Additionally, human samples of debrided burns were provided in a de-identified fashion by the United States Army Institute of Surgical Research Pathology Department. The use of human samples was approved by the institutional regulatory department.
Slides were deparaffinized, cleared in xylene, and rehydrated in preparation for staining. Antibody specificity for S. Smears were then incubated with the following primary antibodies for 1 h at room temperature: S. Following incubation, P. In addition to antibody specific staining, Gram stain was employed in vitro using standard protocol as described by the manufacturer Remel, Lenexa, KS. For staining of normal skin and infected burn biopsies ex vivo, serial tissue sections were obtained as described above.
To verify the presence of P. Gram stain was performed similarly to the smears described above with minor modifications Fig. Briefly, crystal violet was applied to the tissue sections for 5 min at room temperature, and slides were briefly rinsed under running tap water to remove excess crystal violet.
Gram iodine mordant was applied for 2 min to the tissue sections and briefly washed in tap water. To remove any non-specific crystal violet staining, a Gram decolorizer solvent was applied to the slides for 30 s then quickly rinsed under running tap water until the water ran clear. Schematic showing the modified Gram stain procedure. The traditional Gram stain procedure is completed in either case top box.
The major difference in the modified Gram stain procedure occurs during the dehydration process and is the application of alcoholic saffron lower right box , highlighted in red. The yellow contrast in the connective tissue collagen within skin tissue is seen in the low magnification images on the bottom Scale bar 1 mm. Images of entire wound biopsies both porcine and human were obtained using an AxioScan Z1 slide scanner Carl Zeiss, Inc.
Brightfield images were taken, as were fluorescent images using nm P. Gram stain is the most common staining technique used diagnostically within both the clinical setting and in research laboratories to differentiate between Gram positive and Gram negative microorganisms in various types of tissues [ 17 , 26 ].
As seen in Fig. Moreover this distinction is still evident when applied to both large aggregates and individual detached bacteria within biofilms despite the presence of structures within the polymeric matrix such as carbohydrates, lipids and proteins that have the potential to interfere with the stain [ 32 ].
For a more detailed classification of the bacterial strains used in this study, antibodies specific for S. In vitro validation of applied stains. Routine Gram stain shows identification of Gram negative a P. Selected P. Histologically, ex vivo detection of bacterial biofilms has been completed with expensive and time consuming techniques such as electron microscopy and confocal microscopy [ 33 , 34 ]. Light microscopy has been pursued for biofilm localization in conditions such as rhinosinusitis [ 24 , 25 , 35 ].
However, in the case of burn wounds, non-specific staining of connective tissue, cellular debris, and other proteins present within the eschar occurs due to the absorption of the crystal violet dye used in the Gram stain [ 15 ].
Also, since the eschar provides essential nutrients that support bacterial growth, it is common to find colonizing bacteria at this site [ 11 ].
In this study, we employed a porcine model of infected burns to explore a modification of the traditional Gram stain that provides contrast with connective tissue to allow for better visualization of bacteria. Pigs are the ideal choice for studying skin wounds due to similarities to human skin.
For example, pig skin and human skin are similar in their epidermal and dermal thicknesses, collagen content in both papillary and reticular dermal layers, distribution of hair follicles, and healing patterns i.
Initially, immunohistochemistry was performed with the previously verified antibodies to identify P. Figure 3 a, d show that these bacteria are not present within normal, non-burned pig skin. In Fig. Importantly, no reactivity of the P. Similarly, the S. Bacteria localization was also found to be different between the two species. Notably, this distribution of S. Visualization of successful bacterial inoculation ex vivo.
Normal pig skin a , d , P. Note that these antibodies allow for specific labeling of bacteria ex vivo as indicated by arrows pointing out unique bacterial morphologies. Despite the effectiveness of immunohistochemical techniques, the use of antibodies is time-consuming and expensive, and also requires prior knowledge of the bacterial species residing in the tissue of interest.
Moreover, the majority of wound infections encountered are polymicrobial. Following infection with both P. This is, in part, due to the amount of dye retained within the tissue section non-specifically i. A modified Gram stain improves bacterial detection in tissue sections. Serial sections of normal porcine skin a , d , g , P.
Gram stain readily differentiates the clusters as Gram positive or Gram negative bacteria e , f , however there is lack of contrast in the tissue in all samples. This lack of contrast is alleviated in the modified Gram, stain which distinctly enhances the detection of bacterial clusters as Gram negative h or Gram positive i. The Gram stain has also been routinely used for in vitro and clinical samples alike, where cultures from patients are collected for bacterial identification [ 42 ].
Although Gram stain is a fast, effective and inexpensive technique for bacterial differentiation, it does not allow for visualization of connective tissue i. This is primarily attributed to the lack of counterstain e. The Gram stain is able to differentiate between Gram positive and Gram negative microorganisms ex vivo, as seen in Fig.
However, non-specific staining of damaged tissue is also apparent, which can ultimately overestimate bacterial colonization in tissues. These observations lead us to conclude that although the Gram stain is the standard staining technique in vitro, its efficiency ex vivo can lead to subjective interpretation, and also fails to illustrate important aspects of tissue morphology i.
Given the limitations of the two previously described stains we sought to develop an inexpensive and fast addition to the Gram stain that highlights tissue structure. To achieve this, we modified the Gram stain by incorporating alcoholic saffron as a contrasting dye.
Although a similar stain has recently been reported by Roche et al. Alcoholic saffron has been traditionally used in other staining techniques i. While collagen structure is greatly altered following burn injury as evidenced by coagulation seen in Fig.
Importantly, this modification of the Gram stain retains the ability to distinguish both Gram positive Fig. The contrast provided by the alcoholic saffron allows bacteria to be detected deep within the burn eschar when compared to the Gram stain alone Fig. At high magnification, the morphology of the bacteria i. In order to test the clinical applicability of the modified Gram technique, the same staining comparison was performed on surgical biopsies from a de-identified patient Fig.
The selection criteria for the biopsy were for a patient undergoing burn debridement, identified as having a polymicrobial infection.
Of note, dye retention during the Gram stain process led to non-specific staining, especially in the skeletal muscle Fig. This was slightly less apparent in the dermis Fig. However, high magnification of the modified Gram stain clearly demonstrated bacterial morphology within dermal connective tissue Fig. The modified gram stain applied to clinical surgical biopsies. Serial sections of tissue from a patient that underwent burn debridement were stained with three different stains.
Closer inspection of the Gram stain in both skin e and muscle f areas reveal some areas of bacterial morphology that are more apparent where host connective tissue is more sparse.
Closer inspection of the modified Gram stain in both skin h and muscle i illustrates a counterstain that allows for identification of bacterial morphology arrows within connective tissue.
While a wide variety of molecular techniques e. However, histopathology will always have a place for diagnosis of different tissues, and a quick, inexpensive, detailed, and reproducible histological technique for bacteria identification and localization is desired. The modified Gram stain we have described herein improves on the traditional Gram stain by providing contrast of connective tissues. The incubation time in alcoholic saffron for these tissues was found to be optimal at 4 min.
Less time led to lighter staining of the connective tissue, while longer incubation in alcohol began to remove the Gram stain components. This 4 min incubation represents a short addition to the staining process already in clinical use. While in this study we utilized paraffin embedding that takes several days of processing, a flash-freezing strategy could potentially allow for bacterial identification in less than an hour from biopsy collection [ 46 ].
There are potential limitations for implementing this stain. For example, certain species e. However, the applications examined herein were aimed at addressing clinically relevant species of bacteria most often associated with wound infections. As such, these most common species present within chronic non-healing wounds would be detected with the technique described herein.
Additionally, this staining technique does not provide any evidence for the species of bacteria present in tissue, which would need to be addressed with subsequent diagnostic tools. Also, tissues used in the current study have been optimized for infection which may make detection easier compared to a lesser amount of bacteria that may be present in an acute wound.
Determining whether or not wounds that contain fewer bacteria would benefit from this stain is a point of further study. We have described a histological technique that allows for visualization of tissue structures along with detection of Gram positive and Gram negative bacteria. Wash briefly with water and shake off excess. Add about 5 drops of iodine solution to the culture. Let stand for 30 seconds, wash briefly with water and shake off excess.
Tilt slide and decolorize with solvent acetone-alcohol solution until purple color stops running. Be careful not to over-decolorize. Wash immediately within 5 seconds with water and shake off excess. Add about 5 drops of Safranine O. Let stand for one minute, wash briefly with water and shake off excess. Staphylococcus Mostly harmless sphere-shaped, skin bacteria ; Streptococcus rod shaped bacteria: Rheumatic fever, Scarlet fever.
How does your stain look? You can test your reagents against a known negative sample and a known positive sample. The oral bacteria present will show positive and negative in a good stain.
If only one result shows up, either your staining method was flawed or there was a problem with the reagent used. If everything shows negative red you can shorten the decolorization time. If everything shows positive purple , try a slightly longer decolorization period. Is your Gram Stain positive or negative? See if the organisms show Grams-Negative red or Grams-Positive purple. It does so by colouring a portion of a specimen e. The Safranin is the counterstain used in this method.
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