A scanning electron microscope survey was initiated to determine if the previously reported findings of “dinosaurian soft tissues” could be identified in situ within the bones. The results obtained allowed a reinterpretation of the formation and preservation of several types of these “tissues” and their content. Mineralized and non-mineralized coatings were found extensively in the porous trabecular bone of a variety of dinosaur and mammal species across time. They represent bacterial biofilms common throughout nature. Biofilms form endocasts and once dissolved out of the bone, mimic real blood vessels and osteocytes. Bridged trails observed in biofilms indicate that a previously viscous film was populated with swimming bacteria. Carbon dating of the film points to its relatively modern origin. A comparison of infrared spectra of modern biofilms with modern collagen and fossil bone coatings suggests that modern biofilms share a closer molecular make-up than modern collagen to the coatings from fossil bones. Blood cell size iron-oxygen spheres found in the vessels were identified as an oxidized form of formerly pyritic framboids. Our observations appeal to a more conservative explanation for the structures found preserved in fossil bone.

Materials and Methods

Scanning electron microscopes used were JEOL T300, Zeiss Supra and Cambridge S200. Light microscopes were Zeiss Axiomat, Nikon SMZ-U and Unitron. EDS spectra were taken with Kevex Delta 5, EDAX Apollo 40 running under EDAX Genesis/Pegasus and Link LZ5 running under WinEDS.

SEM specimens were prepared by pressure fracturing and selected pieces approximately 10 mm square were fixed to aluminum stubs with high purity carbon tabs. Initial specimens were uncoated to minimize potential disruption of internal contents. Subsequent specimens were gold coated with a Bio-Rad E5000 sputter coater or Denton Desk IV turbo coater with Pd or Pt target. Low dot-pitch element maps were run at 15 kV. The maps of individual elements were combined with a high-resolution secondary electron images to produce a high resolution colored element maps.

Modern biofilms were grown with the following method. Two gallons of water obtained from a local pond was placed in a new five gallon bucket and recirculated with a small pump at room temperature. Five new microscope slides were placed in the bottom with 100 mg of glucose nutrient added to the water. Water samples were taken every two days to monitor microbe population. One slide was removed and examined under the light microscope every few days to monitor biofilm accumulation rate. Slides were allowed to desiccate at room temperature over several days. Microbial communities could clearly be seen in the hydrated biofilms under the light microscope but subsequent examination under SEM showed only a smooth undulating profile.

Fourier Transform Infrared Spectroscopy (FT-IR) was used to investigate the specimens’ molecular structure. A turtle carapace from the Hell Creek formation was selected for spectroscopy because of its proportionally large chambers in the trabecular bone that allowed scraping the coatings loose. Two milligrams of material was ground with 450 milligrams of potassium bromide (KBr) and pressed into a pellet using 8 tons pressure. Modern biofilms grown on microscope slides in pond water were allowed to desiccate for 7 days and 2.5 milligrams were pressed into a KBr pellet as above. A 2.5 milligram sample of desiccated tendon from a chicken was ground with KBr and pelletized. Spectrums were taken on a Nicolet 510P bench at 1 cm⁻¹ resolution with a minimum of 15 scans. Infrared flux was matched within 5% for all specimens and a clean KBr pellet used for background subtraction between specimens. Excel cross correlation routines were used to determine percentage of similarity for spectrums.

Framboids were individually extracted from fractured dinosaur bone fragments with a magnet and transferred to a carbon sticky tab on an SEM stub. EDS was performed on a small section of the sphere at 20 kV in the area shown with the spectrum in figure 1.

Figure 1. EDS spectrum of framboid.

EDS spectrum of framboid showing an iron-oxygen signature. Pt is from coating for SEM. Area in red box was scanned for elements.

 

Multiple specimens were pressure fractured and 10–20 mm fragments selected for demineralization in 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH 8.0) in individual plastic containers at room temperature. Resident times ranged from several days to several weeks depending on specimen resistance. Baths were changed at approximately three day intervals with fresh acid. Remaining structures were either photographed directly in the baths at low magnification 7–75× or removed for higher power imaging.

All specimens for carbon dating were handled under a flow hood with clean sterile gloves and instruments. The specimens were pressure fractured to reveal fresh surfaces. A bone fragment from the Lance formation was microscopically examined and coatings that appeared to have been dislodged were removed for analysis. Fifty milligrams of material were sent to Geochron Labs, Cambridge Mass. for accelerator mass spectrometry (AMS) analysis. The results were 139.01%±0.65 of modern (1950) of ¹⁴C activity.

Figure 2. Well preserved complete bone used in initial investigation.

Exceptionally well preserved small phalange from the Lance formation used for initial survey. No cracks or deformities present. Specimen was pressure fractured and directly examined under the SEM. UWBM 89327 Scale bar, 10 mm.

Figure 3. Iron oxide framboids.

An iron oxide framboid cluster in dinosaur trabecular bone found commonly throughout time and taxa. At approximately 10 microns in diameter they are closely matched in size to red blood cells and typical pyrite framboids. UWBM 89327 Scale bar, 3 µm.

Citation: Kaye TG, Gaugler G, Sawlowicz Z (2008) Dinosaurian Soft Tissues Interpreted as Bacterial Biofilms. PLoS ONE 3(7): e2808. doi:10.1371/journal.pone.0002808

Editor: Anna Stepanova, Paleontological Institute, Russian Federation