How do I enrich for anaerobic bacteria (Megasphaera, Pectinatus, L. acetotolerans)?

These bacteria require anaerobic (oxygen-devoid) growth enrichments, meaning there should be no air exposure for optimal growth. Anaerobic bacteria die easily in an aerobic environment, so it is very difficult to grow and detect these species even with enrichment and PCR. Your enrichment container should contain 50% enrichment solution and 50% sample and leave no room for air exposure—i.e., if you utilize a 50 mL container, you should include 25 mL of FastOrange B solution and 25 mL of your sample.

To enrich, add 50% of sample and 50% of FastOrange B solution to your enrichment container to completely fill up the volume, topping off as necessary to ensure the solution reaches the rim of the container. Transfer your sample and FastOrange B solution using sterile technique. Incubate the container vertically at room temperature (25 °C +/- 2 °C) and do not vent to preserve the anaerobic environment. We recommend using sterile 50 mL Falcon or Eppendorf tubes or glass bottles, utilizing 25 mL of your sample and 25 mL FastOrange B Broth and topping off with sample to ensure that there is no air exposure in the container. The FastOrange B Enrichment Bottles are not recommended for this bacteria enrichment due to pressure buildup resulting from bacterial growth. 

Enriching your samples before running direct PCR is recommended to improve sensitivity but requires extended incubation times. If enriching for L. acetotolerans, incubate the sample for at least 5 days. Sensitivity can increase by enriching up to 10 days. If enriching for Megasphaera and Pectinatus, incubate for a minimum of 7 days. Sensitivity can increase by enriching up to 12 days. Some anaerobic bacteria grow slowly and may have a long growth lag phase if exposed to oxygen during sampling or the enrichment process.