of observable features e.g. microbes
- Traditionally, organisms were grouped by physical resemblances  skeletal structure/ internal
anatomy  but microbes are small, simple morphology this approach is not valid- Not a lot of
observable features.
• Convergent evolution (development of similar functions and structures in unrelated or distantly-related
organisms) e.g. many diverse species have developed spherical cells (cocci) as the optimum shape 
therefore, classification by observable features does not directly suggest lineage within the group, these
features may have just been the cause of convergent evolution.
• Horizontal gene transfer: (movement of genes between sister cells not from parents- moved via
plasmids among cells – therefore cells acquiring different DNA from all around them) features of one
microbe may be acquired from multiple different sources  hard to classify microbes – because usually
their genetic material is contaminated.
• Facultative metabolism: one species may have several different possible ‘lifestyles’ depending on
environment.
E.g. rhodobacter – alternates between 4 different types of metabolism.
Phenetic vs. phylogenetic classifications
• Phenetic (classification based on phenotypes- similar observable features) e.g. cocci or rod
- This method is useful for determinative microbiology.
– Identification of unknown microbes by comparison to features of known microbes
– Still widely applied in clinical microbiology – undertake easy biochemical tests, figure out is it gram
negative/gram positive, aerobic/anaerobic – to identify unknown.
• Phylogenetic methods (the development of the evolutionary history of a species, genus, or group, as
contrasted with the development of an individual ontogeny- from a fertilized ovum to maturity)
Useful for systematic microbiology
– yield a natural classification system
– represent evolutionary relationships
Phenetic classification
• Based solely on similarity of phenotype, independent of evolutionary considerations
• Which are the most informative features to use?
Features that are:
– Stable over time – the microbe always has to manifest certain feature (e.g. not motility – this
changes over time – depends on how energetic the cell is, how old culture is etc.)
– Stable within a taxon – always has to behave the same way with a certain feature (e.g. not cell
orientation – which direction cell is facing – this changes)
– Vary between taxa (eg. not presence of DNA- All bacteria has DNA – although, the sequence
of DNA is useful)
• Phenotypic features only give natural classifications if they are homologous: i.e. have same evolutionary
origins e.g. Duck and platypus bills have different origins/ancestors; example of CONVERGENT EVOLUTION  Phenetic
classification of the two based on bill feature solely would – give an incorrect representation of the relationship of an
organism
Phylogenetic classification
• Reflects the real evolutionary relationships between organisms
• Traditionally, phylogenetic classification is difficult for microbes, due lack of fossil record evidence
• Sequencing of nucleic acids and proteins has overcome this problem  phylogeny that includes
microorganisms
• Comparison of gene sequences from unknowns to online database (GenBank)  identification AND
classification
Methods of microbial identification
• Cell morphology (microscopic): shape, size, arrangement, presence of flagella, spores, or inclusion
bodies
- information is quick to obtain
- theses are easily observable features
- easily accessible via microscope
- Useful for phenetic type classification.
• Colony morphology: (macroscopic): colour, shape, size, edge type, elevation
• Biochemical and physiological tests:
– Carbon sources
– Nitrogen sources
– Energy sources
– Enzyme activities
– Aerobic / anaerobic
– Other electron acceptors
– Temperature and pH optimum
API strip: Analysis of carbon source utilization and enzyme activities, based on colour production from indicator dyes 
comparison of results against a database, can lead to identification of an unknown microbe.
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This method however, is limited by the type of database available for use and the number of known microbes in
the database.
• Carbon & energy sources
• Diversity of microbial nutrition requires new concepts
• Categorized by the sources of carbon and energy
Nutritional Type
Heterotroph e.g. animals, fungi
Photoautotroph e.g. plants
Chemoautotroph (an organism that
obtains energy through the
oxidation of an inorganic
substance) e.g. microbes only.
Carbon Source
Organic compounds
CO2
CO2
Energy Source
Organic compounds
Light
Inorganic
compounds
• Electron Acceptors
• Plants and animals respire using oxygen (O2) as the electron acceptor - this is required to oxidize food
to CO2
Glucose  oxidation  product = CO2
O2  reduction  product = water
• Microbes sometimes respire with O2, but also have other options available – i.e. alternative electron
acceptors e.g. anaerobic respiration.
• Diversity of possible electron acceptors allows microbes to colonize diverse niches – especially,
anaerobic respiration – allowing microbes to survive in deep sea sediments, inside gut
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e.g. anaerobic microbes living in
mangroves reduce sulfate to stinky
H2S.
In humans anaerobic fermentation
in muscles, involves use of organic
compounds to respire e.g. pyruvate
– being converted to lactate during
intense exercise.
Glucose  glycolysis  pyruvate w/ oxygen = pyruvate enters tri-carboxylic acid cycle, and be oxidised
fully to CO2. – Most energy yielding reaction.
 Pyruvate w/o oxygen = pyruvate is reduced to ethanol (waste product) and CO2
(regenerated internal electron acceptor).
• Chemical composition
– Cell wall composition determined via Gram stain: thin or thick peptidoglycan  Gram –ve or Gram +ve
– Cell membrane fatty acids: gas chromatography
Fatty acid chromatogram – characteristic pattern of peaks for diff. fatty acids present in cell membrane –
limited by database of known fingerprint for comparison and identification of the unknown.
– G+C content of DNA: melting temperature
– Whole cell fingerprints: electrophoresis of proteins
Protein electrophoresis – polyachraimide gel, separation via charge and mass.
• Specific stains
• Acid-fast stain differentiates bacteria with mycolic acids in the cell wall from all other bacterial types
• Acid fast cells retain carbol fuchsin dye after acid-alcohol rinse. Other cells counter stain with methylene
blue, stains the ones decolourised.
• Why is it useful? Acid-fast bacteria include important pathogens. E.g. Mycobacterium - tuberculosis and
leprosy
E.g. Acid-fast Mycobacterium cells stain red, human tissues stain blue
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Convenient, cheap and accessible.
• Spore stain reveals endospores in bacteria belonging to the genera Bacillus and Clostridium
• Endospores retain malachite green dye after water rinse. Bacterial cells are counterstained with
safranin.
E.g. Endospores stain green, their host cells (Bacillus) stain red
• Why is it useful? Presence of endospores is diagnostic for many important pathogens, e.g. anthrax
(Bacillus anthracis)
• Phase- Contrast Microscopy
• Allows visualisation of live, unfixed, unstained samples
– Determination of motility (can cells swim?)
– can see natural cell morphology
– can see some internal structures (endospores, storage materials) without need for staining
• How does it work?
– Phase rings in microscope convert differences in refractive index (which we can’t see) into differences
in contrast (which eyes can see)
E.g. Phase-contrast image of Clostridium botulinum (botulism) Bacterial cells are dark, endospores are
bright
Fluorescence microscopy
• High-energy light (e.g. blue) excites fluorochromes in the sample, which emit light of lower wavelength
(e.g. green or red)
• Non-specific fluorochrome: dye binds to all cell types. E.g. acridine orange binds non-specifically to all
DNA
• Specific fluorochrome: dye attached to an antibody which can bind to specific proteins or a short DNA
sequence  binds only to specific cell types
E.g. Fluorescent antibodies can be used to detect whole bacteria protist organelles
Electron microscopy
• Resolution of the light microscope is limited by the wavelength of light – e.g. viruses (~100 nm) are
smaller than visible light wavelengths (~400-700 nm)
• Solution: use electrons (wavelength ~0.1 nm) instead of light to view samples  electron microscope.
• Scanning electron microscope (SEM) bounces electrons off sample surface; transmission electron
microscope (TEM) sends electrons through a thin section
• Sample preparation for EM is intensive (contrast to phase-contrast): fix, dehydrate, coat with heavy
metal atoms to increase contrast – introduction of artifacts may change naturally morphology of cell.
DNA-DNA hybridization
• DNA is the molecule of heredity; therefore DNA-based classification has direct evolutionary significance
• Single-stranded (ss) DNA of two species made by heating
• ss DNA mixed, then cooled to give double-stranded hybrids
• Double-stranded (ds) hybrid heated to melting temp (ss)
• Melting temp indicates stability of hybrid, level of DNA homology, and similarity of the two species
If the two organism are v. similar = strong hybrid – higher melting point
If the two organisms are v. different – weak hybrid – low melting point.
DNA, RNA, protein sequences
• Sequencing nucleic acids and proteins has had a great impact on microbial identification and
classification
– Huge amount of information. E.g. one DNA sequencing machine can read ~ 20,000 base pairs per day
– Availability of universally-present marker genes and proteins e.g. strongly conserved 16S ribosomal
RNA to allow comparisons across ALL species
– Don’t need to culture microbes to get sequence data – amplify sequences by PCR of microbes that can
not be grown in lab.
– Direct evolutionary significance of genes and proteins finally allows phylogenetic classification of
microbes
Ribosomal RNA
• All organisms use ribosomes to translate RNA into protein
• Genes coding for structural components of ribosomes, e.g. ribosomal RNA (rRNA) can be sequenced,
used as markers
• Polymerase chain reaction (PCR) amplification of rRNA genes has revolutionized microbiology
 rapid and reliable identification of unknowns
 detection of ‘unculturable’ microbes in environment
 development of phylogenetic classification system
• Combining rRNA sequence & computational methods gives a phylogeny of all organisms – the Tree of
Life
Tree of life based on rRNA shows three Domains
• Confirmation of endosymbiotic origin of organelles
• Fungi are a coherent group, but algae and protozoa are not
• Vast majority of life’s diversity is microbial
mitochondrion and chloroplast – ribosomal sequence- shows it is clearly bacteria.
What is a microbial species?
• Species concept for animals and plants is easy to define, on basis of sexual reproductive compatibility
• What about microbes that can reproduce asexually?
• Use polyphasic taxonomy: comparison of multiple shared phenotypic and genotypic features