1. Microbial Isolation and Identification Procedures
Prof. Karkaz M. Thalij. PhD

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

3. 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.

4. 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

5. The Importance of studying the Microorganism

6. 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.

7. 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.

8. 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.

9. 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.

13. Identification of Microorganism

14. 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

15. 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

16. Shapes types of bacteria

17. 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

19. 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

20. 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.

23. 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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