Paint It Black: Staining of the Yeast Cryptococcus Neoformans with India Ink

Forman Erwin Siagian *

Department of Parasitology, Faculty of Medicine, Universitas Kristen Indonesia, Jakarta, Indonesia.

*Author to whom correspondence should be addressed.


Abstract

Aims: To rectify the application of negative staining using India Ink dye in order to visualize the opportunistic yeast, Cryptococcus neoformans and its capsule.

Discussion: The yeast C. neoformans is generally considered an opportunistic fungal pathogen due to its propensity to infect immunocompromised hosts, particularly those with HIV (+). This yeast responsible for causing severe disseminated Cryptococcus meningoencephalitis (CM) in humans. The type of clinical sample commonly sent for laboratory analysis of suspected patients is the cerebrospinal fluid (CSF) that usually obtained from spinal tap or lumbar puncture procedure. laboratory analysis to confirm CM initially by direct visualization of Cryptococci via light microscopy. Visualization of capsule, as the hallmark of C. neoformans is easy when appropriate number of yeasts are available in the clinical sample. India Ink is the dye of choice to stain C. neoformans and its capsule. The pathognomonic resemblance of its capsule is that of a halo appearance, whether thin or thick, surrounding the cell made visible by suspending the yeast in India ink preparations.

Conclusion: India ink stain is still widely used for the detection of cryptococci in CSF, particularly in resource-limited laboratory/health service. Its sensitivity can be improved.

Keywords: Meningitis, cryptococcosis, encapsulated, halo, cell, dye, cerebrospinal fluid, fungal infection


How to Cite

Siagian, Forman Erwin. 2024. “Paint It Black: Staining of the Yeast Cryptococcus Neoformans With India Ink”. International Journal of Pathogen Research 13 (3):56-64. https://doi.org/10.9734/ijpr/2024/v13i3286.

Downloads

Download data is not yet available.

References

Ruschel MAP, Thapa B. Cryptococcus meningitis. In: StatPearls Treasure Island (FL): StatPearls Publishing; 2024. Available:https://www.ncbi.nlm.nih.gov/books/NBK525986/

Okurut S, Boulware DR, Olobo J, Meya DB. Landmark clinical observations and immunopathogenesis pathways linked to HIV and Cryptococcus fatal central nervous system co-infection. Mycoses. 2020;63(8):840-853.

Available:https://doi.org/10.1111/myc.13122

Tu A, Byard RW. Cryptococcosis and unexpected death. Forensic Sci Med Pathol. 2021;17(4):742-745.

Available:https://doi.org/10.1007/s12024-021-00400-1

McHale TC, Boulware DR, Kasibante J, Ssebambulidde K, Skipper CP, Abassi M.Diagnosis and management of cryptococcal meningitis in HIV-infected adults. Clin Microbiol Rev. 2023;36: e00156-22.

Available:https://doi.org/10.1128/cmr.00156-22

Cafarchia C, Romito D, Iatta R, Camarda A, Montagna MT, Otranto D. Role of birds of prey as carriers and spreaders of Cryptococcus neoformans and other zoonotic yeasts. Med Mycol. 2006;44(6): 485-92.

Available:https://doi.org/10.1080/13693780600735452

Randhawa HS, Mussa AY, Khan ZU. Decaying wood in tree trunk hollows as a natural substrate for Cryptococcus neoformans and other yeast-like fungi of clinical interest. Mycopathologia. 2001;151 (2):63-9.

Available:https://doi.org/10.1023/a:1010906220888.

Stempinski PR, Gerbig GR, Greengo SD, Casadevall A. Last but not yeast-The many forms of Cryptococcus neoformans. PLoS Pathog. 2023;19(1):e1011048. Available:https://doi.org/10.1371/journal.ppat.1011048.

Denham ST, Brown JCS. Mechanisms of Pulmonary Escape and Dissemination by Cryptococcus neoformans. J Fungi (Basel). 2018;4(1):25. Available:https://doi.org/10.3390/jof4010025.

Wang Y, Pawar S, Dutta O, Wang K, Rivera A, Xue C. Macrophage Mediated Immunomodulation During Cryptococcus Pulmonary Infection. Front Cell Infect Microbiol. 2022;12:859049.

Available:https://doi.org/10.3389/fcimb.2022.859049

de Castro RJA, Marina CL, Sturny-Leclère A, Hoffmann C, Bürgel PH, Wong SSW, et al. Kicking sleepers out of bed: Macrophages promote reactivation of dormant Cryptococcus neoformans by extracellular vesicle release and non-lytic exocytosis. PLoS Pathog. 2023;19(11):e1011841. Available:https://doi.org/10.1371/journal.ppat.1011841

Alanio A. Dormancy in Cryptococcus neoformans: 60 years of accumulating evidence. J Clin Invest. 2020;130(7):3353-3360. Available:https://doi.org/10.1172/JCI136223

Yang C, Huang Y, Zhou Y, Zang X, Deng H, Liu Y, Shen D, Xue X. Cryptococcus escapes host immunity: What do we know? Front Cell Infect Microbiol. 2022;12:1041036. Available:https://doi.org/10.3389/fcimb.2022.1041036

Benedict K, Gold JAW, Dietz S, Anjum S, Williamson PR, Jackson BR. Testing for Cryptococcosis at a Major Commercial Laboratory-United States, 2019-2021. Open Forum Infect Dis. 2022;9(7): ofac253.

Available:https://doi.org/10.1093/ofid/ofac253

Pfennig CL, Brown B. Cerebrospinal Fluid Examination—India Ink Preparation for Cryptococcus Neoformans. In: Knoop KJ, Stack LB, Storrow AB, Thurman R. eds. The Atlas of Emergency Medicine, 5e. McGraw-Hill; 2021. Available:https://accessemergencymedicine.mhmedical.com/content.aspx?bookid=2969&sectionid=250464413

AL-Zubaidy IMK, Shabaa RAH. Evaluation the efficacy of some culture media in melanin production by Cryptococcus neoformans. Materials Today: Proceedings, 2023;80(3):3167-3170, ISSN 2214-7853. Available:https://doi.org/10.1016/j.matpr.2021.07.184

Vidal JE, Boulware DR. Lateral flow assay for Cryptococcus antigen: An important advance to improve the continuum of hiv care and reduce cryptococcus meningitis-related mortality. Rev Inst Med Trop Sao Paulo. 2015;57Suppl19(Suppl19):38-45. Available:https://doi.org/10.1590/S0036-46652015000700008

Holgate JH, Webb J. MICROSCOPY | Transmission Electron Microscopy. Ed(s): Caballero B. Encyclopedia of Food Sciences and Nutrition (Second Edition). Academic Press, 2003; 3928-3934, ISBN 9780122270550. Available:https://doi.org/10.1016/B0-12-227055-X/00780-X

Coovadia YMC, Mahomed S, Dorasamy A, Chang C. A comparative evaluation of the Gram stain and India ink stain for the rapid diagnosis of cryptococcal meningitis in HIV infected patients in Durban, Southern African. Journal of Infectious Diseases. 2015;30:2:61-63, Available:https://doi.org/10.1080/23120053.2015.1054199

Suryowati T. Biochemical aspects of cell staining. Asian Journal of Research in Biochemistry. 2024;14(3):61-71. Available:https://doi.org/10.9734/AJRB/2024/v14i3288

Moyes RB, Reynolds J, Breakwell DP. Preliminary staining of bacteria: Negative stain. Curr Protoc Microbiol; 2009. Available:https://doi.org/10.1002/9780471729259.mca03fs15.

Hughes RB, Smith AC. Capsule Stain Protocols. American Society for Microbiology; 2007.

Available:https://asm.org/ASM/media/Protocol-Images/Capsule-Stain-Protocols.pdf?ext=.pdf

Droste MS, Biel SS, Terstegen L, Wittern KP, Wenck H, Wepf R. Noninvasive measurement of cell volume changes by negative staining. J Biomed Opt. 2005;10(6):064017. Available:https://doi.org/10.1117/1.2138011.

Zhu L, Rajendram M, Huang KC. Effects of fixation on bacterial cellular dimensions and integrity. iScience. 2021;24(4):102348. Available:https://doi.org/10.1016/j.isci.2021.102348.

Tripathi N, Sapra A. Gram Staining. In: StatPearls Treasure Island (FL): StatPearls Publishing; 2024.

Available:https://www.ncbi.nlm.nih.gov/books/NBK562156/

Chedore P, Th'ng C, Nolan DH, Churchwell GM, Sieffert DE, Hale YM, Jamieson F. Method for inactivating and fixing unstained smear preparations of mycobacterium tuberculosis for improved laboratory safety. J Clin Microbiol. 2002;40 (11):4077-80. Available:https://doi.org/10.1128/JCM.40.11.4077-4080.2002.

Petersen J, McLaughlin S. Laboratory exercises in micr cises in microbiology: Disco obiology: discovering the unseen world through hands-on investigation. City University of New York (CUNY)- CUNY Academic Works. CUNY Queensborough Community College; 2016. Available:https://academicworks.cuny.edu/cgi/viewcontent.cgi?article=1015&context=qb_oers

Ogodo AC, Narayana MSSV, Vardhan PS, Gupta RK, Gautam AK, Egbuna C, Kushwaha PP, Singh AK, Kumar S. Chapter 9 - Principles of applied microbiology and biotechnology: Technique for the screening of antimicrobial herbs. Ed(s): Egbuna C, Mishra AP, Goyal MR. Preparation of Phytopharmaceuticals for the Management of Disorders. Academic Press. 2021;185-214. ISBN 9780128202845.

Available:https://doi.org/10.1016/B978-0-12-820284-5.00001-0.

Schramm LL. Emulsions, Foams, and Suspensions: Fundamentals and Applications. Wiley VCH-Verlag- GmbH & Co KgaA Weinheim; 2005. Available:http://repository.ottimmo.ac.id/41/1/Emulsions%2C%20Foams%2C%20and%20Suspensions%20-%20Fundamentals%20and%20Applications.pdf

Kröger M, Schleusener J, Lademann J, Meinke MC, Jung S, Darvin ME. Tattoo Pigments Are Localized Intracellularly in the Epidermis and Dermis of Fresh and Old Tattoos: In vivo Study Using Two-Photon Excited Fluorescence Lifetime Imaging. Dermatology. 2023;239(3):478-493.

Available:https://doi.org/10.1159/000529577.

Boezaart A, Sala-Blanch X, Monzo E, Reina MA. India ink: A time-tested histological marker. Reg Anesth Pain Med. 2019:rapm-2019-100553. Available:https://doi.org/10.1136/rapm-2019-100553.

Kizhakkoottu S, Jayaraj G, HeraldJ S, Don KR, Santhanam A. Inking of gross specimens: A systematic review. Russian Journal of Archive of Pathology. (In Russ., In Engl.) 2021;83(1):49‑52.

Available:https://doi.org/10.17116/patol20218301149

Patil A, Anand R, Mahajan PG. Comparative Study of Acrylic Color and India Ink for Their Use as a Surgical Margin Inks in Oral Squamous Cell Carcinoma. World Journal of Dentistry, 2015;6:26-30.

Rathod S, Munshi A, Agarwal J. Skin markings methods and guidelines: A reality in image guidance radiotherapy era. South Asian J Cancer. 2012;1(1) :27-9. Available:https://doi.org/10.4103/2278-330X.96502.

Khan N, Hou H, Swartz H, Kuppusamy P. Direct and repeated measurement of heart and brain oxygenation using In vivo EPR Oximetry. Qin PZ, Warncke K (eds). book: Electron Paramagnetic Resonance Investigations of Biological Systems by Using Spin Labels, Spin Probes, and Intrinsic Metal Ions, Part B. Academic Press; 2015. Available:https://doi.org/10.1016/bs.mie.2015.06.023.

Davies KJ. Protein damage and degradation by oxygen radicals. I. general aspects. J Biol Chem. 1987;262(20):9895-901.

Shahin A, Bachir W, El-daher MS. Optical properties in the visible range of two different India ink used as biological phantoms. The European Physical Journal Applied Physics. 2020;93(1): 10502.

Available:https://doi.org/10.1051/epjap/2020200234

Boulware DR, Rolfes MA, Rajasingham R, von Hohenberg M, Qin Z, Taseera K, Schutz C, Kwizera R, Butler EK, Meintjes G, Muzoora C, Bischof JC, Meya DB. Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contrast. Emerg Infect Dis. 2014;20:45–53.

Available:https://doi.org/10.3201/eid2001.130906.

McHugh KE, Gersey M, Rhoads DD, Procop GW, Zhang Y, Booth CN, Sturgis CD. Sensitivity of cerebrospinal fluid cytology for the diagnosis of Cryptococcus infections: A 21-Year Single-Institution Retrospective Review, American Journal of Clinical Pathology. 2019;151(2):198–204. Available:https://doi.org/10.1093/ajcp/aqy133

Chammard TB, Lanternier F, Lortholary O. 89 – Cryptococcosis. Ed(s): Ryan ET, Hill DR, Solomon S, Aronson NE, Endy TP. Hunter's Tropical Medicine and Emerging Infectious Diseases (Tenth Edition). Elsevier. 2020;678-681. ISBN 9780323555128. Available:https://doi.org/10.1016/B978-0-323-55512-8.00089-2.

Yogev R. CHAPTER 43 - Chronic Meningitis. Ed(s): Sarah S. Long. Principles and practice of pediatric infectious disease (Third Edition). W.B. Saunders. 2008;291-299, ISBN 9780702034688.

Available:https://doi.org/10.1016/B978-0-7020-3468-8.50049-3.

Soni ZJ, Reubenson G, De Maayer T, Coovadia A, Lazarus EM. Neonatal Cryptococcosis: beware of false-positive results, Journal of the Pediatric Infectious Diseases Society. 2012; 1(3):250–253.

Available:https://doi.org/10.1093/jpids/pis037

Gomerepa SS, Isaa SE, Obasekib DO, Badung BP. BadungAccuracy of Indian ink staining in diagnosis of cryptococcal meningitis in HIV infection in Jos, Nigeria. The Royal Society of Chemistry;2012. Available:https://www.google.com/search?q=Accuracy+of+Indian+ink+staining+in+diagnosis+of+cryptococcal&oq=Accuracy+of+Indian+ink+staining+in+diagnosis+of+cryptococcal&gs_lcrp=EgZjaHJvbWUyBggAEEUYOTIHCAEQIRigATIHCAIQIRigAdIBBzk1NWowajeoAgCwAgA&sourceid=chrome&ie=UTF-8

Luma HN, Temfack E, Halle MP, Tchaleu BC, Mapoure YN, Koulla-Shiro S. Cryptococcus meningoencephalitis in human immunodeficiency virus/acquired immunodeficiency syndrome in douala, cameroon: A cross sectional study. N Am J Med Sci. 2013;5(8):486-91. Available:https://doi.org/10.4103/1947-2714.117318.

Teunissen CE, Petzold A, Bennett JL, Berven FS, Brundin L, Comabella M, et al. A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking. Neurology. 2009;73(22):1914-22.

Available:https://doi.org/10.1212/WNL.0b013e3181c47cc2

Gangadharan SS, Raveendranath S. Cryptococcosis in oncology patients: A case series in a tertiary care cancer centre. Iran J Microbiol. 2023;15(6):821-825. Available:https://doi.org/10.18502/ijm.v15i6.14163

Le T. 45 - Systemic Mycoses,Ed(s): Farrar J, Garcia P, Hotez P, Junghanss T, Kang G, Lalloo D, White N. Manson's Tropical Diseases (Twenty-Fourth Edition). Elsevier. 2024;495-509. ISBN 9780702079597.

Available:https://doi.org/10.1016/B978-0-7020-7959-7.00045-2.

Zerpa R, Huicho L, Guillén A. Modified India Ink Preparation for Cryptococcus neoformans in Cerebrospinal Fluid Specimens. J Clin Microbiol. 1996;34(9):2290-1. Available:https://doi.org/10.1128/JCM.34.9.2290-2291

Morikawa S, Sato A, Ezaki T. A simple, one-step polychromatic staining method for epoxy-embedded semithin tissue sections. Microscopy. 2018;67(6):331–344. Available:https://doi.org/10.1093/jmicro/dfy037

Ibembe IN, Wiggin TR. An Alternative To India Ink Stain. Trop Doct. 2015;45(3):206-7. Available:https://doi.org/10.1177/0049475515587821

Kambugu A, Meya DB, Rhein J, O'Brien M, Janoff EN, Ronald AR, et al. Outcomes of cryptococcal meningitis in Uganda before and after the availability of highly active antiretroviral therapy. Clin Infect Dis. 2008;46:1694–1701. Available:https://doi.org/10.1086/587667

Melly MA, McGee ZA, Horn RG, Morris F, Glick AD. An electron microscopic India ink technique for demonstrating capsules on microorganisms: Studies with Streptococcus pneumoniae, Staphylococcus aureus, and Neisseria gonorrhoeae. J Infect Dis. 1979;140(4): 605-9.

Available:https://doi.org/10.1093/infdis/140.4.605

Yoshikawa M, Noguchi K, Toda T. Effect of particle sizes in India ink on its use in evaluation of apical seal. J Osaka Dent Univ. 1997;31(1-2):67-70.

Mizutani H, Akeda T, Yamanaka K, Isoda K, Gabazza EC. Single step modified ink staining for Tzanck test: quick detection of herpetic giant cells in Tzanck smear. J Dermatol. 2012 ;39(2):138-40.

Available:https://doi.org/ 10.1111/j.1346-8138.2011.01335.x

Kosemehmetoglu K, Guner G, Ates D. Indian ink vs tissue marking dye: A quantitative comparison of two widely used macroscopical staining tool. Virchows Arch. 2010;457(1):21-5.

Available:https://doi.org/10.1007/s00428-010-0941-5