Pierson and Howard developed two techniques for detecting bacteriochlorophyll-containing photosynthetic bacteria using infrared fluorescence microscopy. Purple photosynthetic bacteria contain bacteriochlorophylls that fluoresce in the near infrared (~900 nm), invisible to standard light microscopy. The first technique used hypersensitized Eastman spectroscopic safety film type I-M mounted on a microscope with a mercury arc lamp, filters to isolate exciting wavelengths, and a Kodak Wratten 87C barrier filter. Exposures of 10-15 minutes captured infrared fluorescence images of bacteriochlorophyll-containing organisms including Rhodospirillum rubrum and filamentous bacteria. The second technique employed a Wild M500 infrared image-converter unit that converted infrared fluorescence to visible images viewable through an eyepiece, with shorter exposure times of 2-7 minutes. Both methods successfully distinguished bacteriochlorophyll-containing bacteria from morphologically similar organisms like blue-green algae and heterotrophic bacteria in mixed environmental samples. The film method proved inexpensive and widely applicable but suffered from long exposures and focusing difficulties. The image-converter technique provided faster results and easier observation but required specialized equipment.

Key findings

• Two novel infrared fluorescence microscopy techniques successfully detected bacteriochlorophyll fluorescence at ~900 nm, previously invisible with standard microscopy
• Hypersensitized infrared film and a Wild infrared image-converter unit both enabled identification of photosynthetic bacteria in mixed microbial samples
• These techniques distinguished bacteriochlorophyll-containing filaments from morphologically identical organisms like Phormidium cyanobacteria and Herpetosiphon heterotrophs in environmental samples
• The infrared image-converter provided faster results (2-7 minute exposures) and better focusing compared to film technique (10-15 minute exposures)
• Bacteriochlorophyll fluorescence was eliminated when pigments were extracted with methanol, confirming the fluorescence source