number Done by Corrected by Doctor Hamed Al Zoubi

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1 number 5 Done by Marah Karablieh Corrected by Abdullah Zreqat Doctor Hamed Al Zoubi

2 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: Microscopy Phenotypic (Morphology) Growth media Methods used to identify bacteria Genotypic (Molecular techniques) Biochemical tests (Rapid test methods) Immunological (Serological) tests

3 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

4 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.

5 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 identifythe bacteria that lyse blood completely are called β hemolytic, the ones

6 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

7 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

8 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 -Nucleic acid sequence analysis -PCR (polymerase chain reaction) -rrna analysis -RELP (Restriction Fragment Length Polymorphism) -Plasmid fingerprinting

9 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.

10 Taxonomic Rank: Species Genus Family Order Class Division or Phylum Kingdom or Domain 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.

11 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

12 Classification of Bacteria Classification of bacteria Phenotypic Classification Environmental Reservoirs (modes of transmission) Genotypic Classification Morphology and Gram staining characteristics DNA hybridization, used to designate species Growth requirements and metabolic behavior Genomics 'G+C' content or 'Guanine+Cytosine ratio' rrna sequence analysis (highly conserved) 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.

13 Phenotypic Classification Morphology Cocci Bacilli Curved or Spiral Filamentous -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)

14 Autotrophic* Nutritional requirements Heterotrophic** Aerobic bacteria Growth requirements and metabolic behaviour Gaseous requirements Anaerobes Faculitative anaerobes Psychrophiles Thermal requirements Mesophiles Thermophiles *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)

15 Sorry for any mistakes