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

SHAPE AND SIZES

GRAM STAINING METHODS

Gram +ve

Gram -ve

DIFFERENCES BETWEEN GRAM -/+

*Important to classify between gram -/+

MACROSCOPIC MORPHOLOGY

•Colony appearance

•Speed of growth

•Patterns of growth

COLONY GROWTH FORM

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

BIOCHEMICAL TEST

• Presence/absence of

selective

sugars/proteins/amino acids

API Kit for Rapid Test

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)

Colonies on different media & Microscopic Aspergillus & Penicillium

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

-

+

-

-