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Temporal proteomic profiling of Chlamydia trachomatis-infected HeLa-229 human cervical epithelial cells

Tan GM, Lim HJ, Yeow TC, Movahed E, Looi CY, Gupta R, et al.

Proteomics, 2016 05;16(9):1347-60.
PMID: 27134121 DOI: 10.1002/pmic.201500219

Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography-tandem mass spectrometry (LC-MS(3) ) analysis. C. trachomatis (serovar D, MOI 1)-infected HeLa-229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis-infected HeLa-229 cells indicate complex host-pathogen interactions at early phase of chlamydial infection.

Matched MeSH terms: Staining and Labeling/methods
Fulltext Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration

Leow SN, Luu CD, Hairul Nizam MH, Mok PL, Ruhaslizan R, Wong HS, et al.

PLoS One, 2015;10(6):e0128973.
PMID: 26107378 DOI: 10.1371/journal.pone.0128973

To investigate the safety and efficacy of subretinal injection of human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats.

Matched MeSH terms: Staining and Labeling/methods
Pseudocyst forms of Trichomonas vaginalis from cervical neoplasia

Afzan MY, Suresh K

Parasitol Res, 2012 Jul;111(1):371-81.
PMID: 22398830 DOI: 10.1007/s00436-012-2848-3

Trichomonas vaginalis, a flagellated protozoan parasite causes a variety of adverse health consequences in both men and women. The parasite exists in the trophozoite and the pseudocystic stage. The study reports for the first time that pseudocyst forms of T. vaginalis isolated from cervical neoplasia (CN) patients demonstrated distinct, different and significant in vitro growth profiles when grown in vitro cultures from day 1 up to day 5 (p<0.05, Mann-Whitney test) when compared with the same life cycle stages isolated from non-cervical neoplasia but symptomatic patients (NCN). Pseudocysts from CN and NCN isolates remained viable in distilled water until 3 h 10 min and 2 h 10 min, respectively. The nucleus of pseudocysts in CN isolates using acridine orange and DAPI showed more intense staining revealing higher nuclear content. The FITC-labeled Concanavalin A stained stronger green fluorescence with surface of pseudocysts in CN isolates showing more rough and creased surface with higher numbers of deep micropores with larger numbers of chromatin masses, vacuoles, and hydrogenosomes. The study confirms that pseudocystic stage from CN, despite the uniformity in appearance of being rounded and showing no motility without a true cyst wall under light microscopy, demonstrated different biochemical, surface, and ultrastructural properties. The study provides evidence that phenotypic variant forms of pseudocysts does exist and possibly does play a role in exacerbating cervical cancer.

Matched MeSH terms: Staining and Labeling/methods
Fulltext Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples

Ummul Haninah A, Vasan SS, Ravindran T, Chandru A, Lee HL, Shamala Devi S

Trop Biomed, 2010 Dec;27(3):611-23.
PMID: 21399603 MyJurnal

This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.

Matched MeSH terms: Staining and Labeling/methods
Bone marrow and adipose stem cells can be tracked with PKH26 until post staining passage 6 in in vitro and in vivo

Ude CC, Shamsul BS, Ng MH, Chen HC, Norhamdan MY, Aminuddin BS, et al.

Tissue Cell, 2012 Jun;44(3):156-63.
PMID: 22402173 DOI: 10.1016/j.tice.2012.02.001

Tracking of transplanted cells has become an important procedure in cell therapy. We studied the in vitro dye retention, survival and in vivo tracking of stem cells with PKH26 dye. Sheep BMSCs and ADSCs were labeled with 2, 4 and 8 μmol of PKH26 and monitored for six passages. Labeled BMSCs and ADSCs acquired mean cumulative population doubling of 12.7±0.4 and 14.6±0.5; unlabeled samples had 13.8±0.5 and 15.4±0.6 respectively. Upon staining with 2, 4 and 8 μmol PKH26, BMSCs had retentions of 40.0±5.8, 60.0±2.9 and 95.0±2.9%, while ADSCs had 92.0±1.2, 95.0±1.2 and 98.0±1.2%. ADSCs retentions were significantly higher at 2 and 4 μmol. On dye retention comparison at 8 μmol and 4 μmol for BMSCs and ADSCs; ADSCs were significantly higher at passages 2 and 3. The viability of BMSCs reduced from 94.0±1.2% to 90.0±0.6% and ADSCs from 94.0±1.2% to 52.0±1.2% (p<0.05) after 24h. BMSCs had significant up regulation of the cartilage genes for both the labeled and the unlabeled samples compared to ADSCs (p<0.05). PKH26 fluorescence was detected on the resected portions of the regenerated neo-cartilage. The recommended concentration of PKH26 for ADSCs is 2 μmol and BMSCs is 8 μmol, and they can be tracked up to 49 days.

Matched MeSH terms: Staining and Labeling/methods*
Fulltext Colonial morphotypes and biofilm forming ability of Burkholderia pseudomallei

Koh SF, Tay ST, Puthucheary SD

Trop Biomed, 2013 Sep;30(3):428-33.
PMID: 24189672 MyJurnal

Burkholderia pseudomallei the causative agent of melioidosis, is being increasingly recognized as an important cause of morbidity and mortality in South East Asia. Biofilm formation of B. pseudomallei may be responsible for dormancy, latency and relapse of melioidosis. Based on the colonial morphology of the bacteria on B. pseudomallei selective agar medium, seven distinct morphotypes were identified. This study was conducted to assess the in vitro biofilm produced by B. pseudomallei and to investigate possible correlation between B. pseudomallei morphotypes with biofilm forming abilities of the isolates. Using a standard biofilm crystal violet staining assay, comparison was made between the biofilm forming ability of 76 isolates of B. pseudomallei and Burkholderia thailandensis ATCC 700388. Amongst the blood isolates, 30.2% were considered as high biofilm producers and 27.9% were low producers, 33.3% of the pus isolates were considered as high and 16% low biofilm producers. Most of the isolates were identified as morphotype group 1 which displayed a rough centre with irregular circumference on the agar medium. However, we did not find any correlation of B. pseudomallei morphotypes with biofilm forming abilities (p > 0.05). Additional studies are needed to identify internal and external factors which contribute to the high and low biofilm formation of B. pseudomallei.

Matched MeSH terms: Staining and Labeling/methods
Metabolic glycan labeling and chemoselective functionalization of native biomaterials

Ren X, Evangelista-Leite D, Wu T, Rajab TK, Moser PT, Kitano K, et al.

Biomaterials, 2018 11;182:127-134.
PMID: 30118980 DOI: 10.1016/j.biomaterials.2018.08.012

Decellularized native extracellular matrix (ECM) biomaterials are widely used in tissue engineering and have reached clinical application as biomesh implants. To enhance their regenerative properties and postimplantation performance, ECM biomaterials could be functionalized via immobilization of bioactive molecules. To facilitate ECM functionalization, we developed a metabolic glycan labeling approach using physiologic pathways to covalently incorporate click-reactive azide ligands into the native ECM of a wide variety of rodent tissues and organs in vivo, and into the ECM of isolated rodent and porcine lungs cultured ex vivo. The incorporated azides within the ECM were preserved after decellularization and served as chemoselective ligands for subsequent bioconjugation via click chemistry. As proof of principle, we generated alkyne-modified heparin, immobilized it onto azide-incorporated acellular lungs, and demonstrated its bioactivity by Antithrombin III immobilization and Factor Xa inhibition. The herein reported metabolic glycan labeling approach represents a novel platform technology for manufacturing click-reactive native ECM biomaterials, thereby enabling efficient and chemoselective functionalization of these materials to facilitate tissue regeneration and repair.

Matched MeSH terms: Staining and Labeling/methods
Species identification of intestinal microsporidia using immunofluorescence antibody assays

Al-Mekhlafi MA, Fatmah MS, Anisah N, Azlin M, Al-Mekhlafi HM, Norhayati M

Southeast Asian J. Trop. Med. Public Health, 2011 Jan;42(1):19-24.
PMID: 21323160

Abstract. The species identification of Enterocytozoon bieneusi and Encephalitozoon intestinalis is only possible using transmission electron microscopy (TEM), mo lecular techniques and immunofluorescence antibody assays (IFA). In this study, 50 positive and 50 negative fecal specimens for microsporidial spores using the Weber modified trichrome (WMT) staining technique were examined using IFA-MAbs. Of the 100 specimens examined, the microsporidial spores identified by IFA-MAbs were Enterocytozoon Bieneusi 42 (75%) Encephalitozoon intestinalis 7 (12.5%) and mixed infections 7 (12.5%). The sensitivity and specificity of IFA-MAbs in detecting microsporidial spores were 98% and 86%, respectively. The agreement between the WMT staining technique and IFA-MAbs was statistically significant by Kappa statistics (K = 0.840; p < 0.001). E. bieneusi was the commonest Microsporidia species isolated from the studied population; the presence of microsporidial spores detected by IFA-MAbs should be confirmed by other methods.

Matched MeSH terms: Staining and Labeling/methods
Rapid diagnosis of Helicobacter pylori infection in gastric imprint smears

Kaur G, Madhavan M, Basri AH, Sain AH, Hussain MS, Yatiban MK, et al.

Southeast Asian J. Trop. Med. Public Health, 2004 Sep;35(3):676-80.
PMID: 15689086

The objective of this study was to determine the sensitivity, specificity, positive (PPV), and negative predictive values (NPV) of Diff-Quik-stained gastric imprint cytology smears in the detection of H. pylori compared with histology. Air-dried imprint smears of gastric biopsies from 150 patients were stained by the Diff-Quik method in the endoscopy suite and examined for H. pylori, providing results within minutes. The presence of inflammation and intestinal metaplasia were documented. The same biopsy was processed and stained with H&E and Warthin-Starry stains, and reviewed by a different pathologist blind to the imprint cytology results. Ninety-four of the 150 patients were male with a mean age of 50 years. Based on histology, the H. pylori prevalence was very low at 8%. The sensitivity and specificity of imprint cytology in the detection of H. pylori were 83.3% and 100%, respectively. The PPV and NPV were 100% and 98.6%, respectively. There were two false negatives and no false positives. A combination of imprint cytology and histology achieved 100% sensitivity. Imprint smears did not provide added value over histology with regards to inflammation and metaplasia. Gastric imprint smears stained with Diff-Quik method is a rapid, cheap, and reliable method for the detection of H. pylori and have their best results when complemented with histology.

Matched MeSH terms: Staining and Labeling/methods*
Wintergreen oil: a novel method in Wheatley's trichrome staining technique

Salleh FM, Anuar TS, Yasin AM, Moktar N

J Microbiol Methods, 2012 Oct;91(1):174-8.
PMID: 22986100 DOI: 10.1016/j.mimet.2012.08.004

Permanent staining of faecal smears by Wheatley's trichrome technique has been used by many scientists for the detection of parasites in the past and it was found to be highly sensitive. This study was conducted to evaluate the use of Wintergreen oil in comparison with xylene in Wheatley's trichrome staining technique, as the reference technique. In a blind comparison study, 500 collected faecal samples from aboriginal communities were examined. Wintergreen oil was found to be more superior than xylene as a clearing agent in the Wheatley's trichrome staining of polyvinyl alcohol-fixed faecal smears for the identification of intestinal protozoa. Elimination of toxic, carcinogenic, and fire hazards makes Wintergreen oil the preferred choice in routine parasitology examinations.

Matched MeSH terms: Staining and Labeling/methods*
Evaluation of gram-chromotrope kinyoun staining technique: its effectiveness in detecting microsporidial spores in fecal specimens

Salleh FM, Al-Mekhlafi AM, Nordin A, Yasin 'M, Al-Mekhlafi HM, Moktar N

Diagn Microbiol Infect Dis, 2011 Jan;69(1):82-5.
PMID: 21146718 DOI: 10.1016/j.diagmicrobio.2010.08.028

This study was conducted to evaluate the modification of the usual Gram-chromotrope staining technique developed in-house known as Gram-chromotrope Kinyoun (GCK) in comparison with the Weber Modified Trichrome (WMT) staining technique; as the reference technique. Two hundred and ninety fecal specimens received by the Microbiology Diagnostic Laboratory of Hospital Universiti Kebangsaan Malaysia were examined for the presence of microsporidial spores. The sensitivity and specificity of GCK compared to the reference technique were 98% and 98.3%, respectively. The positive and negative predictive values were 92.5% and 99.6%, respectively. The agreement between the reference technique and the GCK staining technique was statistically significant by Kappa statistics (K = 0.941, P < 0.001). It is concluded that the GCK staining technique has high sensitivity and specificity in the detection of microsporidial spores in fecal specimens. Hence, it is recommended to be used in the diagnosis of intestinal microsporidiosis.

Matched MeSH terms: Staining and Labeling/methods*
Evaluation of formalin-ether sedimentation and trichrome staining techniques: its effectiveness in detecting Entamoeba histolytica/dispar/moshkovskii in stool samples

Anuar TS, Al-Mekhlafi HM, Abdul Ghani MK, Abu Bakar E, Azreen SN, Salleh FM, et al.

J Microbiol Methods, 2013 Mar;92(3):344-8.
PMID: 23361047 DOI: 10.1016/j.mimet.2013.01.010

This study was conducted to evaluate two routinely microscopic diagnostic methods in comparison with single-round PCR assay as the reference technique to detect Entamoeba histolytica/dispar/moshkovskii. Examination was performed on 500 stool samples obtained from Orang Asli communities in different states of Malaysia using formalin-ether sedimentation, trichrome staining and single-round PCR techniques. Ninety-three stool samples were detected E. histolytica/dispar/moshkovskii positive by routine microscopy, while single-round PCR detected 106 positive samples. Additional positives detected by PCR assay were eventually confirmed to be negative by both microscopic techniques. Detection rate of E. histolytica/dispar/moshkovskii was highest in combination techniques (18.6%), followed by trichrome staining (13.4%) and formalin-ether sedimentation (11.2%) techniques. Single-round PCR detected 21.2% of the stool samples. The sensitivity and specificity of formalin-ether sedimentation and trichrome staining techniques compared to the reference technique were 31.1% (95% CI: 29.0-36.0) and 94.2% (95% CI: 89.8-98.9), and 53.8% (95% CI: 46.0-76.2) and 97.5% (95% CI: 92.8-99.1), respectively. However, the sensitivity [59.4% (95% CI: 48.9-78.5)] of the method increased when both techniques were performed together, but the specificity decreased to 92.4% (95% CI: 81.0-98.0). The agreement between the reference technique, trichrome staining and combination techniques were statistically significant by Kappa statistics (trichrome staining: K = 0.592, p < 0.05; combination techniques: K = 0.543, p < 0.05). Hence, the combination technique is recommended to be used as a screening method in the diagnosis of E. histolytica/dispar/moshkovskii infections either for clinical or epidemiological study.

Matched MeSH terms: Staining and Labeling/methods
Comparison of the sensitivity and specificity of p16/Ki-67 dual staining and HPV DNA testing of abnormal cervical cytology in the detection of histology proven cervical intraepithelial neoplasia grade 2 and above (CIN 2+)

Tay TKY, Lim KL, Hilmy MH, Thike AA, Goh ST, Song LH, et al.

Malays J Pathol, 2017 Dec;39(3):257-265.
PMID: 29279588

INTRODUCTION: Human papillomavirus (HPV) testing is used as a means of triaging cervico-vaginal smears with low grade squamous abnormalities or as part of co-testing with cytology. While HPV testing has a high sensitivity, it has a low specificity in detecting cervical intraepithelial neoplasia grade 2 and above (CIN 2+) leading to unnecessary colposcopy referrals. We investigate the accuracy of the p16/Ki-67 dual immunocytochemical stain in determining the presence of CIN 2+ lesions on histology and its potential as a superior biomarker for triage.
METHODS: Liquid based cervico-vaginal cytology specimens with squamous abnormalities and corresponding histology from 97 women with subsequent colposcopy and biopsy were included. The specimens were then subjected to the dual stain and Roche Cobas 4800 multiplex real time PCR HPV DNA testing. The sensitivity and specificity of the dual stain and HPV testing were calculated using CIN 2+ on histology as a reference standard.
RESULTS: The sensitivity and specificity of the dual stain in detecting histology proven CIN 2+ was 93.7% and 76.5% while HPV testing was 85.7% and 14.7% respectively. Of the 44 women with ASCUS or LSIL on cytology, the dual stain also reduced the number of unnecessary colposcopy referrals from 27 to 7 when used as a triage marker compared to HPV testing.
CONCLUSION: p16/Ki-67 dual stain was more sensitive and specific than HPV testing in determining the presence of CIN 2+ on histology. It could triage low grade cervico-vaginal specimens more effectively and potentially help women avoid unnecessary colposcopies. Future studies are needed to further evaluate its role in cervical cancer screening programmes.

Matched MeSH terms: Staining and Labeling/methods
Morphological characteristics of developmental stages of Acanthamoeba and Naegleria species before and after staining by various techniques

Ithoi I, Ahmad AF, Mak JW, Nissapatorn V, Lau YL, Mahmud R

Southeast Asian J. Trop. Med. Public Health, 2011 Nov;42(6):1327-38.
PMID: 22299400

Seven stains were studied to determine the best color and contrast for staining the developmental stages of free living pathogenic Acanthamoeba and Naegleria species. The acid-fast bacilli stain (AFB) produced a blue color without contrast; trichrome-eosin and modified Field's showed various color contrasts; Giemsa, iron-hematoxylin, modified AFB and Gram produced only one color which distinguished the nucleus, nucleolus, cytoplasm, food- and water-vacuoles. The motile organs (acanthopodia, pseudopodia, lobopodia and flagella) were also clearly differentiated but produced a similar color as the cytoplasm. These motile organelles were first induced by incubating at 37 degrees C for at least 15 minutes and then fixing with methanol in order to preserve the protruding morphology prior to staining. The trichrome-eosin and iron-hematoxylin stains showed good color contrast for detecting all three stages, the trophozoite, cyst and flagellate; Giemsa and Gram stained the trophozoite and flagellate stages; the modified Field's and modified AFB stains stained only the trophozoite stage. Depending on the purpose, all these stains (except the AFB stain) can be used to identify the developmental stages of Acanthamoeba and Naegleria for clinical, epidemiological or public health use.

Matched MeSH terms: Staining and Labeling/methods*
Fulltext Identification of Entamoeba histolytica trophozoites in fresh stool sample: comparison of three staining techniques and study on the viability period of the trophozoites

Tan ZN, Wong WK, Nik Zairi Z, Abdullah B, Rahmah N, Zeehaida M, et al.

Trop Biomed, 2010 Apr;27(1):79-88.
PMID: 20562817 MyJurnal

Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.

Matched MeSH terms: Staining and Labeling/methods*
Modified Field's staining--a rapid stain for Trichomonas vaginalis

Afzan MY, Sivanandam S, Kumar GS

Diagn Microbiol Infect Dis, 2010 Oct;68(2):159-62.
PMID: 20846588 DOI: 10.1016/j.diagmicrobio.2010.06.005

Trichomonas vaginalis, a flagellate protozoan parasite commonly found in the human genitourinary tract, is transmitted primarily by sexual intercourse. Diagnosis is usually by in vitro culture method and staining with Giemsa stain. There are laboratories that use Gram stain as well. We compared the use of modified Field's (MF), Giemsa, and Gram stains on 2 axenic and xenic isolates of T. vaginalis, respectively. Three smears from every sediment of spun cultures of all 4 isolates were stained, respectively, with each of the stains. We showed that MF staining, apart from being a rapid stain (20 s), confers sharper staining contrast, which differentiates the nucleus and the cytoplasm of the organism when compared to Giemsa and Gram staining especially on parasites from spiked urine samples. The alternative staining procedure offers in a diagnostic setting a rapid stain that can easily visualize the parasite with sharp contrasting characteristics between organelles especially the nucleus and cytoplasm. Vacuoles are more clearly visible in parasites stained with MF than when stained with Giemsa.

Matched MeSH terms: Staining and Labeling/methods*
Field's stain--a rapid staining method for Acanthamoeba spp

Pirehma M, Suresh K, Sivanandam S, Anuar AK, Ramakrishnan K, Kumar GS

Parasitol Res, 1999 Oct;85(10):791-3.
PMID: 10494803

Acanthamoeba sp. is a free-living amoeba known to cause chronic central nervous system infection or eye infection in humans. Many cases remain undetected for want of a good detection system. We report for the first time a rapid staining method to facilitate the identification of Acanthamoeba sp. using the modified Field's staining technique. A. castellanii, which was used in the present experiment, is maintained in our laboratory in mycological peptone medium (Gibco). The cultures were pooled together and smears were made on glass slides for staining purposes. Different types of stains such as Field's stain, modified Field's stain, Wright's stain, Giemsa stain, Ziehl-Neelsen stain, and trichrome stain were used to determine the best stain for the identification of this amoeba. The concentration of various stains and the duration of staining were varied to provide the best color and contrast for each stain. Acanthamoeba was also obtained from the brain of experimentally infected mice and was stained with various stains as mentioned above to determine the best stain for use in identifying the presence of this parasite in experimentally infected animals. The modified Field's stain gives a very good color contrast as compared with other stains. Furthermore, it takes only 20 s to be carried out using the least number of reagents, making it suitable for both laboratory and field use.

Matched MeSH terms: Staining and Labeling/methods*
Fulltext A P. falciparum NF54 Reporter Line Expressing mCherry-Luciferase in Gametocytes, Sporozoites, and Liver-Stages

Marin-Mogollon C, Salman AM, Koolen KMJ, Bolscher JM, van Pul FJA, Miyazaki S, et al.

Front Cell Infect Microbiol, 2019;9:96.
PMID: 31058097 DOI: 10.3389/fcimb.2019.00096

Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic Plasmodium falciparum (Pf) NF54 line was generated that expresses a fusion of mCherry and luciferase genes under the control of the Pf etramp10.3 gene promoter (line mCherry-luc@etramp10.3). Pf etramp10.3 is related to rodent Plasmodium uis4 and the uis4 promoter has been used to drive high transgene expression in rodent parasite sporozoites and liver-stages. We examined transgene expression throughout the complete life cycle and compared this expression to transgenic lines expressing mCherry-luciferase and GFP-luciferase under control of the constitutive gapdh and eef1a promoters. The mCherry-luc@etramp10.3 parasites express mCherry in gametocytes, sporozoites, and liver-stages. While no mCherry signal was detected in asexual blood-stage parasites above background levels, luciferase expression was detected in asexual blood-stages, as well as in gametocytes, sporozoites and liver-stages, with the highest levels of reporter expression detected in stage III-V gametocytes and in sporozoites. The expression of mCherry and luciferase in gametocytes and sporozoites makes this transgenic parasite line suitable to use in in vitro assays that examine the effect of transmission blocking inhibitors and to analyse gametocyte and sporozoite biology.

Matched MeSH terms: Staining and Labeling/methods*
Fulltext Improvement of malaria diagnostic system based on acridine orange staining

Kimura M, Teramoto I, Chan CW, Idris ZM, Kongere J, Kagaya W, et al.

Malar J, 2018 Feb 07;17(1):72.
PMID: 29415724 DOI: 10.1186/s12936-018-2214-8

BACKGROUND: Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent.
METHODS: The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya.
RESULTS: Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard.
CONCLUSIONS: Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

Matched MeSH terms: Staining and Labeling/methods*
Fulltext Antiproliferative property and apoptotic effect of xanthorrhizol on MDA-MB-231 breast cancer cells

Cheah YH, Nordin FJ, Tee TT, Azimahtol HL, Abdullah NR, Ismail Z

Anticancer Res, 2008 Nov-Dec;28(6A):3677-89.
PMID: 19189649

Xanthorrhizol is a natural sesquiterpenoid compound isolated from the rhizome of Curcuma xanthorrhizza Roxb (Zingerberaceae). Recent studies of xanthorrhizol in cell cultures strongly support the role of xanthorrhizol as an antiproliferative agent. In our study, we tested the antiproliferative effect of xanthorrhizol using different breast cancer cell lines. The invasive breast cancer cell line, MDA-MB-231, was then selected for further investigations. Treatment with xanthorrhizol caused 50% growth inhibition on MDA-MB-231 cells at 8.67 +/- 0.79 microg/ml as determined by sulforhodamine B (SRB) assay. Hoechst 33258 nuclear staining assay showed the rate of apoptosis of MDA-MB-231 cells to increase in response to xanthorrhizol treatment. Immunofluorescence staining using antibody MitoCapture and fluorescein isothiocyanate (FITC)-labeled cytochrome c revealed the possibility of altered mitochondrial transmembrane potential and the release of cytochrome c respectively. This was further confirmed by Western-blotting, where cytochrome c was showed to migrate from mitochondrial fraction to the cytosol fraction of treated MDA-MB-231 cells. Caspase activity assay showed the involvement of caspase-3 and caspase-9, but not caspase-6 or caspase-8 in MDA-MB-231 apoptotic cell death. Subsequently, cleavage of PARP-1 protein is suggested. These data suggest treatment with xanthorrhizol modulates MDA-MB-231 cell apoptosis through the mitochondria-mediated pathway subsequent to the disruption of mitochondrial transmembrane potential, release of cytochrome c, activation of caspase-3 and caspase-9, and the modulation of PARP-1 protein.

Matched MeSH terms: Staining and Labeling/methods

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