Micro-computed tomography with iodine staining resolves the arrangement of muscle fibres

We illustrate here microCT images in which contrast between muscle and connective tissue has been achieved by means of staining with iodine. Enhancement is shown to be dependent on the concentration of iodine solution (I₂KI), time in solution and specimen size. Histological examination confirms that the arrangement of individual muscle fibres can be visualised on the enhanced microCT images, and that the iodine accumulates in the muscle fibres in preference to the surrounding connective tissues. We explore the application of this technique to describe the fibrous structure of skeletal muscle, and conclude that it has the potential to become a non-destructive and cost-effective method for investigating muscle fascicle architecture, particularly in comparative morphological studies.     

Cellulase screening by iodine staining: An artefact

Hydrolysis zones visualized by iodine (KI/I₂) staining of cellulose-agar media after growth of Trichoderma reesei QM6a, RUT C30, QM9136 (cellulase negative) or Thielavia terrestris cultures, or incubation of crude endoglucanases and amylases, were due primarily to degradation of a small amount of starch contaminant in commercial agar and not to cellulolysis as recently suggested. No zones were evident when amylase-digested agar or Gelrite was used as the gelling agent or when purified cellobiohydrolase and endoglucanase were used. Cellulase screening free from artefacts is best obtained by growing cultures on acid-swollen or crystalline cellulose with Gelrite as optimal gelling agent, followed by incubation at elevated temperature to enhance visualization of hydrolysis zones while restricting fungal growth, but without additional staining.     

Quantification of total iodine in intact granular starches of different botanical origin exposed to iodine vapor at various water activities

Iodine has been used as an effective tool for studying both the structure and composition of dispersed starch and starch granules. In addition to being employed to assess relative amylose contents for starch samples, it has been used to look at the molecular mobility of the glucose polymers within intact starch granules based on exposure to iodine vapor equilibrated at different water activities. Starches of different botanical origin including corn, high amylose corn, waxy corn, potato, waxy potato, tapioca, wheat, rice, waxy rice, chick pea and mung bean were equilibrated to 0.33, 0.75, 0.97 water activities, exposed to iodine vapor and then absorbance spectra and LAB color were determined. In addition, a new iodine quantification method sensitive to <0.1% iodine (w/w) was employed to measure bound iodine within intact granular starch. Amylose content, particle size distribution of granules, and the density of the starch were also determined to explore whether high levels of long linear glucose chains and the surface area-to-volume ratio were important factors relating to the granular iodine binding. Results showed, in all cases, starches complexed more iodine as water content increased and waxy starches bound less iodine than their normal starch counterparts. However, much more bound iodine could be measured chemically with waxy starches than was expected based on colorimetric determination. Surface area appeared to be a factor as smaller rice and waxy rice starch granules complexed more iodine, while the larger potato and waxy potato granules complexed less than would be expected based on measured amylose contents. Corn, high amylose corn, and wheat, known to have starch granules with extensive surface pores, bound higher levels of iodine suggesting pores and channels may be an important factor giving iodine vapor greater access to bind within the granules. Exposing iodine vapor to moisture-equilibrated native starches is an effective tool to explore starch granule architecture.     

Mass-transfer based modeling to investigate iodine staining effects for enhanced contrast X-ray computed tomography

Iodine staining combined with X-ray computed tomography (CT) has become a core approach in anatomy, offering three-dimensional and essentially non-destructive imaging of soft tissues. Although there have been rapid advances in methodologies and techniques, the mechanisms underlying diffusible iodine contrast-enhanced CT are not yet fully understood. The protocols for staining samples of differing sizes and tissue types have not yet been justified theoretically. Here we utilize mass transfer modeling to simulate iodine diffusion and predict iodine concentrations within distinct tissue types. We also undertake iodine staining experiments to visualize the detailed anatomy and contrast effects on whole-body avian specimens using different concentrations of iodine solution to compare with model simulation results. The simulations effectively explain most observed concentration changes in differently-sized samples over distinct iodine treatment durations. These results also provide insight into the mechanisms behind the efficacy of solution replenishment for enhancing staining effects. Both consistencies and inconsistencies between our simulation and experimental results regarding iodine concentration in tissues will inform further investigations to optimize iodine staining protocols.     

Automated Feulgen staining with a temperature-controlled staining machine

A Shandon Varistain 24-3 staining machine was modified in order to run automated DNA Feulgen staining. Initial studies showed a strict dependence of the staining intensity (integrated optical density [IOD]) on the temperature of the DNA hydrolysis in 4 N HCl: a difference of 0.5 degrees C around the optimum hydrolysis temperature of 27.5 degrees C resulted in IOD differences of up to 7.8% in epithelial cells and up to 12.0% in lymphocytes. A temperature-controlled stainless steel cuvette, covered with a 4 N HCl-resistant material, was developed and integrated into the machine. Temperature measurements were performed at different positions in the cuvette and on glass slides with copper-constantan electrodes fixed on them; no temperature gradient could be detected within the cuvette. The adjusted temperature of 27.5 degrees C remained constant over 24 hours. The coefficient of variation (CV) of the staining intensity in lymphocytes between different areas on the same slide and between different slides of the same staining cycle was less than 0.6%. The CV between different staining cycles was 5.9%. This system for automated Feulgen staining thus gives reproducible and reliable results and may be introduced into routine diagnostic procedures.     

A guide for optimal iodine staining and high‐throughput diceCT scanning in snakes

Diffusible iodine‐based contrast‐enhanced computed tomography (diceCT) visualizes soft tissue from micro‐CT (µCT) scans of specimens to uncover internal features and natural history information without incurring physical damage via dissection. Unlike hard‐tissue imaging, taxonomic sampling within diceCT datasets is currently limited. To initiate best practices for diceCT in a nonmodel group, we outline a guide for staining and high‐throughput µCT scanning in snakes. We scanned the entire body and one region of interest (i.e., head) for 23 specimens representing 23 species from the clades Aniliidae, Dipsadinae, Colubrinae, Elapidae, Lamprophiidae, and Viperidae. We generated 82 scans that include 1.25% Lugol''s iodine stained (soft tissue) and unstained (skeletal) data for each specimen. We found that duration of optimal staining time increased linearly with body size; head radius was the best indicator. Postreconstruction of scans, optimal staining was evident by evenly distributed grayscale values and clear differentiation among soft‐tissue anatomy. Under and over stained specimens produced poor contrast among soft tissues, which was often exacerbated by user bias during “digital dissections” (i.e., segmentation). Regardless, all scans produced usable data from which we assessed a range of downstream analytical applications within ecology and evolution (e.g., predator‐prey interactions, life history, and morphological evolution). Ethanol destaining reversed the known effects of iodine on the exterior appearance of physical specimens, but required substantially more time than reported for other destaining methods. We discuss the feasibility of implementing diceCT techniques for a new user, including approximate financial and temporal commitments, required facilities, and potential effects of staining on specimens. We present the first high‐throughput workflow for full‐body skeletal and diceCT scanning in snakes, which can be generalized to any elongate vertebrates, and increases publicly available diceCT scans for reptiles by an order of magnitude.     

Nuclear staining with alum hematoxylin

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.     

Permanent Staining with Iodine Vapor

Sections of cooked rice have been stained with iodine vapor, and the method has given greater permanency than any heretofore reported. The method, which permits the use of a counter-stain, has been applied also to starches, starch pastes, uncooked rice flour, and cooked rice flour. The resulting color is of moderate intensity, so that structure is revealed and the birefringence of ungelatinized starch is not impaired. Preparations made more than a year ago have retained their characteristic blue or lavender color.     

The use of elemental iodine to enhance staining of thin sections to be viewed in the electron microscope.

A noticeable increase in contrast is observed when thin sections, stained with Reynolds lead citrate, are subsequently exposed to elemental iodine vapor for 30 seconds. There is no loss of ultrastructural detail, and there is no evidence of harmful iodine contamination of the microscope after prolonged study of such material. It is recommended that this simple procedure be used when other methods of staining have not proved adequate.     

Mitochondrial nucleoid staining with ammoniacal silver

Under controlled conditions, ammoniacal silver (A-S) reaction appears in the nucleoid in the mitochondria of Physarum polycephalum as well as the dense chromatin in the nuclei. The results and the effect of selective extraction of basic proteins on the A-S reaction of the nucleoid and the nucleus suggest that the mitochondrial nucleoid may contain basic protein, probably histone-like protein.     

Gram staining with an automatic machine

This study was undertaken to develop a new Gram-staining machine controlled by a micro-controller and to investigate the quality of slides that were stained in the machine. The machine was designed and produced by the authors. It uses standard 220 V AC. Staining, washing, and drying periods are controlled by a timer built in the micro-controller. A software was made that contains a certain algorithm and time intervals for the staining mode. One-hundred and forty smears were prepared fromEscherichia coli, Staphylococcus aureus, Neisseria sp., blood culture, trypticase soy broth, direct pus and sputum smears for comparison studies. Half of the slides in each group were stained with the machine, the other half by hand and then examined by four different microbiologists. Machine-stained slides had a higher clarity and less debris than the hand-stained slides (p<0.05). In hand-stained slides, some Gram-positive organisms showed poor Gram-positive staining features (p<0.05). In conclusion, we suggest that Gram staining with the automatic machine increases the staining quality and helps to decrease the work load in a busy diagnostic laboratory.