Microbial Isolation and Identification Procedures Prof. Karkaz M. Thalij. PhD kthalij@gmail.com

The Variety of Microorganism Archaea : prokaryotes living in extreme habitats Bacteria: Cyanobacteria and eubacteria Eukarya: Fungi and Algae

Kingdoms of Bacteria Archaebacterial: Lack peptidoglycan in cell walls Have different lipids in their cell membrane Different types of ribosomes. Very different gene sequences. Undersea volcanic vents, acidic hot springs, salty water. They do not require oxygen and can live in extremely salty environments as well as extremely hot environments Archaebacterial can live in extremely harsh environments Called the Ancient bacteria.

Eubacteria: Subdivided into 3 groups: Methanogens Thermoacidophiles Extreme Halophiles Eubacteria: Called the true bacteria Most bacteria are in this group Include photosynthetic Cyanobacteria 3 basic shapes (coccus, bacillus, spirillum) Most are heterotrophic (can’t make their own food) May be aerobic or anaerobic…Identified by Gram staining

The Importance of studying the Microorganism

Isolation of Bacteria Obtaining of Pure Cultures from Mixed Population: A. Streak Plate Method of Isolation B. The Pour Plate Method of Isolation. and The spin plate method. C. The Spreading Plate Method of Isolation.

Media for Microbial Cultivation Use of Specialized Media: which include… 1. Selective media: such as Columbia CNA agar has the antibiotics colistin and nalidixic acid added which inhibit the growth of gram-negative bacteria but not the growth of gram-positives. 2. Differential media: Its contains additives that cause an observable color change in the medium when a particular chemical reaction occurs. 3. Enrichment media: contains additives that enhance the growth of certain organisms.

4. Combination selective and differential media: For example, Eosin Methylene Blue (EMB) agar is selective for gram-negative bacteria. The dyes eosin Y and methylene blue found in the medium inhibit the growth of gram-positive bacteria but not the growth of gram-negatives. In addition, the appearance of typical members of the enterobacteriaceae on EMB agar is as follows: Escherichia coli: large, blue-black colonies with a green metallic sheen. Enterobacter and Klebsiella: large, mucoid, pink to purple colonies with no metallic sheen. Salmonella and Shigella and Proteus: large, colorless colonies. Shigella: colorless to pink colonies. The result was refers to ability of bacterial to fermentation of lactose.

ENUMERATION OF MICROORGANISMS Plate Count :Calculate the number of CFUs per ml of original sample. B. Direct Microscopic Method: Observe the demonstration of the Petroff - Hausser counting chamber. C. Turbidity: Observe your instructor's demonstration of the spectrophotometer.

Identification of Microorganism

Identification of Bacteria was depended on the bacterial species First...Morphological and Microscopic characterizes Bacteria that are motile have appendages called flagella…which Used for Classification A bacteria can have one or many flagella Monotrichous: 1 flagella Lophotrichous: tuft at one end Amphitrichous: tuft at both ends Peritrichous: all around bacteria

BACTERIAL SHAPES AND ARRANGEMENTS Microorganism were different in the processing of shape and arrangements detected. They were depended on the type: The bacterial shapes were arranged at: Bacillus: Rod shaped Coccus: Spherical (round) Vibrio: Comma shaped with flagella Spirillum: Spiral shape Spirochete: wormlike spiral shape

Shapes types of bacteria

COLONY MORPHOLOGY AND PIGMENTATION Below a list of several common chromogenic bacteria: Staphylococcus aureus - gold; water-insoluble Micrococcus luteus - yellow; water-insoluble Micrococcus roseus - pink; water-insoluble Mycobacterium phlei - orange; water-insoluble Serratia marcescens - orange/red; water-insoluble Pseudomonas aeruginosa - green/blue; water-soluble

THE STAIN The Gram Stain. Ziehl–Neelsen staining (Ziehl–Neelsen staining:Concentrated carbolfuchsin; heat three times until vapor is observed Rinse off with water Destain with HCl (3%)/alcohol mixture. Counterstain with dilute carbolfuchsin, 1 minute Rinse off with water) The Capsule Stain Endospore Stain Bacterial Motility

Second…Identification of Bacteria Used some Cultural Testing This contains: Growth ability at different temp. Growth ability at different pH levels. Growth at different concentration of NaCl. Ability to produce the pigments.

Third…IDENTIFICATION OF BACTERIA THROUGH BIOCHEMICAL TESTING The types of biochemical reactions each organism undergoes act as a "thumbprint" for its identification. This is based on the following chain of logic:

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