I. Preparing Anaerobic Cultivation of Wildlife’s Fecal Bacteria

Preparing anaerobic cultivation of wildlife fecal bacteria involves several steps to ensure a proper anaerobic environment and appropriate culture conditions. Here’s a general guideline:

  1. Collection of Fecal Samples:
  • Collect fresh fecal samples from wildlife to ensure a high viability of bacteria.
  • Store samples in sterile containers and keep them cool during transport to the laboratory.
  1. Sample Preparation:
  • Homogenize the fecal samples in a sterile saline solution or appropriate buffer to create a uniform suspension.
  • Depending on the requirements, dilute the samples to obtain the desired concentration of bacteria.
  1. Anaerobic Environment:
  • Anaerobic cultivation requires an environment devoid of oxygen. Use an anaerobic chamber or anaerobic jars with gas packs that create an anaerobic atmosphere.
  • Ensure all materials and media entering the anaerobic chamber are pre-reduced to remove oxygen.
  1. Culture Media:
  • Select appropriate anaerobic culture media. This can include pre-reduced agar plates or broths specific for the bacteria of interest.
  • Media supplements like vitamin K, hemin, or other growth factors may be necessary, depending on the bacterial species.
  1. Inoculation and Incubation:
  • Inoculate the media with the fecal suspension using sterile techniques.
  • Incubate under anaerobic conditions at temperatures suitable for the bacteria you’re cultivating. This is typically around 37°C, but can vary.
  1. Monitoring and Analysis:
  • Regularly check the cultures for growth. This might include visual inspection or using methods like gas chromatography to monitor metabolic products.
  • Once sufficient growth is achieved, you can proceed with identification and analysis of the bacteria using techniques such as 16S rRNA sequencing, MALDI-TOF MS, or biochemical tests.
  1. Safety Precautions:
  • Always follow biosafety guidelines as fecal samples from wildlife can contain pathogens.
  • Use personal protective equipment (PPE) and work in a biological safety cabinet when necessary.

Remember, the specific procedures can vary based on the type of bacteria you are interested in and the wildlife source. Consult relevant literature and safety guidelines for more detailed protocols and ensure all activities comply with ethical and legal regulations regarding wildlife sampling.

II. Transport Media

Transport media are special media used for the safe and stable transfer of clinical specimens, including fecal samples, from the collection site to the testing laboratory. These media are designed to preserve the viability of the microorganisms in the sample without allowing for significant growth or reproduction, ensuring that the sample remains as close to its original state as possible. Here are some common types of transport media:

  1. Amies Transport Medium:
  • A medium commonly used for transporting swab specimens. It helps maintain the viability of a wide variety of organisms.
  1. Cary-Blair Transport Medium:
  • Ideal for transporting fecal samples, especially for the isolation of enteric pathogens. This medium is particularly useful for preserving Salmonella, Shigella, and Campylobacter.
  1. Stuart’s Transport Medium:
  • Similar to Amies, it is often used for throat, vaginal, and wound swabs. It maintains the viability of aerobic and anaerobic organisms.
  1. Buffered Glycerol Saline:
  • Used primarily for fecal specimens, particularly for the isolation of Vibrio species.
  1. Modified Thayer-Martin (MTM) Medium:
  • Used for transporting specimens for Neisseria gonorrhoeae culture.
  1. Regan-Lowe Medium:
  • Used for transporting Bordetella pertussis.
  1. Selenite F Broth:
  • Often used for the enrichment of Salmonella spp. from fecal samples before plating on solid media.
  1. Boric Acid Transport Medium:
  • Used for urine specimens, particularly for urinary tract infections.

These media are formulated to maintain the pH and osmotic balance and often contain substances that inhibit bacterial overgrowth, fungal contamination, and also prevent the cells from dying. The choice of transport medium depends on the type of specimen being collected and the suspected pathogen. It’s important to use the correct transport medium to ensure the viability of the sample for accurate diagnosis and research.

III. Ingredients of Cary-Blair Transport Medium

  1. Sodium Chloride (NaCl)
    • Maintains the osmotic balance in the medium.
  2. Disodium Phosphate (Na₂HPO₄)
    • Serves as a buffering agent to maintain the pH.
  3. Monopotassium Phosphate (KH₂PO₄)
    • Another component of the buffering system.
  4. Calcium Chloride (CaCl₂)
    • Provides essential calcium ions which can be beneficial for certain bacteria.
  5. Agar
    • A gelling agent that thickens the medium slightly without creating a solid gel, helping to trap bacteria and reduce exposure to potentially harmful substances.
  6. Water (H₂O)
    • Used as the solvent for all the components.
  7. Sodium Thioglycollate
    • Acts as a reducing agent to maintain a reduced environment, particularly beneficial for anaerobic bacteria.
  8. pH Indicator (Optional)
    • Substances like bromothymol blue may be added to indicate changes in the medium’s pH.

IV. Preparation of Cary-Blair Transport Medium

  1. Dissolving the Salts
    • Mix the following salts in about 800 mL of distilled water:
      • Sodium Chloride (NaCl): 5.0 g
      • Disodium Phosphate (Na₂HPO₄): 1.1 g
      • Monopotassium Phosphate (KH₂PO₄): 0.6 g
      • Calcium Chloride (CaCl₂): 0.1 g
  2. Adding Agar
    • Add Agar: 5.0 g
    • Heat the mixture gently to dissolve the agar completely.
  3. Adjusting the pH
    • After dissolving the agar, check the pH of the medium.
    • Adjust the pH to 8.4 ± 0.2, if necessary, using a pH meter.
  4. Adding Reducing Agent
    • Once the pH is adjusted, add Sodium Thioglycollate: 1.0 g.
    • Heat again to ensure complete dissolution.
  5. Sterilization
    • Autoclave the mixture at 121°C for 15 minutes.
    • After autoclaving, cool the medium to about 50°C.
  6. Dispensing
    • Pour the medium into sterile tubes or containers.
    • If using a pH indicator, add it before dispensing the medium.
  7. Cooling and Storage
    • Allow the medium to solidify and cool to room temperature.
    • Store the prepared medium at 2-8°C.

Notes

  • Ensure all equipment and containers are sterilized before use.
  • Wear appropriate protective equipment (gloves, lab coat, eye protection) during preparation.
  • The medium should be clear and light amber in color.
  • Discard any unused medium after the expiration date.

V. Using Cary-Blair Transport Medium for Fecal Sample Collection

  1. Gathering Necessary Materials
    • Cary-Blair Transport Medium container
    • Sterile gloves
    • Sterile specimen collection tool (e.g., swab or spatula)
  2. Wearing Protective Gear
    • Put on sterile gloves before handling the specimen to maintain sterility and ensure personal safety.
  3. Collecting the Fecal Sample
    • Using the sterile collection tool, take a small amount of feces.
    • Aim for pea-sized amount or as directed by the testing protocol.
    • Ensure the sample is representative of the feces, avoiding areas with urine or water contamination.
  4. Transferring the Sample
    • Carefully place the fecal sample into the container with Cary-Blair Transport Medium.
    • Avoid overfilling; a small amount is usually sufficient for analysis.
  5. Securing the Container
    • Close the container tightly to prevent leaks and contamination.
  6. Labeling
    • Label the container with the patient’s information, date, and time of collection.
  7. Transporting the Sample
    • Transport the sample to the laboratory as soon as possible.
    • If immediate transport is not possible, store the sample at 2-8°C.
  8. Handling and Disposal
    • Handle all specimens as if they are infectious.
    • Dispose of gloves and other used materials in a biohazard waste container.

Important Notes

  • Do not overfill the container as it can dilute the transport medium and affect the test results.
  • Ensure the fecal matter is in contact with the transport medium.
  • The sample should be processed as soon as possible, ideally within 48 hours of collection.
  • Follow all relevant guidelines and protocols for specimen handling and transport.