This document discusses procedures for preparing and staining microbial smears and slides. It describes how microbes are fixed to slides through air drying or heat, and then stained using simple, differential or special staining techniques. Key points covered include how gram staining classifies bacteria as either gram-positive or gram-negative based on cell wall structure and staining properties, and how acid-fast staining identifies mycobacteria by their lipid-rich cell walls. Negative staining can reveal capsules around cells.
this presentation involves a comprehensive outlines regarding the most common different methods used in diagnostic microbiology to stain bacteria and their structures
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 .
this presentation involves a comprehensive outlines regarding the most common different methods used in diagnostic microbiology to stain bacteria and their structures
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 .
Microbiology is the study of
living organisms of microscopic
size which includes bacteria ,
Fungi , Algae , Protozoa and Viruses. It is concerned with the forms, structure , reproduction , physiology , metabolism and classification.
Principle Of Microbiology
Medical microbiology deals with the causative agent of the infectious disease of the human , the ways in which they produce disease in the body and essential information for diagnosis and treatment.
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.
Microbiology is the study of
living organisms of microscopic
size which includes bacteria ,
Fungi , Algae , Protozoa and Viruses. It is concerned with the forms, structure , reproduction , physiology , metabolism and classification.
Principle Of Microbiology
Medical microbiology deals with the causative agent of the infectious disease of the human , the ways in which they produce disease in the body and essential information for diagnosis and treatment.
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.
Why is it that the Grams stain is not appropriate for acid fast ba.pdfarkleatheray
Â
Why is it that the Gram\'s stain is not appropriate for acid fast bacterium?
Solution
During staining in microbiology, microbes are coloured or dyed so that their structure can be
viewed against a lighter background. There are different staining techniques such as direct(Dye
binds to the cell surface and all the microbes are stained alike),differential(based on their specific
cell surface different microbes get stained differently) special (usually a particular organelle is
targeted)and negative staining(back ground is stained instead of microbes) Gram stain is a
differential stain where the bacteria with a thick peptidoglycan layer around the cells are stained
with violet colour and those with an additional lipopolysaccharide layer will be stained pink. To
understand this lets take a look at Gram staining procedure. First of all bacterial culture is
smeared onto a glass slide and is heat fixed so that during the washes in the procedure the
bacteria stay fixed onto the slide. Primary stain crystal violet is added onto the slide and the dye
seperates into ions which penetrate or enter the cell membranes of both Gram positive and
negative cells. Next iodine is added onto the slide which forms huge complexes with crystal
violet. Then a dye remover usually called as decolouriser such as alchohol or acetone is used to
remove the stain. In Gram negative bacteria the outer most membrane is lost during the treatment
with alchohol whereas in the case of gram positive bacteria the dye is trapped inside the cell
membrane. A secondary stain saffronin is used and which gives pink colour to Gram negative
bacteria. Now coming to acid-fast bacteria, they have a compound called mycolic acid in their
cell walls and are resistant to decolourisation after the acid treatment and alchohol and hence
would not lose colour and appear violet. As we can see these organism can be easily confused
with Gram positive bacteria which also do not lose stain not because they are resistant to acids
but they have the crystal violet dye trapped in their cell membrane..
identification of bacteria- lecture 7.pptxOsmanAli92
Â
he culture media are classified in many different ways: Based on the physical state Liquid media Solid media Semisolid media Based on the presence or absence of oxygen Anaerobic media Aerobic media Based on nutritional factors Simple media Synthetic media Complex
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.
Food hygiene is more than cleanliness ......
Protecting food from risk of contamination, including harmful bacteria, poison and other foreign bodies.
Preventing any bacteria present multiplying to an extent which would result in the illness of consumers or the early spoilage of the food.
Destroying any harmful bacteria in the food by thorough cooking
or processing.
Discarding unfit or contaminated food.
T-Cell Activation
⢠Concept of immune response
⢠T cell-mediated immune response
⢠B cell-mediated immune response
I. Concept of immune response
⢠A collective and coordinated response to the introduction of foreign substances in an individual mediated by the cells and molecules in the immune system.
II. T cell-mediated immune response
⢠Cell-mediated immunity is the arm of the adaptive immune response whose role is to combat infection of intracellular pathogens, such as intracellular bacteria (mycobacteria, listeria monocytogens), viruses, protozoa, etc.
Major Histocompatibility Complex
MHC:
⢠Major Histocompatibility Complex
â Cluster of genes found in all mammals
â Its products play role in discriminating self/non-self
â Participant in both humoral and cell-mediated immunity
⢠MHC Act As Antigen Presenting Structures
⢠In Human MHC Is Found On Chromosome 6
â Referred to as HLA complex
⢠In Mice MHC Is Found On Chromosome 17
â Referred to as H-2 complex
⢠Genes Of MHC Organized In 3 Classes
â Class I MHC genes
⢠Glycoproteins expressed on all nucleated cells
⢠Major function to present processed Ags to TC
â Class II MHC genes
⢠Glycoproteins expressed on macrophages, B-cells, DCs
⢠Major function to present processed Ags to TH
â Class III MHC genes
⢠Products that include secreted proteins that have immune functions. Ex. Complement system, inflammatory molecules
Antigen Processing and Presentation MID
Antigens and âforeignnessâ
⢠Antigens (or, more properly, immunogens) have a series of features which confer immunogenicity.
⢠One of these features is âforeignness.â
⢠So, we can infer that â most often â antigens â ultimately â originate externally.
⢠(There are exceptions, of course. Some cells become transformed by disease [e. g., cancer] or by aging. In such instances, the antigens have an internal origin.)
Extinction of a particular animal or plant species occurs when there are no more individuals of that species alive anywhere in the world - the species has died out. This is a natural part of evolution. But sometimes extinctions happen at a much faster rate than usual. Natural Causes of Extinction.
Difference between In-Situ and Ex-Situ conservation
Conservation of biodiversity and genetic resources helps protect, maintain and recover endangered animal and plant species. There are mainly two strategies for the conservation of wildlife: In-situ conservation and Ex-situ conservation. Although, both the strategies aim to maintain and recover endangered species, they are different from each other. Let us see how they differ from each other!
Evolution Of Bacteria
Bacteria have existed from very early in the history of life on Earth. Bacteria fossils discovered in rocks date from at least the Devonian Period (419.2 million to 358.9 million years ago), and there are convincing arguments that bacteria have been present since early Precambrian time, about 3.5 billion years ago. Bacteria were widespread on Earth at least since the latter part of the Paleoproterozoic, roughly 1.8 billion years ago, when oxygen appeared in the atmosphere as a result of the action of the cyanobacteria. Bacteria have thus had plenty of time to adapt to their environments and to have given rise to numerous descendant forms.
Impact of Environment on Loss of Genetic Diversity and Speciation
Genetic variation describes naturally occurring genetic differences among individuals of the same species. This variation permits flexibility and survival of a population in the face of changing environmental circumstances. Consequently, genetic variation is often considered an advantage, as it is a form of preparation for the unexpected. But how does genetic variation increase or decrease? And what effect do fluctuations in genetic variation have on populations over time?
GENE ENVIRONMENT INTERACTION
Subtle differences in one personâs genes can cause them to respond differently to the same environmental exposure as another person. As a result, some people may develop a disease after being exposed to something in the environment while others may not.
As scientists learn more about the connection between genes and the environment, they pursue new approaches for preventing and treating disease that consider individual genetic codes.
How to store food in hot
The Good News
To maximize benefit of preservation, keep your food as fresh as possible for as long as possible. You can do this, even in the heat, by creating a âcoolerâ made from two basic terra cotta pots, one larger than the other. Put the smaller pot in the larger one, fill the gap with sand, and saturate the sand with water. Then cover it with a cloth. To add additional insulation from the heat, bury the pot up to its rim. The evaporation of moisture from the wet sand will cool the air around the food and help keep it fresh.
What is IUPAC naming?
In order to give compounds a name, certain rules must be followed. When naming organic compounds, the IUPAC (International Union of Pure and Applied Chemistry) nomenclature (naming scheme) is used. This is to give consistency to the names. It also enables every compound to have a unique name, which is not possible with the common names used (for example in industry). We will first look at some of the steps that need to be followed when naming a compound, and then try to apply these rules to some specific examples.
IUPAC Nomenclature
IUPAC nomenclature uses the longest continuous chain of carbon atoms to determine the basic root name of the compound. The root name is then modified due to the presence of different functional groups which replace hydrogen or carbon atoms in the parent structure.
Hybridization describes the bonding atoms from an atom's point of view. For a tetrahedral coordinated carbon (e.g. methane CH4), the carbon should have 4 orbitals with the correct symmetry to bond to the 4 hydrogen atoms.
INTRODUCTION:
Hybrid Orbitals
Developed by Linus Pauling, the concept of hybrid orbitals was a theory created to explain the structures of molecules in space. The theory consists of combining atomic orbitals (ex: s,p,d,f) into new hybrid orbitals (ex: sp, sp2, sp3).
1. Why Firefly give light during night?
2. Why atomic mass and Atomic numbers are given to elements ?
3. Why elements have been characterized and classified into different groups?
4. What is the transition of elements and what they play their role in elements stability?
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
Â
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
Â
RESULTS: Overall life span (LS) was 2252.1Âą1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years â 64.8%, 20 years â 42.5%. 513 LCP lived more than 5 years (LS=3124.6Âą1525.6 days), 148 LCP â more than 10 years (LS=5054.4Âą1504.1 days).199 LCP died because of LC (LS=562.7Âą374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0âN12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0âN12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Â
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
HOT NEW PRODUCT! BIG SALES FAST SHIPPING NOW FROM CHINA!! EU KU DB BK substit...GL Anaacs
Â
Contact us if you are interested:
Email / Skype : kefaya1771@gmail.com
Threema: PXHY5PDH
New BATCH Ku !!! MUCH IN DEMAND FAST SALE EVERY BATCH HAPPY GOOD EFFECT BIG BATCH !
Contact me on Threema or skype to start big business!!
Hot-sale products:
NEW HOT EUTYLONE WHITE CRYSTAL!!
5cl-adba precursor (semi finished )
5cl-adba raw materials
ADBB precursor (semi finished )
ADBB raw materials
APVP powder
5fadb/4f-adb
Jwh018 / Jwh210
Eutylone crystal
Protonitazene (hydrochloride) CAS: 119276-01-6
Flubrotizolam CAS: 57801-95-3
Metonitazene CAS: 14680-51-4
Payment terms: Western Union,MoneyGram,Bitcoin or USDT.
Deliver Time: Usually 7-15days
Shipping method: FedEx, TNT, DHL,UPS etc.Our deliveries are 100% safe, fast, reliable and discreet.
Samples will be sent for your evaluation!If you are interested in, please contact me, let's talk details.
We specializes in exporting high quality Research chemical, medical intermediate, Pharmaceutical chemicals and so on. Products are exported to USA, Canada, France, Korea, Japan,Russia, Southeast Asia and other countries.
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Â
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2â3 criteria; moderate AUD: 4â5 criteria; severe AUD: 6â11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
Â
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Departmentâs official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
For Better Surat #âall #Girl Service â¤85270-49040⤠Surat #âall #Girls
Â
Preparing smears for staining
1. 1 AmjadKhanAfridi
PreparedBy Amjad Khan Afridi
Preparing Smears for Staining
Most initial observations of microorganisms are made with stained preparations. Staining
simply means coloring the microorganisms with a dye that emphasizes certain structures.
Before the microorganisms can be stained, however, they must be fixed (attached) to the
microscope slide. Fixing simultaneously kills the microorganisms and fixes them to the slide. It
also preserves various parts of microbes in their natural state with only minimal distortion .
When a specimen is to be fixed, a thin film of material containing the microorganisms is spread
over the surface of the slide. This film, called a smear, is allowed to air dry. In most staining
procedures the slide is then fixed by passing it through the flame of a Bunsen burner several
times, smear side up, or by covering the slide with methyl alcohol for I minute. Stain is applied
and then washed off with water; then the slide is blotted with absorbent paper. Without fixing,
the stain might wash the microbes off the slide. The stained microorganisms are now ready for
microscopic examination.
Stains are salts composed of a positive and a negative ion, one of which is colored and is known
as the chromoplwre. The color of so-called basic dyes is in the positive ion; in acidic dyes. it is in
the negative ion. Bacteria are slightly negatively charged at pH 7. Thus, the colored positive ion
in a basic dye is attracted to the negatively charged bacterial cell. Basic dyes, which include
crystal violet, methylene blue, malach ite green, and safran in, are more commonly used than
acidic dyes. Acidic dyes are not attracted to mosttypesof bacteriabecause the dye's negative ions
are repelled by the negatively charged bacterial surface, so the stain colors the background
instead. Preparing colorless bacteria against a colored background is called negative staining. It
is valuable for observing overall cell shapes, sizes, and capsules because the cells are made
highly visible against a contrasting dark background . Distortions of cell size and shape are
minimized because fixing is not necessary and the cells do not pick up the stain . Examples of
acidic dyes are eosin, acid fuchsin, and nigrosin.
To apply acidic or basic dyes, microbiologists use three kinds of staining techniques: simple,
differential, and special.
ďź Simple Stains
A simple stain is an aqueous or alcohol solution of a single basic dye. Although different dyes
bind specifically to different part s of cells, the primary purpose of a simple stain is to highlight
the entire microorganism so that cellular shapes and basic structures are visible. The stain is
applied 10 the fixed smear for a certain length of lime and then washed off, and the slide is
2. 2 AmjadKhanAfridi
PreparedBy Amjad Khan Afridi
dried and examined. Occasionally, a chemical is added to the solution to intensify the slain;
such an additive is called a mordant. One function of a mordant is 10 increase the affinity of a
stain for a biological specimen; another is to coal a structure (such as a flage llum) to make it
thicker and easier to see after it is stained with a dye. Some of the simple stains commonly used
in the laboralory are methylene blue, carbolfuchsin, crystal violet, and safranin.
ďźDifferentialStains
Unlike simple stains, differential stains react differently with different kinds of bacteria and thus
can be used to distinguish them. The differential stains most frequently used for bacteria are
the Gram stain and the acid-fast stain.
ďź Gram Stain:
The Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram. It is
one of the most useful staining procedures because it classifies bacteria into two large groups:
gram-positive and gram-negative.
1. A heat -fixed smear is covered with a basic purple dye, usually crystal violet. Because the
purple stain imparts its color to all cells, it is referred to as a primary stain.
2. After a short time, the purple dye is washed off, and the smear is covered with iodine, a
mordant. When the iodine is washed off, both gram- positive and gram-negative
bacteria appear dark violet or purple.
3. Next, the slide is washed with alcohol or an alcohol-acetone solution. This solution is a
discoloring agent. Which removes the purple from the cells of some species but not
from others.
4. The alcohol is rinsed off, and the slide is then stained with safranin, a basic red dye. The
smear is washed again, blotted dry, and examined microscopically.
The purple dye and the iodine combine in the cytoplasm of each bacterium and color it dark
violet or purple. Bacteria that retain this color after the alcohol has attempted to decolorize
them are classified as gram-positive; bacteria that lose the dark violet or purple color after
depolarization are classified as gramnegative . Because gram-negative bacteria are colorless
after the alcohol wash, they are no longer visible. This is why the basic dye safranin is applied; it
turns the gram-negative bacteria pink. Stains such as safranin that have a contrasting color to
the primary stain arc called counterstains. Because grampositive bacteria retain the original
purple stain, they are not affected by the safranin counterstain .
3. 3 AmjadKhanAfridi
PreparedBy Amjad Khan Afridi
As you will see in Chapter 4, different kinds of bacteria react differently to the Gram stain
because structural differences in their cell walls affect the retention or escape of a combination
of crystal violet and iodine, called the crystal violet- iodine (CV-I) complex. Among other
differences, gram-positive bacteria have a thicker peptidoglycan (disaccharides and amino
acids) cell wall than gram-negative bacteria. In addition, gram- negative bacteria contain a layer
of lipopolysaccharide (lipids and polysaccha - rides) as part of their cell wall .When applied to
both gram-positive and gram-negative cells, crystal violet and then iodine readily enter the
cells. Inside the cells, the crystal violet and iodine combine to form CV- 1. This complex is larger
than the crystal violet molecule that entered the cells, and, because of its size, it can not be
washed out of the intact pept idoglycan layer of gram-positive cells by alcohol. Consequently,
gram-positive cells retain the color of the crystal violet dye. In gram-negative cells, however,
the alcohol wash disrupts the outer lipopolysaccharide layer, and the CV- I complex is washed
out through the thin layer of peptidoglycan . As a result, gramnegative cells are colorless until
counterstained with safranin, after which they arc pink.
In summary, gram-positive cells retain the dye and remain purple. Gram-negative cells do not
retain the dye; they are colorless until counterstained with a red dye.
The Gram method is one of the most important staining techniques in medical microbiology.
But Gram staining results arc not universally applicable, because some bacterial cells stain
poorly or not at all. The Gram reaction is most consistent when it is used on young, growing
bacteria.
In summary, gram-positive cells retain the dye and remain purple. Gram-negative cells do not
retain the dye; they are colorless until counterstained with a red dye.
The Gram method is one of the most important staining techniques in medical microbiology.
But Gram staining results arc not universally applicable, because some bacterial cells stain
poorly or not at all. The Gram reaction is most consistent when it is used on young, growing
bacteria.
ďź Acid-Fast Stain:
Another important differential stain (one that differentiates bacteria into distinctive groups) is
the acid-fast stain, which binds strongly only to bacteria that have a waxy material in their cell
walls. Microbiologists use this stain to identify all bacteria in the genus Mycobacterium,
including the two important pathogens .
In the acid-fast staining procedure, the red dye car- bolfuchsin is applied to a fixed smear, and
the slide is gently heated for several minutes. (Heating enhances penetration and retention of
4. 4 AmjadKhanAfridi
PreparedBy Amjad Khan Afridi
the dye.) Then the slide is cooled and washed with water. The smear is next treated with acid-
alcohol, a decolorizer, which removes the red stain from bacteria that are not acid -fast. The
acid-fast microorganisms retain the red color because the carbolfuchsin is more soluble in the
cell wall lipids than in the acid-alcohol. In non- acid-fast bacteria, whose cell walls lack the lipid
components, the carbolfuchsin is rapidly removed during decolorization, leaving the cells
colorless.
The smear is then stained with a methylene blue counterstain. Non-acid-fast cells appear blue
after application of the counterstain.
Special Stains
Special stains are used to color and isolate specific parts of microorganisms, such as endospores
and flagella, and to reveal the presence of capsules.
ďźNegative Staining for Capsules
Many microorganisms contain a gelatinous covering called a capsule.. In medical microbiology,
demonstrating the presence of a capsule is a means of determining the organism's virulence.
the degree to which a pathogen can cause disease.
Capsule staining is more difficult than other types of staining procedures because capsular
materials are soluble in water and may be dislodged or removed during rigorous washing. To
demonstrate the presence of capsules, a microbiologist can mix the bacteria in a solution
containing a fi ne colloidal suspension of colored particles (usually India ink or nigrosin) to
provide a contrasting background and then stain the bacteria with a simple stain, such as
safranin. Because of their chemical composition, capsules do not accept most biological dyes,
such as safranin, and thus appear as halos surrounding each stained bacterial cell.
ďź Endospore (Spore)Staining:
An endospore is a special resistant, dormant structure formed within a cell that protects a
bacterium from adverse environmental conditions. Although endospores arc relatively
uncommon in bacterial cells, they can be formed by a few genera of bacteria.
Endospores cannot be stained by ord inary methods, such as simple staining and Gram sta
ining, because the dyes do not penetrate the wall of the endospore.
The most commonly used endospore stain is the Schaeffer- Fulton endospore stain. Malachite
green, the primary stain, is applied to a heat-fixed smear and heated to steaming for about 5
minutes. The heat helps the stain penetrate the endospore wall. Then the prepa ration is
washed for about 30 seconds with water to remove the malachite green from all of the cells'
5. 5 AmjadKhanAfridi
PreparedBy Amjad Khan Afridi
parts except the endospores. Next, safranin, a counterstain, is applied to the smear to sta in
portions of the cell other than endospores. In a properly prepared smear, the endospores
appear green within red or pink cells. Because endospores are highly refractive, they can be
detected under the light microscope when unstained, but they cannot be differentiated from
inclusions of stored material without a special stain .
ďź Flagella Staining
Bacterial flagella (singular: flagllum) are structures of loco - motion too small to be seen with a
light microscope without staining. A tedious and delicate staining procedure uses a mordant
and the stain carbolfuchsin to build up the diameters of the flagella until they become visible
under the light micro - scope. Microbiologists use the number and arrangement of flagella as
diagnostic aids. Animation Staining .
www.microbiologyplace.com