Rapid Staining Research Articles

Research Techniques Made Simple: Optical Clearing and Three-Dimensional Volumetric Imaging of Skin Biopsies

Skin histology is traditionally carried out using two-dimensional tissue sections, which allows for rapid staining, but these sections cannot accurately represent three-dimensional structures in skin such as nerves, vasculature, hair follicles, and sebaceous glands. Although it may be ideal to image skin in a three-dimensional manner, it is technically challenging to image deep into tissue because of light scattering from collagen fibrils in the dermis and refractive index mismatch owing to the presence of differing biological materials such as cytoplasm, and lipids in the skin. Different optical clearing methods have been developed recently, making it possible to render tissues transparent using different approaches. Here, we discuss the steps involved in tissue preparation for three-dimensional volumetric imaging and provide a brief overview of the different optical clearing methods as well as different imaging modalities for three-dimensional imaging.

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue.

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

Leukocyte Image Segmentation Based on Adaptive Histogram Thresholding and Contour Detection

Aims: The proposed method falls into the category of medical image processing. Background: Computer-aided automatic analysis systems for the analysis and cytometry of leukocyte (White Blood Cells, WBCs) in human blood smear images are a powerful diagnostic tool for many types of diseases, such as anemia, malaria, syphilis, heavy metal poisoning, and leukemia. Leukocyte segmentation is a basis of its automatic analysis, and the segmentation accuracy will directly influence the reliability of image-based automatic leukocyte analysis. Objective: This paper aims to present a leukocyte segmentation method, which improves segmentation accuracy under rapid and standard staining conditions. Methods: The proposed method first localizes leukocytes by color component combination and Adaptive Histogram Thresholding (AHT), and crops sub-image corresponding to each leukocyte. Then, the proposed method employs AHT to extract the nucleus of leukocyte and utilizes image color features to remove image backgrounds such as red blood cells and dyeing impurities. Finally, Canny edge detection is performed to extract the entire leukocyte. Accordingly, cytoplasm is obtained by subtracting nucleus with leukocyte. Results: Experimental results on two datasets containing 160 leukocyte images show that the proposed method obtains more accurate segmentation results than their counterparts. Conclusion: The proposed method obtains more accurate segmentation results than their counterparts under rapid and standard staining conditions.

Utility of rapid brillant cresyl blue stained wet mount preparation in routine fine needle aspiration cytology

Fine Needle Aspiration Cytology is the preliminary investigation employed for superficial and deep swelling in the body. It provides early information regarding the lesions before performing biopsy and aid in the treatment plan of the patient. However sample inadequacy from representative lesion may render patients to repeat aspirations and may also cause delay in reports. There is an ever increasing demand for rapid diagnosis in cytology. The present study was conducted to assess the utility of brilliant cresyl blue as a rapid stain in fine needle aspiration cytology. To assess the utility of rapid Brilliant cresyl blue (BCB) stained wet mount preparation in routine fine needle aspiration cytology. The study was carried out in the Cytology laboratory, Govt. Medical College Thiruvananthapuram and PG Pathology Lab, Department of MLT, Govt. Medical College Thiruvananthapuram for a period of six months. A total of 50 cases referred to cytology laboratory for FNAC were included in this study. A wet mount preparation with Brilliant cresyl was prepared immediately after aspiration and examined for cellular adequacy and cytomorphology, which was then compared with the routine Papanicolau stained smear . In situations for rapid diagnosis, and to assess the cellular adequacy of a lesion Brilliant cresyl blue staining method is very helpful. Most of the cases diagnosed as malignant on Brilliant cresyl blue staining correlate with the Pap stain. All the cases diagnosed as benign on rapid brilliant cresyl blue stain were also diagnosed as benign with Pap stain. The result clearly indicate that the brilliant cresyl blue stained wet mount preparation can be used as a rapid supplementary diagnostic procedure in FNAC at least for urgent reporting and sample adequacy assessment.

Cervical cytochemistry: a potential adjunct to cytomorphology for detection of abnormal cells in cervical cancer screening

Background: Pap smear examination has been universally used as an effective screening tool for early detection of cervical carcinoma. The aim of this study was to assess the utility of Cervical Acid Phosphatase staining as an adjunct to routine Pap smear testing to improvethe sensitivity and specificity of routine Pap smear examination for cervical cancer detection.
 Materials and Methods: Cervical smears were taken from patients attending the gynecology department and a few cervical cancer screening programmes. One set of slides were alcohol fixed and stained with rapid pap stain and another set of slides were fixed in a special fixative and stained with Cervical Acid Phosphatase -Pap stain. The nuclear features of these Cervical Acid Phosphatase stained dysplastic cells was studied on Pap stain to diagnose cervical intraepithelial lesion/ malignancy.
 Results: Out of 489 cases included in the study 6 cases were diagnosed with intraepithelial lesion/ malignancy. On Cervical Acid Phosphatase -Pap stain 2 of the cases diagnosed as inflammatory smears on pap stain showed Cervical Acid Phosphatase positivity and thus were re evaluated. Mild nuclear atypia was observed in the Cervical Acid Phosphatase positive cells and these cases were diagnosed as Low grade squamous intraepithelial lesion and later biopsy proven to be Cervical intraepithelial Neoplasia I. Therefore Cervical Acid Phosphatase -Pap test was 100% sensitive and specific for cervical cancer detection.
 Conclusions: With 100% sensitivity Cervical Acid Phosphatase -Pap test satisfies the criteria of an efficient screening test.

Handling of Thyroid FNA Samples During the COVID-19 Pandemic

Handling of Thyroid FNA Samples During the COVID-19 Pandemic

Rapid stain biomarker: A novel tool for forensic detection of semen (effect of time, different fabrics and vaginal secretions on its reliability)

Abstract Background The identification of semen stain is one of the most common human stains that can provide crucial information for crime scene reconstruction and forensic investigation. In sexual assault cases semen identification helps to prove victim’s allegations it also provides a material for DNA analysis that generate the genetic profile of the alleged suspect. The rapid Stain Identification of Human Semen (RSIDTM-Semen) bioassay is designed to detect specifically the presence of human semenogelin. Test development is completed within 10 minutes and can detect as little as 2.5 nL of human semen and it does not cross-react with other human or nonhuman tissues. The detection protocol can be completely integrated into the procedures for DNA extraction and analysis. Aim To assess the efficacy of RSIDTM – Semen strip test for the detection of human semen under some different variables (different fabrics, different time intervals and mixed with vaginal secretions) in order to be used in the routine forensic work of sexual assault cases and for further personal identification. Methodology: Semen samples were collected from four male participants. Vaginal specimens were collected from four female participants on cotton, linen or nylon-tipped swabs (2 swabs from each female). Each semen sample was divided into two portions; one used for semen only test group and the other mixed with vaginal secretions for the mixed test group. One of the fabrics tipped vaginal swab was mixed with semen for the mixed test group and the other used as a positive control group to test the sensitivity and specificity of the RSIDTM – Semen strip. The semen samples were deposited over different fabrics at the same time. All the samples were left to dry for 15 minutes at room temperature then extracted and analyzed according to the protocol designed for The Rapid Stain Identification Test (RSIDTM – Semen). The collected samples were studied as two test groups and one control group. Each of the previous groups, was categorized into 4 subgroups (a, b, c and d) according to the time interval of semen extraction (zero (on the spot), 2, 4, 6 and 10 days respectively). Results Semen could be identified in 100% of tested samples of the semen only group as well as of the combined semen and vagina group over cotton and linen fabrics at all the different tested time intervals. However, semen extracted from nylon fabric was identified in tested samples of the semen only group and of the combined semen and vagina group only at zero time only and couldn’t be identified at the rest of tested time intervals. Conclusion the current study evidenced that the new RSIDTM-semen kit is a reliable method for semen identification over different types of fabrics even in the presence of vaginal secretions. It also resists environmental factors up to 10 days.

Clinical Pattern of Inflammatory bowel disease in central reigon, Saudi Arabia

Abstract Background The identification of semen stain is one of the most common human stains that can provide crucial information for crime scene reconstruction and forensic investigation. In sexual assault cases semen identification helps to prove victim’s allegations it also provides a material for DNA analysis that generate the genetic profile of the alleged suspect. The rapid Stain Identification of Human Semen (RSIDTM-Semen) bioassay is designed to detect specifically the presence of human semenogelin. Test development is completed within 10 minutes and can detect as little as 2.5 nL of human semen and it does not cross-react with other human or nonhuman tissues. The detection protocol can be completely integrated into the procedures for DNA extraction and analysis. Aim To assess the efficacy of RSIDTM – Semen strip test for the detection of human semen under some different variables (different fabrics, different time intervals and mixed with vaginal secretions) in order to be used in the routine forensic work of sexual assault cases and for further personal identification. Methodology: Semen samples were collected from four male participants. Vaginal specimens were collected from four female participants on cotton, linen or nylon-tipped swabs (2 swabs from each female). Each semen sample was divided into two portions; one used for semen only test group and the other mixed with vaginal secretions for the mixed test group. One of the fabrics tipped vaginal swab was mixed with semen for the mixed test group and the other used as a positive control group to test the sensitivity and specificity of the RSIDTM – Semen strip. The semen samples were deposited over different fabrics at the same time. All the samples were left to dry for 15 minutes at room temperature then extracted and analyzed according to the protocol designed for The Rapid Stain Identification Test (RSIDTM – Semen). The collected samples were studied as two test groups and one control group. Each of the previous groups, was categorized into 4 subgroups (a, b, c and d) according to the time interval of semen extraction (zero (on the spot), 2, 4, 6 and 10 days respectively). Results Semen could be identified in 100% of tested samples of the semen only group as well as of the combined semen and vagina group over cotton and linen fabrics at all the different tested time intervals. However, semen extracted from nylon fabric was identified in tested samples of the semen only group and of the combined semen and vagina group only at zero time only and couldn’t be identified at the rest of tested time intervals. Conclusion the current study evidenced that the new RSIDTM-semen kit is a reliable method for semen identification over different types of fabrics even in the presence of vaginal secretions. It also resists environmental factors up to 10 days. Background: Inflammatory bowel disease (IBD) is A chronic inflammatory disorder of the gastrointestinal tract (GIT). IBD comprises Crohn’s disease (CD) and Ulcerative Colitis (UC). Patients with CD and UC may present with variety of clinical presentations according to the activity of the disease. The treatment of IBD varies according to degree of activity. The demographics and clinical patterns of IBD have not been adequately studies in Saudi Arabia. Thus, the current study investigated the clinical presentation, course and outcome of IBD in a hospital in the Central region, Saudi Arabia. Methodology This prospective study included 92 patients from December 1, 2017, to August 31, 2018. The study constituted clinical history, screening through surveys that consisted of open ended and closed ended questions, and physical and laboratory assessment. Result There was no significant gender difference presents by male to female (M: F) 20:21 in UC and 23:28 in CD. The study showed that the most affected age group was the 4th decade in age group 31 to 40 years old represent that 56%. The most common presenting symptoms in active ulcerative colitis and Crohn’s disease were diarrhea, abdominal pain , weight loss, headache and fever. The study patients presented in different stages according to Montreal classification with the highest prevalence in Montreal stage two by 16.30% in UC and 22.82% in CD. Among patients with diarrhea, the vast majority due to infectious cause followed by UC the CD. Arthralgia’s, arthritis and ophthalmic manifestations were the most frequent extra-intestinal manifestations. Conclusion As progression of IBD in Saudi Arabia the health organization should research more in IBD and the cause of increase it’s incidence

Effect of mild blast-induced TBI on dendritic architecture of the cortex and hippocampus in the mouse

Traumatic brain injury (TBI) has been designated as a signature injury of modern military conflicts. Blast trauma, in particular, has come to make up a significant portion of the TBIs which are sustained in warzones. Though most TBIs are mild, even mild TBI can induce long term effects, including cognitive and memory deficits. In our study, we utilized a mouse model of mild blast-related TBI (bTBI) to investigate TBI-induced changes within the cortex and hippocampus. We performed rapid Golgi staining on the layer IV and V pyramidal neurons of the parietal cortex and the CA1 basilar tree of the hippocampus and quantified dendritic branching and distribution. We found decreased dendritic branching within both the cortex and hippocampus in injured mice. Within parietal cortex, this decreased branching was most evident within the middle region, while outer and inner regions resembled that of control mice. This study provides important knowledge in the study of how the shockwave associated with a blast explosion impacts different brain regions.

Optimal fluorescent-dye staining time for the real-time detection of microbes: a study of Saccharomyces cerevisiae.

To provide information on the time-dependent behaviour of microbe staining by fluorescent dyes in the order of seconds, which is important in terms of the recent rapid and online techniques for microbe measurements and/or environmental microbe analysis. For combinations of yeast (Saccharomyces cerevisiae) and typical dyes, including DAPI (4',6-diamidino-2-phenylindole) and Auramine-O, a suspension of yeast cells in ultrapure water was injected into a dye solution in a micro cuvette placed inside a spectrofluorometer and the fluorescence intensity of the resulting solution was measured at 1s intervals, starting immediately after the mixing and continued until the time for the maximum intensity using various concentrations of yeast and dyes. The relaxation time τ, which corresponds to ~63·2% of the maximum fluorescence intensity, was shown to decrease to below 1s with increasing DAPI concentration, whereas it remained constant for 2-3s with increasing Auramine-O concentration, for example at a yeast concentration of 100µgml-1 . For the conditions of yeast >10µgml-1 , DAPI >1µgml-1 and Auramine-O >0·1µgml-1 , τ could be adjusted to below 5s to achieve a rapid and stable staining. Design and operating conditions for rapid and online measurements of microbes can be optimized.

Ultrafast Toluidine Blue Staining for Rapid On-Site Evaluation of Cytological Smears

Rapid on-site evaluation (ROSE) is one of cytopathology’s “unique selling propositions.” The quality, speed, and ease of handling of the staining used is a critical factor for the efficacy of the ROSE procedure. Here, we describe a modification of rapid toluidine blue staining that can be performed within 25 s, provides excellent nuclear morphology, and is compatible with subsequent Papanicolaou staining of the slides. Furthermore, exposure to hazardous chemicals is minimized, as no organic solvents other than the alcohol-based fixative and glycerin for temporary mounting and coverslipping are required. We have used this protocol successfully in our ROSE practice and have not observed any discrepancies between toluidine blue- and permanent Papanicolaou-stained slides.

Comparison of Five Cytochemical Stains used for Fine Needle Aspiration Cytology of Breast and Lymph Node Lesions: A Comparative Study

Introduction: Fine Needle Aspiration Cytology (FNAC) is an established diagnostic tool aiming at an early diagnosis of mass lesions which in turn aids in planning further management of the patients. The need for minimal turnaround time with good quality of cell morphology has encouraged the development of different staining techniques. Quality Index (QI) of cytochemical stains is evaluated with a scoring system based on morphological features. Aim: To find rapid staining methods which were economical without compromising on the quality of FNAC smears. QI of five stains namely Papanicolaou stain (Pap), Haematoxylin and Eosin (H&E), May Grunwald Giemsa (MGG), Modified Ultrafast Papanicolaou (MUFP) and Rapid Economic Acetic acid Pap stain (REAP) were studied and compared. Materials and Methods: This was a prospective study conducted in the Department of Pathology, Kempegowda Institute of Medical Sciences, Bengaluru, Karnataka, India, from August 2011 to July 2013. FNAC of 80 mass lesions including 44 of breast lesions and 36 lymph nodes were included in this study. A minimum of five smears were made in each case and stained with Pap, H&E, MGG, MUFP and REAP. The QI of stain was assessed by the scores given for background of the smear, overall staining, and preservation of cell morphology, nuclear characteristics, cytoplasmic details and air drying artifacts. Statistical analysis of the data was done using chi-square test and Statistical Package for the Social Sciences (SPSS) version 15.0. Results: Pap stain showed maximum QI score followed by H&E, REAP, MGG and MUFP in case of breast lesions. In cases of lymph node highest QI was again with Pap followed by H&E, REAP, MUFP and MGG. Good cytomorphology with lesser expenditure and turnaround time was obtained by REAP and MUFP stains. Conclusion: Pap stain is still the best for cell morphology whereas MUFP and REAP are good, economical and with lesser turnaround time and can be used in routine cytopathology.

Interaction of Cyclooxygenase-2 with Helicobacter pylori Induces Gastric Chronic Nonresolving Inflammation and the Formation of Syndrome of Internal Block of Static Blood in Helicobacter pylori-Related Gastric Diseases.

Cyclooxygenase-2 (COX-2) is an inducible enzyme stimulated by various inflammatory factors (IFs). Chronic gastritis is a classic model of “inflammation-cancer transformation” and Helicobacter pylori-related gastric diseases (HPGD) are specific ones of this model. Traditional Chinese Medicine (TCM) syndromes could play a predictive role in gastric histopathological evolution. To search for early warning evidence about “inflammation-cancer transformation,” this study is about to explore interaction of COX-2 with Helicobacter pylori (Hp) in HPGD with different TCM syndromes. All included subjects underwent endoscopy and biopsy. Hp infection was detected by rapid urease test and methylene blue staining. Histopathological characteristics and COX-2 expression in gastric mucosa (GM) were, respectively, observed by hematoxylin-eosin and Elivision™ plus. SPSS 18.0 and Stata 11.0 statistical software packages were used for statistical analysis. Results of immunohistochemical staining in this study showed COX-2 expression in Hp-positive patients was stronger than that in Hp-negative ones. Spearman' analysis indicated that degrees of both Hp infection and COX-2 expression were positively correlated with those of gastric inflammation and inflammatory activity. Compared with the relative normal group, both severe dysplasia group and gastric carcinoma group had more severe Hp infection and COX-2 expression. Compared with the nonsyndrome, syndrome of internal block of static blood (IBSB) had higher scores in semiquantitative analysis of COX-2 protein expression among TCM groups. Moreover, multivariate logistics regression analysis suggested that patients with Hp infection could increase the risk of IBSB. These results indicated that COX-2 interacting with Hp could play an important role in transforming gastric chronic nonresolving inflammation into carcinoma in subjects with HPGD, as well as inducing the formation of IBSB. HPGD together with IBSB could be an early warning evidence for GM with histopathological evolution from benign to malignant.

Evaluation of Role of Intraoperative Cytology Technique in Diagnosis and Management of Cancer.

Context:Intraoperative pathological assessment provides valuable information in a patient's diagnosis and management. Touch imprint/crush cytology is simple, rapid, and cost-effective. Also, imprint smears give excellent cytomorphology.Aims:To assess the utility and feasibility of intraoperative cytology technique as a rapid and reliable method for diagnosis and to compare sensitivity, specificity and diagnostic accuracy with histopathology.Materials and Methods:Cytology smears were collected intraoperatively from 52 cases of suspected/proven malignancy. From tumors, surgical margins, lymph nodes; crush, imprint, and scrape smears were prepared. Smears were taken from specimens before formalin fixation and stained with rapid ultrafast Papanicolaou stain. The slides were compared with the histopathology report which is the gold standard.Statistical Analysis:Sensitivity, specificity, and diagnostic accuracy were used for statistical analysis.Results:Intraoperative cytology report could be given in time for the surgeon to modify margins of resection and the extent of lymph node dissection. It helps the surgeon to modify surgery. The diagnostic test evaluation showed satisfactory results.Conclusion:Intraoperative imprint/crush cytology is a simple, inexpensive, rapid, accurate cytodiagnostic technique in the diagnosis of cancer where frozen section facilities are not available. It can also be used to assess the clearance of surgical margins.

Examination of the rapid Masson trichrome staining method

Examination of the rapid Masson trichrome staining method

Fluorescent Staining of Arbuscular Mycorrhizal Structures Using Wheat Germ Agglutinin (WGA) and Propidium Iodide.

The colonization of a host plant root by arbuscular mycorrhizal (AM) fungi is a progressive process, characterized by asynchronous hyphal growth in intercellular and intracellular spaces, leading to the coexistence of diverse intraradical structures, such as hyphae, coils, arbuscules, and vesicles. In addition, the relative abundance of intercellular and intracellular fungal structures is highly dependent on root anatomy and the combination of plant and fungal species. Lastly, more than one fungal species may colonize the same root, adding a further level of complexity. For all these reasons, detailed imaging of a large number of samples is often necessary to fully assess the developmental processes and functionality of AM symbiosis. To this aim, the use of rapid and efficient staining methods that can be used routinely is crucial.We herein present a simple protocol to obtain high detail images of both overall intraradical fungal colonization pattern and fine morphology, in AM root sections of Lotus japonicus. The procedure is based on tissue clearing, fluorescent staining of fungal cell walls with fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), and the combined counterstaining of plant cell walls with propidium iodide (PI). The resulting images can be acquired using traditional or confocal fluorescence microscopes and used for qualitative and quantitative analyses of fungal colonization, of particular interest for the comparison of mycorrhizal phenotypes between different experimental conditions or genetic backgrounds.

An integrated microfluidic system for rapid, automatic and high-throughput staining of clinical tissue samples for diagnosis of ovarian cancer

Accurate cancer diagnostic methods are of urgent need. Since traditional immunohistochemistry (IHC)-based approaches, while reliable, are labor-intensive and require well-trained technicians, we developed an integrated microfluidic platform capable of labeling ovarian cancer biomarkers (i.e. aptamer) within formalin-fixed, paraffin embedded tissues via molecular probes. Both aptamer-based 1) fluorescent staining and 2) IHC staining of clinical tissue samples could be automated in the microfluidic system in only 2-3 h (40-50% faster than conventional approaches) with <0.5 mL of reagents, signifying that this device could serve as a promising diagnostic tool for ovarian cancer.

Fluorescein dye as a novel cost-effective approach for staining raw specimens in ophthalmic pathology

Purpose:To study the usefulness of sodium fluorescein dye for staining raw specimens in ophthalmic pathology.Methods:Laboratory-based observational study. Eye specimens received in the ocular pathology department of a tertiary eye care center in northeast India were included in the study after obtaining the informed consent. The study period was from 2016 to 2019. Specimens received were a corneal button, lid, orbital tissues, enucleated eyeballs, eviscerated eye, explanted intraocular lens (IOLs), optic nerve and ocular parasites. Sections of the gross specimens were stained with sodium fluorescein (C20H12O5NA) dye. The average duration of tissue-stain contact time was 45 s. The sections were analyzed under the compound microscope. The intensity of illumination of the microscope was modulated to obtain high contrast digital photographs.Results:26 corneal buttons with or without limbal tissue specimens were analyzed with fluorescein staining procedure; limbus with its pigmented cells were seen in the enucleated eyeballs. 33 enucleated eyes (retinoblastoma [RB] (n = 24), phthisical eyes (n = 4), choroidal melanomas (n = 2), and others (n = 3) were included in the study cohort. In these 33 enucleated eyes, vitreous were also examined for the presence of hyalocytes and other pathological cells. Retinal pigment epithelial cells were also seen (n = 11). RB seeds were seen with fluorescein stain and documented in 14 specimens. The RB seeds were mostly in vitreous (n = 9) and subretinal space (n = 5). Fat cells (n = 8) from orbital tissues and sebaceous cells (n = 5) from frozen section specimens were also observed and documented.Conclusion:This study highlights a novel method of rapid staining of gross ophthalmic pathology specimens.

A comparison between rapid stain identification-blood (RSID-blood) and faecal occult blood test (FOB)to identify human blood on various suspicious stains

A comparison between rapid stain identification-blood (RSID-blood) and faecal occult blood test (FOB)to identify human blood on various suspicious stains

Morris water maze overtraining increases the density of thorny excrescences in the basal dendrites of CA3 pyramidal neurons

The hippocampus plays a fundamental role in spatial learning and memory. Dentate gyrus (DG) granular neurons project mainly to proximal apical dendrites of neurons in the CA3 stratum lucidum and also, to some extent, to the basal dendrites of CA3 pyramidal cells in the stratum oriens. The terminal specializations of DG neurons are the mossy fibers (MF), and these huge axon terminals show expansion in the CA3 stratum oriens after the animals undergo overtraining in the Morris Water Maze task (MWM). However, to our knowledge there are no reports regarding the possible changes in density of post-synaptic targets of these terminals in the basal dendrites of CA3 neurons after overtraining in the MWM. The purpose of this work was to study the density of thorny excrescences (TE) and other dendritic spine types (stubby, thin, and mushroom) in the CA3 stratum oriens in animals overtrained in the MWM for three consecutive days and in animals trained for only one day. Seven days after MWM training, the animals were sacrificed, and their brains removed and processed for rapid Golgi staining to visualize the different types of dendritic protrusions. Our results revealed that the relative quantity of stubby, thin, and mushroom dendritic spines did not change, regardless of amount of training. However, a significant increase in the density of TE was detected in the overtrained animals. These results strongly suggest that spatial water maze overtraining induces an increased density of MF–TE connections, which might be functionally relevant for long-term spatial memory formation.