Isolation-and-Cultivation-of-Microorganism

PART 1: Isolation and Cultivation of Microorganisms

Principles of Bacterial Cultivation

  • Grow and isolate all bacteria present in a clinical specimen.

  • Determine which bacteria are likely causing infection versus contaminants.

  • Obtain sufficient growth of relevant bacteria for identification, characterization, and susceptibility testing.

Key Concepts

Cultivation

  • Process of growing microorganisms by collecting specimens from the infection site (in vivo) and growing them in an artificial laboratory environment (in vitro).

  • Involves increasing the microorganism population by meeting their nutritional and physical requirements.

Culture Medium

  • Nutrient material for growing microorganisms in the lab.

  • Composition includes:

    • Carbon

    • Nitrogen

    • Sulfur

    • Phosphorus

    • Hydrogen

    • Oxygen

    • Buffer

  • Inhibitory agents are used to facilitate isolation of desired organisms while suppressing undesired organisms.

Nutrients

  • Extracellular substances that provide cells with materials for building protoplasm and energy generation.

Culture Types

  • Pure (Axenic) Culture: Composed of one species only.

  • Mixed Culture: Composed of more than one species.

  • Stock Culture: Several species, one species per medium, stored for at least a year.

Agar

  • Sulfated polymer derived from red algae.

  • Cooling Temperature: 55℃ – 60℃ for distribution into petri plates; Volume: 20-25mL.

Uses of Culture Media

  • Growth and maintenance of microbial cultures.

  • Favor production of specific compounds.

  • Study microbial action on medium constituents.

Classification of Culture Media

According to Physical State

  1. Liquid Medium (Broth)

    • Contains no agar; nutrients are in liquid.

    • Examples: Nutrient Broth, Brain Heart Infusion, Trypticase Soy Broth (TSB), Thioglycolate.

  2. Semi-solid Medium

    • Contains 0.5% to 1% agar; consistency between liquid and solid.

    • Used for determining organism motility.

    • Example: Sulfide Indole Motility (SIM) Medium.

  3. Solid Medium

    • Contains 1.5% to 3% agar; supports colony growth.

    • Examples: Triple Sugar Iron (TSI) Agar, MacConkey Agar, Blood Agar Plate (BAP).

According to Composition

  1. Synthetic or Defined Medium

    • Chemically defined; all components known.

    • Example: BG-11 medium for cyanobacteria.

  2. Non-synthetic or Complex Medium

    • Not all components defined; includes peptones and extracts.

    • Examples: Nutrient Broth, MacConkey.

  3. Tissue Culture Medium

    • Contains living tissues; used for obligate intracellular bacteria.

    • Examples: Hela 229, McCoy cells.

According to Dispensing or Distribution Method

  1. Plated Media: Distributed into sterile petri dishes.

  2. Tubed Media: Distributed in sterile test tubes; includes slant and butt varieties.

According to Use

  1. Simple Media

    • Routine use; supports non-fastidious bacteria.

    • Examples: Nutrient Agar, Potato Dextrose Agar.

  2. Enrichment Media

    • Enhances growth of particular organisms while suppressing normal flora.

    • Examples: Alkaline Peptone Water, Thioglycolate.

Selective Media Characteristics

  • Differential Media: Visualizes metabolic differences between groups.

  • Selective Media: Supports growth of specific bacteria while inhibiting others.

PART 2: General Steps in Preparation of Culture Medium

  1. Determine needed volume.

  2. Calculate component amounts.

  3. Adjust pH if necessary.

  4. Dispense in appropriate containers.

  5. Sterilize using an autoclave.

Inoculation Techniques

  • Streaking: Most common, using swabs and sterile loops.

  • Inoculating Tubed Media: Techniques differ for liquid, slant, and butt media.

  • Inoculating Plated Media: Includes streak plate and pour plate techniques.

Grading of Growth on Plate

  • 4+: Heavy growth throughout; up to 4th quadrant.

  • 3+: Moderate growth up to 3rd quadrant.

  • 2+: Light growth in 2nd quadrant.

  • 1+: Rare growth in 1st quadrant only.

PART 3: Anaerobic Cultivation

Facilitation Techniques

  • Culture medium with reducing agents (Thioglycollate).

  • Anaerobic chambers or gas-pak systems to eliminate oxygen.

Specimen Collection Methods

  • Aspirated with sterile needles/syringes; use anaerobic transport devices and avoid dry swabs.

Selective Media for Anaerobic Bacteria

  1. Kanamycin-Vancomycin Blood Agar - Anaerobic gram-negative rods.

  2. Neomycin-Vancomycin Blood Agar - Fusobacterium, Veilonella.

  3. Nagler Agar - Clostridia.