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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