sb20103 microbe diversity: microbe identification methods
TRANSCRIPT
SB20103 MICROBE DIVERSITYWeek 4: MICROBIAL IDENTIFICATION METHODS
Jaya Seelan Sathiya Seelan PhD
Institute for Tropical Biology and Conservation, UMS
OBJECTIVE
• To introduce students to the basictools and techniques used to identifyand characterize microorganisms.
OUTLINE
• Identification of Bacteria
• Identification of Fungi
• Identification of Algae
• Identification of Virus
IDENTIFICATION OF BACTERIA
• Methods used to identify bacteria to the level of genus and species
• Phenotypic methods• Morphology
• Physiology or biochemistry
• Immunologic method• Serological analysis
• Genotypic techniques• Genetic markers (18S/16S ribosomal gene) for
identifying bacteria
PHENOTYPIC METHODS
•Microscopic morphology
•Macroscopic morphology
•Physiological/Biochemical characteristics
•Chemical analysis
MICROSCOPIC MORPHOLOGY
•Cell shape and size
•Gram stain reaction
•Acid fast reaction
•Special structures
PHYSIOLOGICAL/BIOCHEMICAL CHARACTERISTICS
• Traditional bacterial identification
• Diagnostic tests for determining the presence of specific enzymes and assessing nutritional and metabolic activities
• Examples
• Fermentation of sugars
• Capacity to metabolize complex polymers
• Production of gas
• Presence of enzymes
• Sensitivity to antimicrobial drugs
CHEMICAL ANALYSIS
• Analyzing the types of specific structural substances that the microorganism contains
• Examples
• Chemical composition of peptides in the cell wall
• Lipids in membranes
• Antibiogram
• Where do you find these
bacteria?
• As a microbiologist, how
do you interpret this
chart?
• Can you ID the species
level?
Critical thinking
GENOTYPIC METHODS
•Primary advantage over phenotypic methods:
•Hybridization
•Nucleic Acid Sequencing and rRNA Analysis
•Polymerase Chain Reaction (PCR)-Multiplex PCR
IMMUNOLOGIC METHODS
•Antibody response to antigens
•Blood testing- often easier than testing for the microbe itself
• Serology
• Laboratory kits available for immediate identification of a number of pathogens
• Characteristics of antibodies
can reveal the history of a
patient’s contact with
microorganisms or other
antigens
Serology
• Deals with in vitro diagnostic testing of the serum
FUNGI IDENTIFICATION
Single cell-Yeasts
- Basidiomycetes
Unicellular-Chytrid fungi
Multicellular
Ascomycetes
HOW TO IDENTIFY FUNGI?
• Morphology: under different media; growth forms, colony appearance, growth rates, spore shapes and sized
• Physiology: pH, Temperature and etc.
• Chemical: Secondary metabolites, toxins
• Phylogeny: Very important and up to date (Fungal Barcode DNA, ITS)
IDENTIFICATION OF ALGAE
•Color
•Type of photosynthetic food reserve
•Flagella type, cell wall structure and composition
•Morphology vs phylogeny methods
Where do you look for Algae?
IDENTIFICATION OF VIRUS
Morphology: Helical/ Icosahedral
shapes
Microscopy in cell cultures
Immunofluorescence
(IF) Assay
• Real-time RT-PCR assays for influenza virus detection
• Immunofluorescence Foci Assay (IFA)
• Immunoblotting
Methods
• Hemagglutination Assay (HA)Lecture: Virus (more details)
GAME: WHAT AM I?
• In this game, each group will be given a microbe (card) to discuss.
• Identify your microbe
• You may find all the relevant information from the web.
• Identify their scientific name, where you can find them, their role and etc. (Be concise)
• Describe the identification methods.
• Be creative!
Learning objective
• Appreciate the vast
abundance and
diversity of microbes
• Able to classify microbes
based on their
characters
• Understand that most
microbes are not just
harmful
EXAMPLE
• Gram-negative (bacteria)• Facultative anaerobic• Rod shape• Genus/species: Escherichia coli• Found in lower intestine; in soil• Harmless: producing vitamin K2• Pathogenic to human: gastroenteritis, urinary tract infections, and neonatal meningitis• E.coli #0157:H7, that produces a toxin called the Shiga toxin (classified as a bioterrorist agent)
• Have been used in vaccine development, bioremediation, production of biofuels, lighting, and production of immobilized enzymes
Identification method: E.coli
Rapid test-EMB Agar- glowing colonies
Indole test (+)
• Glucose fermentation with gas production, urea and H2S
• Lactose
• Sucrose; mannitol cellobiose
• Citric Acid
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