The Dichotomous Key...

Are these filaments strongly Gram positive ( 1 ) or Gram variable or weakly Gram positive ( 2 ) or Gram negative ( 3 ) ...?

Each filamentous microorganism present is separately evaluated for Gram staining and Neisser staining reaction by observing the stained smears at 900 - 1,000 x using transmitted light (not phase contrast). The position and length of filamentous microorganisms in the wet mount and the presence or absence of attached growth should be carefully noted so that the same filament types can be examined in the stained smears. Care is required in this observation because some filamentous microorganisms change size upon drying and staining (e.g. Type 0092 appears much wider when Neisser stained than in wet mounts. The Gram stain requires much practice. Reagents should be reasonably fresh (3 - 6 months) and, if possible, should be tested on fresh cultures of known Gram reaction. The decolorization step should be controlled precisely to avoid over - decolorization. Also, large flocs do not decolorize fully, so the Gram reactions inside large flocs should be ignored.
Score the Gram reaction as positive, negative, or variable. Most filamentous microorganisms observed in activated sludge are Gram negative. Nostocoida limicola and Type 0041 and 0675 most often are Gram positive but can be Gram variable or Gram negative. Type 1851 stains weakly Gram positive, and generally is observed as a chain of Gram positive "beads". Thiothrix I, Beggiatoa spp., Type 021N, and Type 0914 generally stain Gram negative, but may stain Gram positive when they contain substantial intracellular sulfur deposits. Microthrix parvicella and Nocardia spp. are generally strongly Gram positive. Neisser staining is a straightforward technique. Score as negative, positive (entire trichome is stained), or negative with Neisser - positive granules.
Type 0092 (light purple) and N.limicola (dark purple) stain entirely Neisser - positive. M.parvicella and Nocardia spp. stain Neisser - negative but generally contain Neisser - positive intracellular granules. Beggiatoa spp., Thiothrix spp., and Type 0041, 0675, 021N, 0914 and 1863 may contain Neisser - positive granules (infrequently). In addition, H.hydrossis and Type 0675 and 0041 may have a Neisser - positive trichome "covering" when present in activated sludge that is nutrient - deficient.