Evaluation of immunocytochemistry on destained giemsa stained smears as an alternative to conventional technique.

Introduction: Protocol for immunocytochemical (ICC) staining in May-Grünwald Giemsa (MGG)-stained smears has been difficult to establish. It is the need of the hour to be able to use prestained slides for ICC in specific cases to deliver timely diagnoses and reduce inconvenience to patients. Aims and Objectives: To evaluate and compare the use of MGG-stained smears for the purpose of ICC, after de-staining and saline rehydration to that of routine standard ICC. Materials and Methods: A prospective study was conducted on 40 FNAC samples: 25 cases of breast disease and 15 cases of reactive lymphoid hyperplasia known to express pancytokeratin and leukocyte common antigen (LCA)/CD45, respectively. Air-dried smears of each case were stained by standard MGG stain and after the report was dispatched, one smear was selected and sent for ICC. The smears were analyzed to determine the overall result and grade each smear semi-quantitatively with respect to staining-intensity, stain-localization, staining-uniformity, counter-staining, and background-staining. Observations and Results: The proposed protocol was inferior to conventional ICC in all the parameters, more pronounced in pancytokeratin than LCA/CD45. Only 8% of air-dried smears stained for pancytokeratin showed optimal stain intensity (as opposed to 44% of wet-fixed smears), whereas only 14.3% of air-dried smears were optimally stained for LCA (as opposed to 85.7% of wet-fixed smears). Conclusion: The proposed protocol of de-stained Giemsa smears as an alternative to conventional technique for ICC was unsuccessful in giving satisfactory results.

Could RPMI-1640 Medium be Used in the Diagnosis and Isolation of Cutaneous Leishmaniasis Lacking NNN Medium?

Laboratory diagnosis of leishmaniasis is based on culture, microscopic examination, serological and molecular methods. The gold standard method is to see amastigotes in microscopic examination and to grow promastigotes in Novy, MacNeal, Nicolle (NNN) medium. NNN medium is frequently used for culture all over the world. In our study, it was aimed to investigate whether the use of RPMI-1640 medium is an appropriate method in cases where the gold standard NNN medium is not available for the diagnosis of cutaneous leishmaniasis (CL). Smears were prepared from the needle aspiration fluid sample from the patient who applied to Manisa Celal Bayar University Faculty of Medicine and had lesions suspicious of CL, and were stained with Giemsa for the presence of amastigotes. The samples taken were directly inoculated into RPMI-1640 broth and incubated at 26 °C for the presence of promastigotes. On consecutive days after incubation, it was checked for promastigote growth. Genotyping of the grown isolate was performed with primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region with the help of real-time polymerase chain reaction. The amastigote form was observed in the microscopic examination of the needle aspiration fluid sample smear preparations taken from the patient. On the other hand, promastigote growth was observed in RPMI-1640 broth from the 3rd day. In addition, the isolate obtained from the CL patient was determined to be Leishmania tropica as a result of the species determination made by genotyping. It is thought that this study is important in terms of suggesting an alternative medium for the diagnosis of leishmaniasis in laboratories where the gold standard NNN medium is easily accessible. RPMI-1640 medium, which is easily obtained and prepared in parasitology laboratories, can help in the diagnosis of the disease and treatment follow-up, Leishmania spp. isolation and drug resistance studies.

Detection of Helicobacter pylori in Gastric Biopsies Among Sudanese Patients with Confirmed Gastric Disorders Using Three Diagnostic Methods.

BACKGROUND: Helicobacter pylori (H. pylori) infection represents one of the most common chronic bacterial infections in developing countries. However, in Sudan, the infection is not well diagnosed with standard laboratory methods in many parts of the country. This study aimed to detect H. pylori in gastric biopsies of patients with gastric disorders, using three diagnostic methods. MATERIALS AND METHODS: A cross-sectional study was conducted among 100 patients in Gezira state, central Sudan. Giemsa stain for histopathological examination (HPE), rapid urease test (RUT), and polymerase chain reaction (PCR) techniques were performed to detect H. pylori from the gastric biopsy samples as per standard assays. RESULTS: Most of the patients were males (66%), from rural areas (72%) and in the age group 31 to 50 years. H. pylori were identified in 85% of the samples by at least one of the three tests. The highest positivity was detected by HPE (83%), followed by PCR (67%) and RUT (63%), while 59% were positive by the three diagnostic methods. PCR showed higher sensitivity (80.72% vs. 73.49%) and specificity (100% vs. 88.24%) than RUT. Positive predictive values were reported as 100% for PCR and 96.83% for RUT. Considering PCR as a gold standard method, HPE revealed higher sensitivity (100%) than RUT (88.06%). On the contrary, RUT showed higher specificity (87.88%) than PCR (51.52%). There were no significant associations between H. pylori infection patients' gender (p = 0.747). Loss of weight (p = 0.007) and nausea (p = 0.032) were significantly associated with H. pylori infection. CONCLUSIONS: There was a high prevalence of H. pylori infection in central Sudan. This highlights the need to analyze epidemiological status, virulence factors, and strain characteristics to control disease transmission. PCR is a reliable and valuable technique in detecting H. pylori infection from gastric biopsy samples.

Probing multifunctional azure B conjugated gold nanoparticles with serum protein binding properties for trimodal photothermal, photodynamic, and chemo therapy: Biophysical and photophysical investigations.

Multimodal or combination therapy has been considered as a powerful approach for treatment of complex diseases like cancer. The fascinating physicochemical and optoelectronic properties of gold nanoparticles make them potential candidate for cancer therapeutic and diagnostic applications. Herein, we design a multifunctional nanosystem by conjugating a photosensitizer, Azure B (AB) with citrate reduced gold nanoparticles (CI-Au NPs) through non-covalent interactions. The conjugation of AB with CI-Au NPs was confirmed through UV-Visible absorption spectroscopy and Fourier Transform Infra-red (FT-IR) spectroscopy. The morphology, size, and charge of the prepared nano-conjugates (AB@CI-Au NPs) were determined by transmission electron microscopy (TEM), Dynamic light scattering (DLS), and zeta potential measurements. The proficiency of AB@CI-Au NPs for cancer photo-therapies was demonstrated by evaluating their potential for photothermal heating and singlet oxygen generation in solution upon Visible laser (635 nm, 500 mW/cm2) irradiation. The AB@CI-Au NPs display superior heating efficiency than CI-Au NPs alone or free AB, and offer better photostability as well as singlet oxygen generation rate compared to free photosensitizer. The interaction of AB@CI-Au NPs with Calf thymus DNA (Ct-DNA) and transport protein Bovine Serum Albumin (BSA) were studied using various biophysical techniques including Circular dichroism, UV-Visible and fluorescence spectroscopic studies. AB@CI-Au NPs show intercalative binding with Ct-DNA by inducing local perturbations in double helical structure and hence can exert chemotherapeutic action by targeting DNA. AB@CI-Au NPs also display moderate binding with BSA without any adverse effect on BSA structure, which is desirable for significant biodistribution and pharmacokinetics of AB@CI-Au NPs. Also, in vitro cytotoxicity of the AB@CI-Au NPs with and without laser irradiation (635 nm, 500 mW/cm2) was demonstrated using the hepatocellular carcinoma (HepG2) cell lines. Our findings through photophysical and biophysical studies strongly recommend the exploitation of AB@CI-Au NPs nanoconjugates as a multifunctional probe for trimodal anticancer therapy.

Comparison of special stains (Giemsa stain and Modified Toluidine Blue stain) with immunohistochemistry as gold standard for the detection of H. pylori in gastric biopsies.

BACKGROUND AND STUDY AIMS: Helicobacter pylori (H. pylori) has been extensively implicated in the etiology and pathogenesis of gastric ulcers and carcinomas, which has necessitated its efficient and cost-effective identification in gastric biopsy samples. We have sought to compare various staining methods, namely, Hematoxylin and Eosin (H&E), Giemsa, and Modified Toluidine Blue (MTB), with immunohistochemistry (IHC; gold standard) in terms of efficacy, staining costs, and duration of performing the stains in settings with limited resources. PATIENTS AND METHODS: Gastric biopsy specimens of 50 patients who presented with gastritis symptoms were stained with H&E, Giemsa, MTB, and IHC. The sensitivity, specificity, positive/negative predictive values, and the receiver operating characteristic curve were calculated for each stain. RESULTS: In all, 32 cases of 50 were positive for H. pylori on IHC. The specificity for both H&E and Giemsa was 88.8%, whereas it was lower for MTB (83.3%). The sensitivity of H&E was much lower (46.8%) compared with the other stains (90.6% Giemsa, 93.7% MTB). The most inexpensive and time-consuming stain was H&E followed by MTB and Giemsa. CONCLUSION: When H&E is used alone for H. pylori detection, a significant number of cases may be overlooked, especially mild inflammatory cases and when coccoid forms of the bacteria are present. This study proposes the use of either Giemsa or MTB as reliable alternatives for IHC in resource-limited settings to combat the high prevalence of H. pylori in the developing world.

Detection of Helicobacter pylori in oral squamous cell carcinoma using modified Giemsa stain: A new diagnostic approach.

Context: Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. It is mainly known to be caused by tobacco in various forms and also due to viral, fungal infection and poor oral hygiene, etc. Poor oral hygiene leads to colonization of pathogenic bacteria including Helicobacter pylori. It seems that the presence of H. pylori might be a risk factor for developing oral cancer. The successful attempt was made to detect H. pylori in diagnosed specimens of OSCC using Warthin-starry and Giemsa stains in our department. The modified Giemsa stain is the method of choice because it is sensitive, cheap, easy to perform, faster, and reproducible. Aim: The aim of this study is to detect H. pylori in various grades of OSCC using modified Giemsa stain. Subjects and Methods: Thirty cases of various grades of OSCC were selected from the archives of the department. Five-micrometer-thick paraffin-embedded tissue sections of these cases were taken and stained with modified Giemsa and were studied under ×100 magnification. Results: All the tissue sections studied were positive for H. pylori bacteria. Conclusions: Our study showed a significant presence of H. pylori in histological sections of OSCC, and it seems likely that the presence of H. pylori might be a risk factor for the developing oral lesions such as oral cancer. Early detection and eradication of H. pylori in the oral cavity, especially in high-risk patients, might prevent its harmful consequences.

Modified Giemsa Stain: A solution to improve the quality of hypercellular bone marrow smears.

INTRODUCTION: Morphology is still the cornerstone of the diagnosis of haematological malignancies where the quality of staining plays a major role. The hypercellularity of a smear interferes with the quality of staining. We compared a newly modified giemsa stain (MGS) as a routine method for staining hypercellular bone marrow smears over the conventional Leishman method and the May Grunwald Giemsa method (MGG) in a completely randomized block study. METHODS: Quality of staining, cost, labour intensiveness and turnaround time of the new method was compared with MGG and Leishman methods for bone marrow smears of acute leukaemia using 30 cases. Bone marrow smears were blindly reviewed for the degree of staining of nuclei, cytoplasm and granules on a scale of 0 to 4 and statistically analysed by Minitab 16. RESULTS: A significant difference (p < .05) was revealed for the quality of staining of nuclei, cytoplasm and granules, and it was found that the new MGS was superior to the MGG and Leishman method. The cost of staining was lowest in the new MGS and the staining time of the new MGS was 76% less than MGG and 9.09% greater than the Leishman method. In terms of turnaround time, the new MGS is somewhat similar to the Leishman method and is user-friendly with a lesser number of steps to follow, compared with MGG. CONCLUSION: Our study showed that the new MGS is overall far superior, cost-effective and user-friendly compared with other conventional staining methods when used for hypercellular bone marrows.

Diagnostic study of trypanosomiasis of cats in Mosul, Iraq.

Background: Trypanosomiasis is a zoonotic parasitic disease endemic in Iraq but with limited information about its occurrence in cats. Aim: This study was designed to detect Trypanosoma spp. in cats using microscopic examination by Giemsa stain and conventional polymerase chain reaction (PCR) technique in Mosul, Iraq. Methods: A total of 120 blood samples from cats were microscopically examined using Giemsa stain. Only 35 positive blood samples were examined by the conventional PCR technique. Hematological changes were also reported. Results: The infection rate of Trypanosoma spp. was 34.2% (41 out of 120). Results of conventional PCR technique for the positive 35 blood samples indicated 31.4% as Trypanosoma spp. and 20% Trypanosoma evansi. This study showed that the infection in younger cats was significantly more than in older cats, with significant differences between females and males. Affected cats suffered from fever, dullness, pale mucous membranes, emaciation, muco-purulent ocular discharge, anorexia, incoordination, and anemia. Results of the blood picture indicated increase in total leukocyte count and decrease in hemoglobin concentration, packed cell volume, and total red blood cells. Conclusion: Trypanosoma spp. and T. evansi infection in Mosul of Iraq is reported for the first time in cats, and younger cats were more affected than older cats.

Karyotype diversity in cultivated and wild Indian rice through EMA-based chromosome analysis.

India is known for its diverse cultivated and wild rice germplasm. In today's crop improvement programmes, wild relatives are much-needed genetic repository of valuable traits. Analysis of genetic diversity at the chromosomal level is one cost-effective tool to unlock foundational information related to genetics and plant breeding. Presently, enzymatic maceration and air-drying method (EMA) has been applied for the first time in six cultivated and nine wild Indian rice (diploid and tetraploid). EMA method following Giemsa staining has yielded large numbers of cytoplasm free metaphase plates with distinct chromosome morphology. Detailed analysis has revealed karyotype diversities in terms of total chromatin length (TCL), chromosome morphology and location of sat chromosomes within and between the studied species. Most of the cultivated rice has gained additional amount in TCL during the period of domestication in comparison to their progenitor Oryza nivara. Morphological clarity of the small chromosomes of rice was much required and has helped to identify individual chromosomes in the diverse karyotypes. Diversity in landmark SAT chromosomes is another important observation, not reported previously in Indian rice. Present study has shown that in most of the O. sativa members, the 10th pair contains SAT except one where 6th pair is satellited. On the other hand, diversity of SAT in diploid and tetraplod wild species has been recorded on 5th, 7th and 8th chromosome pairs and on 9th, 12th, 22nd and 23rd chromosome pairs, respectively. Karyomorphometric indices has helped to construct dendrogram to elucidate intraspecies and interspecies relationships. Untapped genetic diversity recorded in Indian rice through chromosomal analysis will be useful to the breeders and genome researchers.

Malaria diagnosis in a malaria non-endemic high-resource country: high variation of diagnostic strategy in clinical laboratories in the Netherlands.

BACKGROUND: Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination of other methods should be used and when. METHODS: Three questionnaires were sent between March 2018 and September 2019 to laboratories subscribing to the external quality assessment scheme for the diagnosis of blood and intestinal parasites of the Dutch Foundation for Quality Assessment in Medical Laboratories in order to investigate how much variation in the laboratory diagnosis of malaria between different clinical laboratories is present in the Netherlands. RESULTS: The questionnaires were partially or fully completed by 67 of 77 (87%) laboratories. Only 9 laboratories reported 10 or more malaria positive patients per year. Most laboratories use a different diagnostic strategy, within office versus outside office hours depending on the screening assay result. Within office hours, 62.5% (35/56) of the responding laboratories perform an immunochromatographic test (ICT) in combination with microscopic examination of thick and thin blood films without additional examinations, such as Quantitative Buffy Coat and/or rtPCR analysis. Outside office hours 85.7% (48/56) of laboratories use an ICT as single screening assay and positive results are immediately confirmed by thick and thin blood films without additional examinations (89.6%, 43/48). In case of a negative ICT result outside office hours, 70.8% (34/48) of the laboratories perform microscopic examination of the thick film the next morning and 22.9% (11/48) confirm the negative ICT result immediately. Furthermore, substantial differences were found in the microscopic examinations of thick and thin blood films; the staining, theoretical sensitivity of the thick film and determination of parasitaemia. CONCLUSIONS: This study demonstrated a remarkably high variation between laboratories in both their diagnostic strategy as well as their methods for microscopic examination for the diagnosis of malaria in a clinical setting, despite existing national and international guidelines. While the impact of these variations on the accuracy of the diagnosis of malaria is yet unknown, these findings should stimulate clinical laboratories to critically review their own diagnostic strategy.

Morphological features of breast cancer circulating tumor cells in blood after physical and biological type of isolation.

BACKGROUND: Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Different isolation tech-niques based on their biological or physical features were established. Currently, the most widely used methods for visualization after their separation are based on immunofluorescent staining, which does not provide the information on the morphology. MATERIALS AND METHODS: The aim of this study was to evaluate how two different separation techniques affect cell morphology and to analyse cell morphology with techniques used in routine cytopathological laboratory. A direct side-by-side comparison of physical (Parsortix®) and biological (MACS®) separation technique was performed. RESULTS: In the preclinical setting, both isolation techniques retained the viability and antigenic characteristics of MCF7 breast cancer cells. Some signs of degeneration such as cell swelling, cytoplasmic blebs, villous projections and vacuolization were observed. In metastatic breast cancer patient cohort, morphological features of isolated CTCs were dependent on the separation technique. After physical separation, CTCs with preserved cell morphology were detected. After biological separation the majority of the isolated CTCs were so degenerated that their identity was difficult to confirm. CONCLUSIONS: Taken together, physical separation is a suitable technique for detection of CTCs with preserved cell morphology for the use in a routine cytopathological laboratory.

Cutaneous Leishmaniasis with Mucosal Involvement

Leishmaniasis is a protozoan parasitic disease transmitted to humans by infected female sand flies. Turkey has received more than three million immigrants from Syria because of the civil war and political instability. This study reported cases of two patients, who were from Syria and lived in Hatay, with cutaneous leishmaniasis and mucosal involvement. Two patients presented to the infectious diseases clinic with a complaint of facial lesions and were subsequently referred to the parasitology department laboratory. Smears were prepared from the lesions, stained with Giemsa and examined under a microscope. Moreover, aspirates taken from the patients' lesions were inoculated into the modified Novy-MacNeal-Nicolle medium. The diagnosis was made when amastigotes were detected in both smears. Proliferation of promastigotes was observed in one of the clinical specimens inoculated on the medium. By PZR-RFLP, Leishmania tropica were detected in the isolate. Both patients were treated with amphotericin B. One patient was treated again with a pentavalent antimony compound because of the recurrence of the lesion.

Insecticide-treated net ownership, utilization and knowledge of malaria in children residing in Batoke-Limbe, Mount Cameroon area: effect on malariometric and haematological indices.

BACKGROUND: Insecticide-treated nets (ITNs) are the most widely used interventions for malaria control in Africa. The aim of this study was to assess the ownership and utilization of ITNs and the knowledge of malaria and their effects on malariometric and haematological indices in children living in the Mount Cameroon area. METHODS: A community-based cross-sectional study involving a total of 405 children aged between 6 months and 14 years living in Batoke-Limbe was carried out between July and October 2017. A semi-structured questionnaire was used to document demographic status, knowledge on malaria and ITN ownership and usage. Venous blood sample was collected from each child to determine the prevalence and intensity of parasitaemia by Giemsa-stained microscopy and full blood count by auto haematology analysis to obtain white blood cell (WBC) and red blood cell (RBC) counts, haemoglobin (Hb) level, haematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). A multilinear regression model was used to determine the relationship between haematological parameter as dependent variable and the independent variables. RESULTS: The overall prevalence of parasitaemia, anaemia, knowledge about malaria, ITN ownership, usage and effective usage was 46.7%, 54.7%, 40.7%, 78.8%, 50.9% and 29.9%, respectively. The prevalence of parasitaemia was significantly higher (P < 0.001) in children who ineffectively utilized ITNs (54.9%) than effective users (27.3%). Having knowledge of malaria, negatively correlated with WBC counts (P = 0.005), but positively correlated with Hb levels (P < 0.001), RBC counts (P < 0.001), Hct (P < 0.001), MCV (P < 0.001) and MCH (P < 0.001). ITN use positively correlated with WBC counts (P = 0.005) but negatively with Hb levels (P = 0.004), RBC counts (P = 0.006), and MCH (P < 0.001). Meanwhile, parasitaemia negatively correlated with Hb levels (P = 0.004), RBC counts (P = 0.01), Hct (P = 0.04) and MCHC (P = 0.015). CONCLUSION: There is need for more sensitization on the benefits of using the ITNs to meet up with the intended and expected impact of the free distribution of ITNs.

The "Dextran-Magnetic Layered Double Hydroxide-Fluorouracil" Drug Delivery System Exerts Its Anti-tumor Effect by Inducing Lysosomal Membrane Permeability in the Process of Cell Death.

The "dextran-magnetic layered double hydroxide-fluorouracil" (DMF) drug delivery system is a new type of pharmaceutic preparation that can cause cancer cell oncosis. In the present study, we used different experimental methods such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cycle assay, reactive oxygen species (ROS) assay, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI), Giemsa stainings, transmission electron microscopy, immunofluorescence staining and Western blotting to study the mechanism of expansion death by using Hydroxychloroquine (HCQ) as a positive control and 5-Fluorouracil (5-Fu) as reference. The results showed that DMF exhibited a better anti-tumor effect than 5-Fu in the process of cell death, and the pharmacological mechanism of 5-Fu was changed by its preparation DMF. The mechanism of cancer cell death induced by DMF was similar to that of HCQ. But DMF intervention did not cause a large amount of accumulation of mitochondrial reactive oxygen species, and the location of lysosomotropic LysoTracker Red (LTR) staining induced by DMF was closer to the nucleus or nuclear membrane. Lysosomal membrane permeability (LMP) and its subsequent the explosive death of cancer cells may be mainly related to the direct action of DMF with different organelles.

The photodynamic and intrinsic effects of Azure B on mitochondrial bioenergetics and the consequences of its intrinsic effects on hepatic energy metabolism.

BACKGROUND: The present study aimed to characterize the intrinsic and photodynamic effects of azure B (AB) on mitochondrial bioenergetics, as well as the consequences of its intrinsic effects on hepatic energy metabolism. METHODS: Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. RESULTS: AB interacted with mitochondria regardless of photostimulation, but its binding degree was reduced by mitochondrial energization. Under photostimulation, AB caused lipid peroxidation and protein carbonylation and decreased the content of reduced glutathione (GSH) in mitochondria. AB impaired mitochondrial bioenergetics in at least three distinct ways: (1) uncoupling of oxidative phosphorylation; (2) photoinactivation of complexes I and II; and (3) photoinactivation of the FoF1-ATP synthase complex. Without photostimulation, AB also demonstrated mitochondrial toxicity, which was characterized by the induction of lipid peroxidation, loss of inner mitochondrial membrane integrity, and uncoupling of oxidative phosphorylation. The perfused rat liver experiments showed that mitochondria were one of the major targets of AB, even in intact cells. AB inhibited gluconeogenesis and ureagenesis, two biosynthetic pathways strictly dependent on intramitochondrially generated ATP. Contrariwise, AB stimulated glycogenolysis and glycolysis, which are required compensatory pathways for the inhibited oxidative phosphorylation. Similarly, AB reduced the cellular ATP content and the ATP/ADP and ATP/AMP ratios. CONCLUSIONS: Although the properties and severe photodynamic effects of AB on rat liver mitochondria might suggest its usefulness in PDT treatment of liver tumors, this possibility should be considered with precaution given the toxic intrinsic effects of AB on mitochondrial bioenergetics and energy-linked hepatic metabolism.

The vibronic absorption spectrum and electronic properties of Azure B in aqueous solution: TD-DFT/DFT study.

The vibronic absorption spectrum of Azure B (AB) in an aqueous solution is calculated using the time-dependent density functional theory (TD-DFT). The results of calculations are analyzed using all hybrid functionals supported by Gaussian16, the 6-31++G(d,p) basis set, and the IEFPCM and SMD solvent models. The solvent model IEFPCM gave significantly underestimated values of λmax in comparison with the experiment. This is a manifestation of the TD-DFT "cyanine failure". However, the SMD model made it possible to obtain good agreement between the calculation results and experimental data. The best fit was achieved using the X3LYP functional. According to our calculations, the shoulder in the visible absorption spectrum of AB has a vibronic origin. However, the calculated shoulder is weaker than the experimental one. Explicit assignment of two water molecules, which form strong hydrogen bonds with a dye molecule, leads to a shift of the calculated absorption spectrum to longer wavelengths by approximately 17 nm but does not lead to an improvement in its shape. Comparative analysis of the calculated vibronic absorption spectra of Azure B with those obtained earlier for Azure A and methylene blue showed that the presence and intensity of the short-wavelength shoulder are determined by the location of the bands of higher vibronic transitions relative to the band of the 00 â†’ 00 main transitions. Photoexcitation leads to an increase in the dipole moment of the dye molecule. An insignificant photoinduced electron transfer was found in the central ring of the chromophore of the dye molecule.