Tuesday, April 30, 2019

Staining-the basic concept


Staining
Microorganism are very minute, generally they are colorless and transparent so it is very difficult to focus through microscope. The use of special types of microscopy such as phase contrast and dark field techniques is design to improve visualization; however these techniques require special microscopes and training. In common purpose we use light microscope for direct observation of bacteria, to observe through this we have to increase the contrast of the organisms against their background is necessary, to do this microorganism should be colored against their background i.e  application of staining techniques.

Staining: It is the process of coloring of microbial cells and their parts with the application of a dye or dyes to a fixed smear.
Stains or dyes: Yhey are coloring compounds, chemically they are salt as they contain acidic or basic part of stain. Microbiologists mostly use aniline or synthetic dyes. Each dye molecule has two functional groups, the auxochrome and chromophore.
       Auxochrome: It ionizes and gives the molecules the ability to react with substrate.
       Chromophore: It is site of unsaturation and absorbs specific wavelength of light. The color of the solution obtained is that of the  unabsorbed light which transmitted through it.

The synthetic dyes be classified as Acidic, Basic and Neutral dyes depending on whether the coloring bearing compound is a cation and an anion.
       Acidic dyes: Anionic dyes, which react with substrate group which ionize to produce positive charge such as corboxyl, phenolic or sulfydryl etc. Anionic dyes are usually found as sodium. Eg: Nigrosin, Congo red, Eosin, Acid fuchsin etc.
       Basic dyes: Cationic dyes, which react with substrate group which ionize to produce negative charge such as ammonium ions. Anionic dyes are usually found as chlorides or sulfates. Eg: Methylene Blue, Crysal Violet, Safranin etc.
       On the basis of number of dyes and staining techniques used, staining are of following types;
       1:   Simple staining: In this staining only single dye is used. Morphology of bacteria, yeast and mold are observed. Generally, positively charged chromophores are used in this staining. Methylene blue, Basic fuchsin, Crystal violet etc are used.
       2:            Negative Staining: It is the techniques where the background is stained leaving the bacterial cell unstained due to electrostatic repelling between same charged cell and stain used. Heat fix is avoided so the accurate size, shape and arrangement can be measured and studied. Bacteria which are hard to stain like Spirochaetes, Mycobacterium and Nocardia are used in this techniques. Capsule staining is one of this techniques. Stains nigrosin, congored, eosin, acid fuchsin are used.
       3:            Differential Staining: In this techniques more than one dye is used. It is applied in categorization of bacteria into two groups. It stains specific structure of cell eg: flagella, spore, capsule etc. Gram staining categorizes bacteria into two groups i.e gram positive and gram negative. 


Wednesday, April 3, 2019

BMC RESEARCH (OPEN ACCESS)


Article published on BMC

Rijal et al. Tropical Medicine and Health (2019) 47:21 https://doi.org/10.1186/s41182-019-0148-7

Micro-stratification of malaria risk in Nepal: implications for malaria control and elimination

Komal Raj Rijal1, Bipin Adhikari2, Nabaraj Adhikari1, Shyam Prakash Dumre3, Mayur Sharma Banjara4, Upendra Thapa Shrestha1, Megha Raj Banjara1, Nihal Singh5, Leonard Ortegea6, Bibek Kumar Lal7, Garib Das Thakur7 and Prakash Ghimire1,5*

* Correspondence: prakashghimire@gmail.com

1Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal
2Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
3Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan.
4World Health and Research Centre, Kathmandu, Nepal.
5World Health Organization (WHO), Country Office, Kathmandu, Nepal
6Global Malaria Program, World Health Organization Headquarters, Geneva, Switzerland.
7Epidemiology and Disease Control Division (EDCD), Department of Health Services, Ministry of Health and Population, Kathmandu, Nepal.

ABSTRACT
Background: A significant reduction in malaria cases over the recent years in Nepal has encouraged the government to adopt a goal of malaria-free nation by 2025.Nevertheless, to achieve this goal, it is critical to identify the epidemiological burden of malaria by specific regions and areas for an effective targeted intervention. The main objective of this study was to estimate the risk of malaria at Village Development Committee (VDC) level in Nepal based on disease, vector, parasite, and geography.

Methods: In 2012, the micro-stratification of malaria risk was carried out in 75 districts of Nepal. Instruments such as a questionnaire, case record forms, and guidelines for malaria micro-stratification were developed and pre-tested for necessary adaptations. Village Development Committee (VDC)-wise malaria data were analyzed using exploratory statistics and were stratified by geographical variables that contributed to the risk of malaria. To understand the transmission risk at VDC level, overlay analysis was done using ArcGIS 10. To ensure transparent, reproducible, and comprehensible risk assessment, standard scoring method was selected and utilized for data from 2009 to 2011. Thus identified, three major variables (key determinants) were given weights (wt.) accordingly to stratification of the malaria risk (disease burden, 0.3wt.; ecology/vector transmission, 0.5wt.; and vulnerability-population movement, 0.2wt.).
Malaria risk in a VDC was determined based on the overall scores and classified into four categories: no risk, low risk, moderate risk, and high risk.

Results: Analyzing the overall risk based on scoring of the total VDCs (n = 3976), 54 (1.36%), 201 (5.06%), 999 (25.13%), and 2718 (68.36%) were identified as high-, moderate-, low-, and no-risk categories for malaria, respectively. Based on the population statistics, 3.62%, 9.79%, 34.52%, and 52.05% of the countrys total population live in high-risk, moderate risk, low-risk, and no-risk VDCs for malaria, respectively. Our micro-stratification study estimates are 100,000 population
at high risk. Regional distribution showed that the majority of the high-risk VDCs were identified in the Far- and Midwestern regions (19 and 18 VDCs) followed by Central and Western regions (10 and 7 VDCs) with no high-risk VDCs in the Eastern region. Similarly, 77, 59, 27, 24, and 14 VDCs of the Central, Mid-western, Western, Eastern, and Far-western regions, respectively, were found under moderate malaria risk. Of the low-risk VDCs, 353, 215, 191, 148, and 92 were respectively from the Central, Eastern, Western, Far-western, and Mid-western regions.

Conclusions: The current micro-stratification study provides insights on malaria risk up to the VDC level. This will help the malaria elimination program to target interventions at the local level thereby ensuring the best utilization of available resources to substantially narrowed-down target areas. With further updates and refinement, the microstratification approach can be employed to identify the risk areas up to smaller units within the VDCs (ward and villages).

Keywords: Malaria, Micro-stratification, Elimination, Intervention, Nepal

Bacteria in Photos

Bacteria in Photos