Gram staining is used to classify bacteria based on differences in cell wall composition. It involves staining with crystal violet and iodine, then decolorizing and counterstaining. Gram-positive bacteria retain the crystal violet stain while gram-negative bacteria take up the counterstain. Acid-fast staining is used to identify mycobacteria by using heat to bind primary stain within acid-fast cell walls. Capsule staining uses India ink's dark background to contrast unstained capsules around stained cells. Spore staining employs malachite green and heat to permanently stain endospores within decolorized vegetative cells.
Staining methods are helpful for presumptive identification of Microbes like Gram stain helps to identify Gram positive and Gram negative bacteria, similarly Z-N stain helps to identify acid fast bacilli and India ink preparation capsule observation of Cryptococcus neoformans.
Capsule is an layer around the bacteria cell which gives bacteria the protection and pathogenicity. Staining such an layer is difficult with the normal stains so it is necessary to stain the background and the cell itself which makes the capsule appear colourless.
Acid fast staining is differential staining technique which differentiate bacteria into two group- acid fast bacteria and non acid bacteria. It used to identify acid-fast organisms such as members of the genus Mycobacterium .
Staining methods are helpful for presumptive identification of Microbes like Gram stain helps to identify Gram positive and Gram negative bacteria, similarly Z-N stain helps to identify acid fast bacilli and India ink preparation capsule observation of Cryptococcus neoformans.
Capsule is an layer around the bacteria cell which gives bacteria the protection and pathogenicity. Staining such an layer is difficult with the normal stains so it is necessary to stain the background and the cell itself which makes the capsule appear colourless.
Acid fast staining is differential staining technique which differentiate bacteria into two group- acid fast bacteria and non acid bacteria. It used to identify acid-fast organisms such as members of the genus Mycobacterium .
Staining is a technique used to enhance contrast in samples, generally at the microscopic level.Staining and fluorescent tagging can serve similar purposes. Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis.
Gram staining Gram positive and gram negative bacteria can be distinguished b...AgraniPaudel
Gram Staining Gram-positive and Gram-negative bacteria Principle of Gram stain, Heat fixing, Gram's iodine, Hans Christian gram, Carl Weighert, Smear making, heat fixing, Precautions for Gram staining, Gram staining distinguish bacteria on the basis of their cell wall composition.
INTRODUCTION TO MICRO LAB, STAINING TECHNIQUES & MORPHOLOGY OF BACTERIADrBhavikapatel
This PPT is helpful to understand first practical to 2nd year MBBS student.
I have added 2 video in this PPT to understand staining techniques properly.
Reference: 1 Gram stain video: Dr.G Bhanu prakash animated medical videos
2. Zn stain video: sridhar Rao
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
2. GRAM STAINING:PRINCIPLES
• Gram staining is used to determine gram status to classify
bacteria broadly. It is based on thecomposition of their
cell wall. Gram staining uses crystal violet to stain cell
walls, iodine as a mordant,and a fuchsin or safranin
counterstain to mark all bacteria. Gram status is important
in medicine; the presence or absence of a cell wall will
change the bacterium's susceptibility to some antibiotics.
Gram-positive bacteria stain dark blue or violet.
• Their cell wall is typically rich with peptidoglycan and
lacks the secondary membrane and lipopolysaccharide
layer found in Gram-negative bacteria
3. STEPS
• Crystal violet acts as the primary stain. Crystal violet mayalso
be used as a simple stain because it dyes the cell wallof any
bacteria.
• 2. Gram’s iodine acts as a mordant (Helps to fix the primary
dye to the cell wall).
• 3. Decolorizer is used next to remove the primary stain (crystal
violet) from Gram Negative bacteria (those with LPS imbedded
in their cell walls). Decolorizer is composed of an organic
solvent, such as, acetone or ethanol or a combination of both.)
• 4. Finally, a counter stain (Safranin), is applied to stain those
cells (Gram Negative) that have lost the primary stain as a
result of decolorization
6. GRAM POSITIVE
• Gram-positive bacteria are those that are stained dark
blue or violet by Gram staining. This is in contrast to
Gram-negative bacteria, which cannot retain the crystal
violet stain, instead taking up the counter stain (safranin
or fuchsine) and appearing red or pink. Grampositive
organisms are able to retain the crystal violet stain
because of the high amount of peptidoglycan in the cell
wall. Gram-positive cell walls typically lack the outer
membrane found in Gram-negative bacteria.
7. GRAM NEGATIVE BACTERIA
• Gram-negative bacteria are those bacteria that
• do not retain crystal violet dye in the Gram
• staining protocol. In a Gram stain test, a counter
• stain (commonly safranin) is added after the crystal
• violet, coloring all Gram-negative bacteria with a red
• or pink color. The test itself is useful in classifying
• two distinct types of bacteria based on the structural
• differences of their cell walls. On the other
• hand, Gram-positive bacteria will retain the crystal
• violet dye when washed in a decolorizing solution.
8. • On most Gram-stained
• preparations, Gram-negative
• organisms will appear red or
• pink because they are
• counterstained. Due to
• presence of higher lipid
• content, after alcoholtreatment,
• the porosity of the
• cell wall increases, hence the
• CVI complex (Crystal violet -
• Iodine) can pass through.
• Thus, the primary stain is not
• retained.
9. ACID FAST STAIN
• Acid-fast cells contain a
large amount of lipids and
waxes in their cell walls
primarily mycolic acid Acid
fast bacteria are usually
members of the genus
Mycobacterium or
Nocardia
• Therefore, this stain is
important to identify
Mycobacterium or
10. ZIEHL-NEELSON STAIN
Primary stain binds cell wall mycolic acids
Intense decolorization does not release primary stain from
the cell wall of AFB
Color of AFB-based on primary stain
Counterstain provides contrasting background
11. PROCEDURE
• Deparaffinize and hydrate to distilled water.
• 2. *Carbol-fuchsin solution, microwave 80 power, 45
seconds, allow
• slides to stand in hot solution for 5 minutes. Filter
solution once a
• week.
• 3. Wash in running tap water.
• 4. 1% Acid alcohol until light pink and color stops
running.
• 5. Wash in running tap water for 5 minutes..
• 6. Rinse in distilled water.
• 7. Working methylene blue for 30 seconds.
• 8. Rinse in water.
• 9. Dehydrate, clear, and coverslip.
12. CAPSULE STAINING:INDIA INK
• Capsules stain very poorly with reagents used in simple staining
and a capsule stain can be, depending on the method, a
misnomer because the capsule may or may not be stained.
• Negative staining methods contrast a translucent, darker
colored, background with stained cells but an unstained
capsule. The background is formed with india ink or nigrosin or
congo red. India ink is difficult to obtain nowadays; however,
nigrosin is easily acquired.
• A positive capsule stain requires a mordant that precipitates the
capsule. By counterstaining with dyes like crystal violet or
methylene blue, bacterial cell wall takes up the dye. Capsules
appear colourless with stained cells against dark background.
13. PROCEDURE
• Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on the slide.
• Congo Red is easier to see, but it does not work well with some strains. India Ink generally works,
but it has tiny particles that display Brownian motion that must be differentiated from your
bacteria. Nigrosin may need to be kept very thin or diluted.
• Using sterile technique, add a loopful of bacterial culture to slide, smearing it in the dye.
• Use the other slide to drag the ink-cell mixture into a thin film along the first slide and let stand for
5-7 minutes.
• Allow to air dry (do not heat fix).
• Flood the smear with crystal violet stain (this will stain the cells but not the capsules) for about 1
minutes. Drain the crystal violet by tilting the slide at a 45 degree angle and let stain run off until it
air dries .
• Examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by
clear zones surrounding the cells.
14.
15. SPORE STAIN:SCHAEFFER-FULTON METHOD
• A differential staining technique (the Schaeffer-Fulton method) is used to distinguish
between the vegetative cells and the endospores. A primary stain (malachite green)
is used to stain the endospores. Because endospores resist staining, the malachite
green will be forced into (i.e, malachite green permeate the spore wall) the
endospores by heating. In this technique heating acts as a mordant.
• There is no need of using any decolorizer in this spore staining as the primary dye
malachite green bind relatively weakly to the cell wall and spore wall .In fact If
washed well with water the dye come right out of cell wall however not from spore
wall once the dye is locked in. Water is used to decolorize the vegetative cells.
• As the endospores are resistant to staining, the endospores are equally resistant to
de-staining and will retain the primary dye while the vegetative cells will lose the
stain. The addition of a counterstain or secondary stain (safranin) is used to stain
the decolorized vegetative cells.
16. PROCEDURE
• Prepare smears of organisms to be tested for presence of endospores on a clean microscope
slide and air dry it.
• Heat fix the smear.
• Place a small piece of blotting paper (absorbent paper) over the smear and place the slide
(smear side up) on a wire gauze on a ring stand.
• Heat the slide gently till it starts to evaporate
• Remove the heat and reheat the slide as needed to keep the slide steaming for about 3-5
minutes. As the paper begins to dry add a drop or two of malachite green to keep it moist, but
don’t add so much at one time that the temperature is appreciably reduced.
• # DO NOT OVERHEAT. The process is steaming and not baking.
• After 5 minutes carefully remove the slide from the rack using a clothespin
• Remove the blotting paper and allow the slide to cool to room temperature for 2 minutes.
• Rinse the slide thoroughly with tap water (to wash the malachite green from both sides of the
microscope slide).
• Stain the smear with safranin for 2 minutes.
17. the vegetative cells
should appear pink/red
(i.e. color of counter
stain),
the vegetative cells
that contain
endospores should
stain pink while the
spores should be seen
as green ellipses
within the cells.
