General Purpose Media (BNO) for Growing Fastidious Gram Negative (FGN) Bacteria

Abstract

General purpose media for growing fastidious pathogenic Gram negative (FGN) bacteria are limited to non-selective blood and chocolate agar. These suffer from excessive contamination with fungi, Gram positive, and Gram negative bacteria, especially Pseudomonas spp. MacConkey agar can be modified to grow Haemophilus, Helicobacter, Campylobacter and other FGN without the use of antibiotics. BNO100 and BNO70 media were based on MacConkey medium. BNO1-4 were Blood Agar Base containing crystal violet and no bile salts. All media were made as chocolate agar with 8% defibrinated sheep blood, yeast extract 0.5%, glycerol 0.5% (v/v), vitamins, and agar. Campylobacter jejuni ATCC29428 and Haemophilus influenzae were able to grow on all described media including BNO100. Helicobacter pylori ATCC43504 grew on all BNO media but not BNO100. Most BNO media were inhibitory for the growth of several Gram positive bacteria; Streptococcus pneumoniae, Staphylococcus epidermidis, Bacillus atrophaeus QUBC16, Staphylococcus aureus including three MRSA clinical isolates. C. jejuni and H. pylori were successfully grown on BNO media containing Dent’s, Skirrow’s, or Line’s selective antibiotic-supplements. MacConkey based BNO Chocolate agar media may be used as general purpose selective media for Gram negative bacteria including FGNs. We recommend the utilization of BNO70 and BNO2 for research and clinical sample processing involving FGN bacteria. The antibiotic-free BNO media reduced contamination with Gram positive and fungi. The incorporation of selective antibiotics and antifungal agents was compatible with the formulated BNO media; whereas Pseudomonas continued to pause a serious contamination problem.