basic skills in diagnostic bacteriology
Identification of Microorganisms
• For many students and professionals the most pressing topic in microbiology is how to identify unknown specimens. • Why is this important?
• Labs can grow, isolate and identify most routinely encountered bacteria within 48 hrs of sampling.
• The methods microbiologist use fall into three categories: ♣Phenotypic- morphology (micro and macroscopic) ♣Immunological- serological analysis ♣Genotypic- genetic techniques Dr.T.V.Rao MD
• The successful identification of microbe depends on: ♥ Using the proper aseptic techniques. ♥ Correctly obtaining the specimen. ♥ Correctly handling the specimen ♥ Quickly transporting the specimen to the lab. ♥ Once the specimen reaches the lab it is cultured and identified. Dr.T.V.Rao MD 3
• Identification measures include:
♣ Microscopy (staining) ♣ growth on enrichment, selective, differential or characteristic media ♣ specimen biochemical test (rapid test methods) ♣ immunological techniques ♣ molecular (genotypic) methods.
• After the microbe is identified for clinical samples it is used in susceptibility tests to find which method of control is most effective. Dr.T.V.Rao MD 4
• Successful identification depends on how the specimen is collected, handled and stored. • It is important that general aseptic procedures be used including sterile sample containers and sampling methods to prevent contamination of the specimen. • E.g. Throat and nasopharyngeal swabs should not touch the cheek, tongue or salvia. • What other precautions must be taken when collecting specimens?
• After collection the specimen must be taken promptly to the lab and stored appropriately (e.g. refrigeration). Dr.T.V.Rao MD 6
Phenotypic Methods of Identification
• Microbiologists use 5 basic techniques to grow, examine and characterize microorganisms in the lab. • They are called the 5 ‘I’s: inoculation, incubation, isolation, inspection and identification. • Inoculation: to culture microorganisms a tiny sample (inoculum) is introduced into medium (inoculation). • Isolation involves the separating one species from another. Dr.T.V.Rao MD
Phenotypic methods of Identification
• Incubation: once the media is inoculated it is incubated which means putting the culture in a controlled environment (incubation) to allow for multiplication. • After incubation the organisms are inspected and identified phenotypically, immunologically or genetically. Dr.T.V.Rao MD 9
5 “I” s
identification Dr.T.V.Rao MD
• ‘Old fashioned’ methods via biochemical, serological and morphological are still used to identify many microorganisms. • Phenotypic Methods • Microscopic Morphology include a combination of cell shape, size, Gram stain, acid fast rxn, special structures e.g. endospores, granule and capsule can be used to give an initial putative identification. Dr.T.V.Rao MD 11
• Macroscopic morphology are traits that can be accessed with the naked eye e.g. appearance of colony including texture, shape, pigment, speed of growth and growth pattern in broth. • Physiology/Biochemical characteristic are traditional mainstay of bacterial identification. • These include enzymes (catalase, oxidase, decarboxylase), fermentation of sugars, capacity to digest or metabolize complex polymers and sensitivity to drugs can be used in identification. Dr.T.V.Rao MD 12
– enhancement of size using ocular and objective lenses.
• Ocular: eyepiece (10X) • Objective: 4X – 100X
– allows for visualization of bacteria, fungi, and parasites, not viruses...