Overview of Isolation and Identification of Bacterial Strain’s from Air, Soil and Water

Around our environment, lots of microbial species (e.g. bacteria, fungi and protozoa) are widely distributed. These microorganisms have both beneficial and harmful effects on human being as well as other organism present in the ecosystem. In order to evaluate their beneficial part and level harmfulness we need to isolate, characterize and identified the bacterial community spreaded around us and subsequently necessary to go further for assessment whether they advantageous or injurious for ecosystem and especially with respect to human being. In this short article I am sharing you all about:

“How we go for isolation and identification of bacterial species from air, soil and water sample?”

How we go for microbial isolation from environment or specific area?

Sample collection

Sample could be collected from air, soil and water to study. Sample should be collected in sterile container with the help of sterile tools to prevent the contamination of another microbe. However this is quit disputed matter that how we maintain the complete sterilized condition during sample collection? But by using the sterilized tools and container we can maintain much more aseptic condition rather than doing it casually. Sample must be coded in order remember about (e.g. types of sample, source, types of microorganisms bacteria or fungi, purpose of its application; such as in case of producing specific compound or enzyme). Soil sample should be collected from 4-6 inch below of the soil surface.

Culture of Bacterial Consortium Present in Sample (Air/ Soil/ Water)

How we did?

Master Plate of Bacterial Consortium

Sample was collected and brought to our microbiology lab. Sample should be open under aseptic condition (inside laminar air flow). Nutrient Agar Media (NAM) plates were prepared to grow bacterial consortium. Serial dilution was carried out to dilute the concentration of microbes present in sample. Later NAM plates were aseptically inoculated (or spreaded) 1.0 ml of 10-3, 10-6 and 10-9 serially diluted sample and incubated in 370C for 24 hrs.

Pure Culture of Each Bacterial Colony

After incubation period, each bacterial colony from master plate of bacterial consortium was streaked in separate NAM plates and agar slants and incubated at in 370C for 24 hrs. NAM plates were used for further morphological and biochemical analysis. Agar slants were stored at 40C as pure culture stock.

Morphological Characterization

  • Size (diameter; in mm or cm)
· Shape (e.g. baccilus, cocobaccilus, fusiform baccilus, coccus, spirillum, vibrio and spirochete)

· Colony shape (e. g. punctiform, round, filamentous and irregular)

· Color (pigmentation e.g. red and yellow colonies)

· Elevation (e.g. raised, convex, flat, umbonate and crateriform)

· Margin (e.g. smooth, filamentous, curled, wavy. lobate, undulate and filiform)

· Gram stain (e.g. gram positive and gram negative)

  • Spore stain

Biochemical Characterization

Ø For Gram Positive Bacteria

  • Catalase Test
  • Mannitol Salt Agar (MSA)
  • Blood Agar Plates (BAP)
  • Streak-stab technique
  • Taxos P (optochin sensitivity testing)
  • Taxos A (bacitracin sensitivity testing)
· CAMP (Christie, Atkins, and Munch-Peterson) Test

  • Bile Esculin Agar
  • Nitrate Broth
  • Spirit Blue agar
  • Starch hydrolysis test
  • Motility Agar
  • Coagulase Test
Ø For Gram Negative Bacteria

  • Oxidase Test
· Sugar (e.g. glucose, lactose and mannitol) broth with Durham tubes

  • Methyl Red & Voges-Proskauer (MR-VP)
  • Kliger’s Iron Agar (KIA)
  • Nitrate Broth
  • Motility Agar
  • MacConkey agar
  • Simmon’s Citrate Agar
  • Urease test
  • Sulfur Indole Motility Media (SIM)

Molecular Characterization

  • Isolation of bacterial genomic DNA
· Amplification of the 16S rRNA genes (18S rRNA gene in case of fungi) by universal primer

  • DNA sequencing

Bioinformatics Database Synchronization and Analysis

Alignment of DNA sequence result with the sequences in existing GenBank database using the Basic Local Alignment Search Tool (BLAST) search program at the National Centre for Biotech Information (NCBI) website.

About Author / Additional Info:
Research Scholar
Department of Microbiology
Gov. ERR Science College,
Bilaspur (C.G.)
Research Interest: Microbial biotechnology