Culture Media Preparation and Sterilization

Media Preparation and Sterilization

• Objectives: Learn proper culture media preparation, aseptic technique in microbiology labs, importance of isolating bacteria, and cultivation/isolation techniques.

• Key Words: Agar, sterilization, aseptic techniques, inoculum, Five I's (Inoculation, Incubation, Isolation, Inspection, and Identification), colony (bacterial characteristics), culture.

• Media: A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of microorganisms or cells. It provides biophysical and biochemical factors for bacterial growth.

• Why Media is Needed: Microorganisms require nutrients, energy sources, and environmental conditions for growth and reproduction. Laboratory conditions must mimic these needs.

• Media Composition:

  • Hydrogen donors & acceptors (1-15 g/L)
  • Carbon source (1-20 g/L)
  • Nitrogen source (0.2-2 g/L)
  • Inorganic nutrients (e.g., S, P, 50mg/L)
  • Trace elements (0.1-1 μg/L)
  • Growth factors (amino acids, purines, pyrimidines, occasionally 50mg/L, vitamins 0.1-1 mg/L)
  • Solidifying agent (e.g., agar 10-20 g/L)
  • Solvent (usually distilled water)
  • Buffer chemicals

• Media Preparation: Required chemicals are dissolved in distilled water using Erlenmeyer flasks or tubes. pH is adjusted by adding NaOH or HCl. Liquid medium is measured (15ml in 100ml of media in a 250ml flask). The mixture is plugged with non-absorbent cotton and covered with brown paper. Autoclaving (15 lbs/inch² at 121°C for 15 minutes) sterilizes the media. The cooled media is poured into petri plates or tubes to solidify.

• Nutrient Agar Preparation: 28g of nutrient agar powder is suspended in 1 liter of distilled water. Heat while stirring to dissolve fully. Autoclave at 121°C for 15 minutes. Allow to cool but not solidify. Pour into sterile plates. Replace lids and store in fridge.

• Nutrient Agar Uses: Isolation and purification of cultures, producing bacterial lawns for antibiotic sensitivity tests.

• Types of Media (by chemical composition):

  • Synthetic/chemically defined media: Prepared by mixing pure chemicals of known composition (e.g., Czapek Dox medium).
  • Semi-synthetic/undefined media: Exact chemical composition is unknown (e.g., potato dextrose agar, MacConkey agar).

• Types of Media (by consistency):

  • Solid: (5-7% agar, or 10–20% gelatin) used for making slants or slopes or agar stabs.
  • Liquid: (no agar) used for making broths
  • Semi-solid: (0.5-0.7% agar) used to check motility, transport fragile organisms.

• Types of Media (by function):

  • Simple/Basal Media: Contain basic nutrients for most bacteria (e.g., nutrient broth, nutrient agar, peptone water).
  • Enriched Media: Contain extra nutrients to support fastidious bacteria (e.g., blood agar, chocolate agar).
  • Selective Media: Contain substances that inhibit the growth of unwanted microorganisms but allow the target bacteria to grow (e.g., Thayer-Martin Agar, EMB agar).
  • Differential Media: Contain substances that allow differentiation of bacteria based on their metabolic activity through color changes (e.g., MacConkey agar, XLD agar).
  • Transport Media: Support the viability of organisms during transport (e.g., thioglycollate broth, Stuart transport).
  • Indicator Media: Use indicators to detect specific metabolic activities (e.g., Wilson-Blair agar, McLeod's medium).
  • Anaerobic Media: Suppress oxygen and provide a reduced environment to support the growth of anaerobic bacteria (e.g., thioglycollate medium).

Cultivation of Bacteria

• Microbial Culture: A method of multiplying microorganisms in predetermined media under controlled laboratory conditions.

• Culture Purposes: Determine organism type and abundance, isolate bacteria, maintain stock cultures, antibiotic testing, create lab antigens. Solid media is also used for separating bacteria.

Culture Media Details

• Culture Media Needs: Proper media must provide similar environmental and nutritional conditions to the organism's natural habitat (water, carbon/energy source, nitrogen source, trace elements, growth factors).

Pure Culture

• Pure Culture Definition: A population of cells or organisms growing in the absence of other species; all cells are genetic clones.

• Requirements for Pure Culture: solid medium, sterile container, aseptic technique to separate the individual cells and to multiply the bacterium, which results in the creation of colonies.

• Agar: A polysaccharide extracted from marine algae used to solidify nutrient solutions. Its properties make it ideally suited for creating solid media.

Methods of Aseptic Technique

• Principles

  • Protect specimens from contamination.
  • Protect yourself and the lab from contamination.

• Methods

  • Sterilization: Removing all microorganisms (vegetative and spores). Methods include autoclaving, dry heat (hot air oven or flaming), filtration, or radiation.
  • Disinfection: Removal mostly of pathogenic microorganisms.
  • Antisepsis: Preventing or inhibiting the growth of microorganisms, but not killing them.

• Items to sterilize: Culture media, lab fluids, reagents, containers, equipment.

• Tips for Aseptic Technique: Disinfect the work area. Flame inoculating loops. Flame container openings. Do not put container lids on the benchtop. Work swiftly!

Types of Culture Methods

• Streak culture: Used for isolating bacteria from clinical samples. A loop is used to deposit small amounts of sample from a mixed culture on the surface and spread thinly in parallel lines.

• Lawn culture: A method of growing bacterial colonies on a thin uniform layer using a liquid suspension. Commonly used for serological, antibiotic testing.

• Stroke culture: Done in tubes containing agar slants/slopes. It provides a pure growth of the bacterium for purposes like slide agglutination tests.

• Stab culture: Prepared by puncturing a suitable medium (gelatin or glucose agar) with a long straight charged wire.

• Pour plate culture: A method used to estimate viable bacterial counts in a suspension by mixing the innoculum with the molten agar and pouring into sterile petri dishes.

• Spread plate culture: Adding a sample to the surface of an agar medium and spreading it evenly using a spreader.

Macroscopic Examination of Bacterial Colonies

• Characteristics: Observing and documenting the macroscopic characteristics of colonies on agar plates. These may include shape (circular, rhizoid, irregular), margin (entire, undulate), elevation (flat, raised), size, texture, appearance, pigmentation, and optical property.

Next Week's Lab

• Quiz
• hands-on activity
• Staining techniques in microbiology will be discussed.

Learn about culture media preparation and sterilization techniques. Understand aseptic techniques in microbiology labs. Covering the Five I's. Also learn the importance of isolating bacteria, and various cultivation/isolation techniques.