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TRANSCRIPT
number 5
Done by Marah Karablieh
Corrected by Abdullah Zreqat
Doctor Hamed Al Zoubi
Bacterial Identification and Classification
First: Identification
The successful identification of microbiological agent depends on:
1- Proper aseptic techniques.
2- Correctly obtaining the specimen, if someone has the symptoms of UTI
(Urinary Tract Inflammation), you don’t need a CSF (Cerebrospinal Fluid)
test, you need a midstream specimen of urine, for example.
3- Correctly handling the specimen.
4- Quickly transporting the specimen to the lab, some samples grow at the
room temperature, which will give a wrong indication that the patient
has some sort of infection (False positive).
5- Once it reaches the lab, it’s cultured and identified.
After the microbe is identified, it is used in susceptibility tests to find out the
effective control measure.
Methods used to identify bacteria fall into these three categories:
Methods used to identify bacteria
Phenotypic
(Morphology)
Microscopy
Growth media
Biochemical tests
(Rapid test methods)
Genotypic
(Molecular techniques)
Immunological
(Serological) tests
➢ Phenotypic methods
1-Microscopic morphology: -Remember Staining-
Includes a combination of cell shape, size, Gram stain, acid fast stain and
special structures (e.g. endospores, granules, capsules) can be used to give an
initial putative identification.
Done by using:
• Simple stain
• Gram stain
• Acid fast stain (Ziehl-Neelsen stain)
• Special stains
2-Macroscopic morphology or growth media:
“Macro” means something seen with the naked eye, but how can I see
bacteria?! –Remember Culturing-
Bacterial cultivation, which means isolation of bacteria from specimens.
-Principles of Cultivation:
• Nutritional requirements: as we took before, some bacteria are
fastidious –they need complex, unusual, unique requirements for
growth- and some are non-fastidious –need simple requirements-.
• Streaking for isolation
• Streaking for quantitation
The tool used to collect bacteria is called a “Loop”, first you need to heat it
for sanitization… then you collect bacteria and streak it (تفردها) on the agar
plate, you should heat the loop after every streak… the aim of this process is to
have single colonies.
Note: It’s important to have pure colonies, so if you have a plate with two
colonies, one is dry and the other is mucoid, you should culture these colonies
separately
-Colony Characteristics: you can recognize this with your naked eye, e.g.
texture, shape, pigment, growth pattern.
• Colony form: pinpoint, circular, filamentous, irregular
• Colon elevation: flat, raised, complex
• Colon margin: smooth, irregular
-Types of culture media:
I. Basal media
Used for culture of bacteria that DO NOT need enrichment of the media,
e.g. Nutrient broth, nutrient agar, peptone water.
II. Enriched media
By adding blood, serum or egg, e.g. blood agar, chocolate agar,
Lowenstein-Jensen media*.
*it’s used to identify Mycobacterium tuberculosis, and it contains malachite
green.
III. Selective media
Contains agents that inhibit the growth of all agents except that being
sought (dyes, bile salts, alcohols, acids, antibiotics) e.g. SSA*, Mannitol salt
agar**.
*Salmonella Shigella agar –only these two types can grow on it-, notice the
black dotes (H2S) on the Salmonella specimen
**Staphylococcus aureus produces the yellow colour
IV. Differential media:
An indicator is included in the medium, and a particular organism causes
changes in this indicator (examples on indicators: blood, neutral red,..) e.g.
blood agar* and MacConkey agar**.
*Some bacteria do hemolysis, so you put them on blood agar to identify-
the bacteria that lyse blood completely are called β hemolytic, the ones
that lyse partially are called α hemolytic, and those which don’t lyse at all
are non-hemolytic or γ hemolytic.
**This culture media is both selective and differential, it isolates gram
negative bacteria, the bacteria ferment lactose, and as a result the colour of
agar changes into pink
V. Transport media
These media are used when specimen cannot be cultured soon after
collection. e.g. Cary-Blair medium, Amies medium, Stuart medium.
VI. Storage media
Media used for storing the bacteria for a long period of time.
3- Biochemical Tests
The microbe is cultured in a media with a special substrate and tested for an
end product. Prominent biochemical tests include:
• Enzymes: Catalase, oxidase, decarboxylase…
• Fermentation of sugars
• Acid or gas production
• Hydrolysis of gelatine
Other biochemical tests of interest include:
• Indole test
• Methyl Red/ Vogues-Proskauer
• Citrate utilization
• Coagulase test
• H2S production (TSA)
• Urease test
• Phenylalanine deaminase test
Note: Spiral bacteria that cause ulcer in stomach protect themselves from
the acidic pH by secreting the enzyme urease, which hydrolyse urea into
carbon and ammonia –raises the surrounding pH-
Urease test Phenylalanine deaminase test Citrate utilization
Rapid tests: biochemical system for the identification of Enterobacteriaceae,
it consists of 20 tests that are converted to a digital profile.
➢ Immunological (Serological) Tests
The immune system defends against antigens by producing antibodies. These
antibodies are particles that attach to the antigens and deactivate them. When
you test your patient’s blood, you can identify the type of antibodies and
antigens that are in his blood sample and identify the type of infection he has.
Agglutination tests:
-Direct whole pathogen agglutination assays
-Particle agglutination tests
-Latex beads or RBCs coated with Ag
ELISAs: Enzyme-Linked ImmunoSorbent Assay
IFAs: Indirect Fluorescent Antibody
➢ Genotypic methods
These methods of identification include the use of:
-Nucleic acid probes -PCR (polymerase chain reaction)
-Nucleic acid sequence analysis -rRNA analysis
-RELP (Restriction Fragment Length Polymorphism) -Plasmid fingerprinting
Bacterial Identification using Vitek
• Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)
• Mass spectrometric method
• Rapid identification
• This offers the analysis of whole bacterial cultures for unique mass
spectra from charged macromolecules
• Growing database
Second: Classification
Taxonomy: the science of classification of organisms
Bacterial taxonomy consists of three separate, but interrelated areas:
1-Classification: the arrangement of organisms into groups (taxa) on the basis
of similarities or relationships.
2-Nomenculature: is the assignment of names to the taxonomic groups
according to the international rules
3-Identification: the practical use of classification scheme to determine the
identity of an isolate as a member of an established taxon or as a member of a
previously unidentified species.
Taxonomic Rank:
Example:
Species: the basic and the most important taxonomic group in bacterial
systemic, the boundaries of species are rather difficult to define precisely,
however; the boundaries of some genra are sharply defined. For example, the
genus Bacillus and the genus Escherichia.
Species
Genus
Family
Order
Class
Division or Phylum
Kingdom or Domain
Typing
WHY?
a) Different bacterial species often exhibit different population structures.
b) Recombination e.g. Transformation.
c) Highly recombining populations are termed panmicticm in contrast to
clonal populations where recombination is infrequent.
d) Typing identifies a recognizable subdivision of species that serves as a
reference marker against which other isolates of the same species can
be compared.
HOW?
a) Classifying ranks below subspecies, such as biovars, servars, phagovars,
and bacteriocin or PCR, are often used to indicate groups of strains that
can be distinguished by some special character, such as antigenic
makeup, reactions to bacteriophage, etc.
b) Such ranks have no official standing in nomenclature but often have
great practical usefulness.
PRACTICAL USEFULNESS
Common reasons for microbial typing are to:
a) Identify common or point sources
b) Discriminate between mixed strain infections
c) Distinguish re-infection from relapse
Biovars: biological/physiological variations- some bacteria need amino acids, other don’t
Serovar: Serological variation – bacteria vary in their antigens
Phagovar: bacteria vary in their susceptibility and resistances to various bacteriophages
Bacteriocin: Some bacteria are sensitive to certain substances, others are not… so we use this substance as a toxin to kill the bacteria that is sensitive to it
PCR: Polymerase Chain Reaction- the one we use the most
Classification of Bacteria
Before getting into details, let’s talk about Adansonian classification first:
In most systems of bacterial classification, the major groups are distinguished
by fundamental characters, such as cell shape, Gram stain reaction and spore
formation.
Genera and Species are usually distinguished by properties such as
fermentation reactions, nutritional requirements and pathogenicity.
Similar coefficient when shared positive characters are considered, a
matching coefficient when both negative and positive shared characters
(matches) are taken into account.
Classification of bacteria
Phenotypic Classification
Morphology and Gram staining characteristics
Growth requirements and metabolic behavior
Environmental Reservoirs (modes of
transmission)Genotypic Classification
DNA hybridization, used to designate species
Genomics
'G+C' content or 'Guanine+Cytosine
ratio'
rRNA sequence analysis
(highly conserved)
Phenotypic Classification
Morphology
-Some correlation between morphology and diseases:
Spiral bacteria—Treponemes, Borrelias, and Leptospiras tend to cause
systemic diseases
Pathogenic Filamentous bacteria -- Actinomyces, Nocardia, and Mycobacteria
tend to cause chronic diseases
Gram positive bacteria—Staphylococcus, Streptococcus more likely to cause
skin infections
-Growth requirements and metabolic behaviour (on the next page)
Cocci
Bacilli
Curved or Spiral
Filamentous
*Autotrophic: an organism that synthesize organic compounds
**Heterotrophic: an organism that require organic compounds
Naming microorganisms
- Binomial nomenclature (Scientific):
Give each microbe 2 names:
o Genus: noun, ALWAYS capitalized
o Species: adjective, lowercase
o Both italicized or underlined
o Examples: Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis),
Escherichia coli (E. coli)
Growth requirements and
metabolic behaviour
Nutritional requirements
Autotrophic*
Heterotrophic**
Gaseous requirements
Aerobic bacteria
Anaerobes
Faculitative anaerobes
Thermal requirements
Psychrophiles
Mesophiles
Thermophiles
Sorry for any mistakes