Can anyone explain to me what I am seeing? I cannot tell if I have imaged the production of cell capsules or a biofilm, or if I have something more interesting. I am familiar with the theory of multi-phase spp. (rod to cocci/cyst), but not a gram-positive rod with a gram-negative cocci/cyst phase. I also am always hunting for bacterial symbionts, but the conserved structures found in these stains are new to me. Please contribute if you have any knowledge about such mysteries! Details and photos below.
In vitro culture of gram-positive rod, obligate anaerobe, thus far no sporulation, two morphologies present on BAP (1: clear, smooth, flat, irregular, lobate, ~4-5 mm @ 48 hrs and 37 C; 2: clear margin with white center, smooth, umbonate, round, entire to spreading, ~2-3 mm @ 48 hrs and 37C). First streak zone of initial inoculation appeared to be of mixed culture (clear smooth lawn with rare raised, clear bumps), suspected two GPR spp (Clostridium or Bacillus spp).
Both colony morphologies present the same cell morphologies under gram stain: uniform gram-positive rods (some anaerobic staining variability) with gram-negative granule (for lack of a better word) attached at the rod's side or point of division. Additionally, this granule appears to take on a consistent "D" shaped structure when attached to cells that have formed filaments, or when it is found in free space on the slide.
I am familiar with some KOH limitations on anaerobes (false-positives for Bacteriods, Bacillus cereus, B. macerans), but KOH reaction seems indicative of gram-positive/negative mixed culture (KOH test positive from streak zones of potential mixed culture, but negative from isolated colonies on same culture plate). Occasional cells that look like true GNR present which are shorter and not as thick as GPR present, with almost a serif look to each end (like an angular barbell or I-beam).
Please forgive resolution of attached images. Thank you.
that pink color structures attached to the bacteria could be biofilm as you are suspecting, but I wonder why don't you amplify the 16s rRNA, and identify to the species level, what bacteria do you have ?
good luck
If you did not grow the bacteria anaerobically they will not be Clostridium. Both Clostridium and Bacillus will develop endospores on most media if you let the plates incubate for more days.
While it is probably not a cell capsule, did you do a negative stain or capsule stain to see if any of the bacteria have a capsule?
Thank you for taking the time to reply and help answer my question. The organism(s) in question is strictly anaerobic, and I have had no classic endospore production as seen in Gram or Acid Fast staining over 7 days of incubation. I do not carry a capsule specific stain, unfortunately.
I will obtain a molecular ID at some point in the near future, but wanted to get the question out here in case the structures documented were recognized by anyone here as typical for a particular species. My work is clinically based, so I only extract DNA for molecular ID and research purposes once every 2-3 months.
More updates to come....
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