This document provides instructions for smear preparation, drying and fixing smears, and performing Gram staining. It discusses:
1) How to make smears from different specimen types such as purulent fluids, cultures, and swabs.
2) Drying smears by air drying or gentle heat fixation to preserve microorganisms.
3) Fixation methods including heat, alcohol, and chemical fixatives and their purposes and techniques.
4) An overview of Gram staining including the principles, reagents used, and steps to distinguish Gram-positive from Gram-negative bacteria under the microscope.
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.
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
Gram stain is technique used to differntiate gram positive and gram negative bacteria.
If you like this slideshare then please do share and follow me.
You can visit my blogs:pranav2705.blogspot.com
Hereby you can get all about bacterial staining.
MICROBIAL STAINING
introduction
Microbial Staining - giving color to microbes. Because microbes are colorless and highly transparent structures.
Staining process in which microbes are getting color.
STAINES / DYES
Staines / dyes - organic compounds which carries either positive charges or negative charges or both .
Based on the charges
Basic stain / dyes :- stain with + ve charge .
Acidic stain / dyes - stain with -ve charge .
2. Based on function of stain :
Neutral stain / dyes - stain with both charges .
Simple staining only one dye is used differentiation among bacteria is impossible Eg . Simple Staining.
Differential staining- more than one dye is used- Differentiation among bacteria is possible- Eg. Gram's staining, Acid - fast staining.
Special staining - more than one dye used - Special structures are seen. Eg. Capsule staining , Spore staining .
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
Gram stain is technique used to differntiate gram positive and gram negative bacteria.
If you like this slideshare then please do share and follow me.
You can visit my blogs:pranav2705.blogspot.com
Hereby you can get all about bacterial staining.
MICROBIAL STAINING
introduction
Microbial Staining - giving color to microbes. Because microbes are colorless and highly transparent structures.
Staining process in which microbes are getting color.
STAINES / DYES
Staines / dyes - organic compounds which carries either positive charges or negative charges or both .
Based on the charges
Basic stain / dyes :- stain with + ve charge .
Acidic stain / dyes - stain with -ve charge .
2. Based on function of stain :
Neutral stain / dyes - stain with both charges .
Simple staining only one dye is used differentiation among bacteria is impossible Eg . Simple Staining.
Differential staining- more than one dye is used- Differentiation among bacteria is possible- Eg. Gram's staining, Acid - fast staining.
Special staining - more than one dye used - Special structures are seen. Eg. Capsule staining , Spore staining .
Similar to smear prep & Gram staining-new.pptx (20)
micro teaching on communication m.sc nursing.pdfAnurag Sharma
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.
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
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
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 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 N0—N12, 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 N0—N12 (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.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
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 Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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
Title: Sense of Smell
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 primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists
smear prep & Gram staining-new.pptx
1. SMEAR PREPARATION &
GRAM STAINING
Dr.priyadharshini.S ,
1st year postgraduate ,
Department of microbiology ,
Chettinad hospital and research institute ,
2. SMEAR PREPARATION
SMEAR should be spread evenly covering an area of about 15 - 20mm diameter on a slide.
The technique used to make smears from different specimens are as follows:
■ Purulent specimen: using a sterile wire loop, make a thin preparation.donot centrifuge a purulent fluid eg c
s.f containing pus cells.
■ Non purulent fluid specimen: centrifuge the fluid and make a smear from a drop of the well mixed
sediment.
■ Culture: Emulsify a colony in sterile distilled water and make a thin preparation on a slide, when a broth
culture transfer a loop ful to a slide and make a thin preparation.
■ Sputum : use a piece of clean stick to transfer and spread purulent and caseous material on a slide. Soak
the stick in a phenol Or hypochlorite disinfectant before discarding it.
■ Swabs: Roll the swab Ona slide. This is particularly important when looking for intracellular bacteria such as
N.gonorrhoea ( urtheral, cervical, eye swab). Rolling the swab avoids damaging the pus cells.
■ Faeces: Use apiece of clean stick to transfer pus and mucus to a slide decontaminate the stick before
discarding it.spread to make a thin preparation.
3. DRYING AND FIXING SMEARS
■ After making a smear,leave the slide in a safe place For the
smear to air dry,
■ When a smear requires urgent staining, it can be dried
quickly using gentle heat
■ The purpose of fixation is to preserve microorganisms and to
prevent smear being washed from slides during staining.
■ Smears are fixed by heat, alcohol, or occasionally by other
chemicals.
4. HEAT FIXATION:
■ This is widely used but can damage organisms and alter their
staining reactions especially when excessive heat is used. Heat
fixation also damages leucocytes and is therefore unsuitable for
fixing smears which may contain intracellular organisms such as
N. Gonorrhoeae and N. Meningitidis.
■ When used, heat fixation must be carried out with care. The
following technique is recommended:
■ Allow the smear to air-dry completely.
■ Rapidly pass the slide, smear uppermost, three times through the
flame of a spirit lamp or pilot flame of a Bunsen burner.
■ Note: After passing the slide through the flame three times, it
should be possible to lay the slide on the back of the hand without
the hand feeling uncomfortably hot. When this cannot be done,
too much heat has been used.
5. ALCOHOL FIXATION
■ This form of fixation is far less damaging to microorganisms than heat.
■ Cells especially pus cells, are also well preserved.
■ Alcohol fixation is therefore recommended for fixing smears when looking for gram
negative intracellular diplococci.
■ Alcohol fixation is more bactericidal than heat ( eg: M.tuberculosis is rapidly killed in
sputum smears after applying 70% alcohol)
■ A method of alcohol fixation is as follows:
1. Allow the smear to air dry completely.
2. Depending on the Type of smear, alcohol fix as follows:
– For the detection of intracellular gram negative diplococci (N.gonorrhoeae or
6. CHEMICAL FIXATIVES
■ Other chemicals are some times necessary to fix smears which contains
particularly dangerous organisms to ensure all the organisms are killed
eg 40g/l potassium permaganate is recommended for fixing smears
which may contain Anthrax bacilli.
■ Formaldehyde vapour is sometimes recommended for fixing smears
which may contain MYCOBACTERIUM species.
■ Formaldehyde fixed smears however tend to stain poorly and the
chemical itself is toxic with an injurious vapour.
8. CORNER STONE OF BACTERIAL INDENTIFICATION
■ Danish physician Hans Christian Gram developed the Gram
staining method in 1884.
■ Gram staining is still the cornerstone of bacterial
identification and taxonomic division. Besides demonstrating
the morphology of bacteria ,the gram stain can be used to
divide most bacterial species into 2 groups.
■ Those that take up the basic dye ( crystal violet) is GRAM
POSITIVE
■ Those that allow the crystal violet dye to washout easily with
decolourizer (Alcohol or Acetone) is GRAM NEGATIVE
■ Gram-positive bacteria- stains Darkpurple
■ Gram-negative bacteria-stains red/pink
9. PRINCIPLES OF GRAM STRAINING
■ CELL WALL THEORY:The differences in Gram-positive and Gram-negative bacteria
cell wall composition account for the Gram staining differences. Gram-positive cell wall
contains a thick layer of peptidoglycan with numerous teichoic acid cross-linking,
which resists decolorization.
■ PH THEORY: In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions
that penetrate through Gram-positive and Gram-negative cell walls. The CV+ interacts
with negatively charged componeIn contrast, the nts of bacterial cells, staining the cells purple.
When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine
(CV-I) complexes within the cytoplasm and outer layers of the cell.
■ The decolorizing agent (ethanol or an ethanol and acetone solution) interacts with the
lipids of both gram-positive and gram-negative bacteria membranes.
■ The outer membrane of the Gram-negative cell is lost from the cell, leaving the thin
peptidoglycan layer exposed. With ethanol treatment, gram-negative cell walls become
leaky and allow the large CV-I complexes to be washed from the cell.
■ The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell
is dehydrated by the addition of ethanol. Thus ethanol treatment traps the large CV-I
complexes within the cell.
■ After decolorization, the gram-positive cell remains purple. In contrast, the gram-
10. Cell wall of Gram-positive and Gram-negative
Bacteria
11.
12. All cocci are gram positive
EXCEPT:
1. Meningococci
2. Gonococci
3. Veilonella
4. Moraxella
All bacilli are gram negative
Except:
1. MYCOBACTERIUM
2. ANTHRACIS BACILLUS
3. CLOSTRIDIUM SP
4. DIPTHERIAE CORYNEBACTERIUM
5. NOCARDIA
6. ACTINOMYCETES
7. LISTERIA
8. DIPTHEROIDS
13. STEPS OF GRAM STAINING
■ Fixation of clinical materials made on a slide from bacterial culture
or specimen is air dried and then heat fixed.
■ STEP 1:Application of the primary stain (crystal violet) for 1
minute. Then he slide is rinsed with water.Crystal violet is a dark
blue to purple dye. It stains all cells blue/purple.
■ STEP 2:Application of mordant: The Gram’s iodine solution
(mordant) is Poured over the slide for 1 minute.then the slide is
rinsed with water.Grams iodine form a crystal violet-iodine (CV-I)
complex; all cells continue to appear blue.
■ STEP 3:Decolorization : Pouring few drops of decolourizer to the
smear eg: acetone for 1-2sec or ethyl alcohol 20-30sec or Acetone
alcohol for 10sec.slide is immediately rinsed with water.The
decolorization step distinguishes gram-positive from gram-negative
cells. The organic solvent such as acetone or ethanol extracts the blue
dye complex from the lipid-rich, thin-walled gram-negative bacteria
to a greater degree than from the lipid-poor, thick-walled, gram-
14. Note: Decolorization is the most crucial step of gram staining.if the
decolourizer is Poured for more times even gram positive bacteria lose
color (Over Decolorization)
And if poured less timethe gram negative bacteria donot lose the
primary color of primary stain properly ( under Decolorization)
■ STEP 4:
Application of counterstain (safranin) is added for 1
minute.then the slide is rinse with water. The red dye
safranin stains the decolorized gram-negative cells
red/pink; the gram-positive bacteria remain blue.
The slide is dried and then examined under oil
immersion objective.
15. • After performing a gram stain, the technician should first determine whether
the Gram stain is adequate. In an appropriately stained biological specimen,
the nuclei of neutrophils are red. If the nuclei are blue, the decolorization is
insufficient.
Results
■ Gram-negative bacteria will stain pink/red and
■ Gram-positive bacteria will stain blue/purple.
16. REAGENTS:
violet dye:
■ Crystal violet or methyl violet is used as a concentration of 0.5 to
2 %.
1. Crystal violet 10g
2. Absolute alcohol 100ml
3. Distilled water 1litre
■ Dissolve the dye in the alcohol, filter through filter paper and
add to the water.
17. ■ However, gram positive staining can be strengthened by the addition of sodium
bicarbonate or ammonium oxalate as in the following solutions:
1. Kopeloff & Beerman’s stain :
solution A: methylviolet 10g
Distilled water 1litre
Solution B: sodium bicarbonate 50g
. Distilled water 1 litre
Shortly before use mix 30 volumes of solution A with 8 volume of solution B
It is a disadvantage of this mixture that it tends to precipitate within a few days and
so cannot be kept.
2. Preston & Morrells stain:
. Crystal violet 20g, methylated spirit 200ml, Ammonium oxalate
1% in water 800 ml
18. IODINE SOLUTION
■ To obtain iodine in aqueous solution, potassium iodide or sodium hydroxide
must be added.
■ The more alkaline solution with sodium hydroxide is thought to give slightly
stronger gram positive staining.
■ Gram’s iodine (lugol’s):
. Iodine 10g
Potassium iodide 20g
Distilled water 1 litre
Kopeloff & Beerman’s iodine:
Iodine 20 g
Sodium hydroxide (4%NAOH) 100ml
Distilled water 900ml
19. DECOLOURIZER:
■ ACETONE:
1. this is the fastest and most specific decolourizer.
2. It is applied to the smear for only 2-3 Seconds and to sections for a
second or 2 longer.
3. It’s rapidity is an advantage when only a single slide is to be stained.
4. However the short period of exposure is difficult to control when many
slides have to be stained simultaneously.
20. ABSOLUTE ALCOHOL(100%ETHANOL)
■ This acts more slowly than acetone and should be applied and reapplied
for about 1 min until on tilting the slide from slide to slide .
ACETONE ALCOHOL:
■ This is a mixture of 1 volume of acetone with 1 volume of 95% ethanol.
■ It requires for about 10 Seconds.
IODINE ACETONE:
■ Preston & morrell have shown that the addition of a small concentration
of iodine to acetone slows it’s rate of Decolorization without reducing its
specificity.
■ With 0.35%iodine the exposure can be lengethed from about 2 seconds to
30 seconds or longer.
21. COUNTERSTAIN
1. Dilute carbol fuchsin:
■ This stain when applied gives the strongest red staining
■ However the colouration may be so dark that some gram negative bacteria mY be
difficult to distinguish from gram positive ones.
■ It’s is better to use one of the following weaker counterstain.
1. BASIC FUCHSIN COUNTERSTAIN : It is applied for 10-30 sec this is
recommended for general use.
2. NEUTRAL RED COUNTERSTAIN: applied for 2-4 min this is recommended
for demonstrating gonococci and other intracellular gram negative bacteria.
3. SAFRANIN COUNTERSTAIN: Safranin 0.5 % in distilled water
22. Reporting Gram smears
The report should include the following information:
■ Numbers of bacteria present, whether many, moderate, few,
or scanty
■ Gram reaction of the bacteria, whether Gram-positive or
Gram-negative
■ Morphology of the bacteria, whether cocci,
diplococci, streptococci, rods, or coccobacilli. Also, whether
the organisms are intracellular.
■ Presence and number of pus cells
■ Presence of yeast cells and epithelial cells.
23. USES OF GRAM STAIN:
■ To differentiate bacteria into gram positive & gram negative
■ For identification: gram staining from bacterial culture gives an idea to put the
corresponding biochemical tests for further identification of bacteria.
■ To start empirical treatment: gram stain from the specimen gives a preliminary
clue about the bacteria present ( based on the shape and gram staining property
of the bacteria) so that the empirical treatment with broad spectrum antibiotics
can be started early before the culture report is available.
■ For Fastidious organisms: such as Haemophilus which takes time to grow in
culture, gram stain helps in early presumptive identification.
■ Yeasts: in addition to stain the bacteria, gram stain is useful for staining certain
fungi such as Candida and Cryptococcus ( appear gram positive)
■ Quality of specimen: gram stain helps on screening the quality of the sputum
specimen before processing it for culture. Presence of More pus cells and less
epithelial cells indicates good quality specimen.
24. MODIFICATIONS OF GRAM STAINING:
■ KOPELOFF & BEERMAN’S MODIFICATION:
Primary stain and counter stain used are methylviolet and basic fuchsin
respectively.
■ JENSEN’S MODIFICATION:
This method involves use of absolute alcohol as decolourizer and neutral
red as counterstain
It is useful for meningococci and gonococci.
■ BROWN &BRENN MODIFICATION:
This is used for Actinomycetes.
25. LIMITATIONS
■ The sensitivity of the Gram stain procedure is low. Sometimes, you
may fail to see the organism in Gram Stain smear, but the same
clinical specimen may yield organisms when cultured. To be visible
on a slide, organisms that stain by the Gram method must be present
in about 104 to 105 organisms per milliliter of centrifuged fluid.
■ Gram staining technique is not recommended for spirochetes and
mycobacteria. Mycobacteria stain weakly with gram stain, and
bacteria such as Mycoplasma, Rickettsiae, Chlamydiae do not take
up the dyes used in Gram stain or are too small to be seen with light
microscopy.
■ Not all bacteria can be seen in the Gram stain. This is the list of
medically important bacteria that can be not seen in Gram-stain.
26. Nearly all clinically important bacteria can be visualized using the
Gram staining technique, the only exceptions being those
organisms;
• That exists almost exclusively within host cells (intracellular
bacteria) (e.g., Chlamydia)
• Those that lack a cell wall (e.g., Mycoplasma)
• Those of insufficient dimensions to be resolved by light
microscopy (e.g., spirochetes)
27. Name Reason
Alternative
Microscopic
Approach
Chlamydiae, including
C. trachomatis
Intracellular; very small
Inclusion bodies in the
cytoplasm
Legionella pneumophila
Poor uptake of red
counterstain
Prolong time of
counterstain
Mycoplasma
pneumophila
Poor uptake of red
counterstain
None
Mycobacterium, includin
g
M. tuberculosis
Too much lipid in cell
wall so dye cannot
penetrate
Acid-fast stain
Rickettsiae Intracellular; very small
Giemsa or other tissue
stains
Treponema pallidum Too thin to see
Dark-field microscopy or
28. QUALITY
COUNTROL
■ Always check new batches of stain and reagents for correct staining
reactions using a smear containing known Gram-positive and Gram-
negative organisms.
Variations in Gram Reaction
■ Various factors influence the results of Gram staining. Sometimes the
result might be entirely different than you have anticipated.
■ Gram-positive bacteria may lose their ability to retain crystal violet
and stain Gram negatively for the following reasons:
– cell wall damage of bacteria due to antibiotic therapy or excessive
heat fixation of the smear.
– over- decolorization of the smear
– use of an old Iodine solution that is yellow in color instead of
brown (always store in a brown glass or other light opaque
containers).
– preparation of smears from old culture.
A thick smear will require more decoloration than a thin smear
29. Organism
Classic
Presentation
Variant
Presentation
Comments
Streptococccus
pneumoniae
Gram-positive, lancet-
shaped, diplococci
Elongated cocci,
resembling short bacilli
May be misinterpreted
as mixed organisms;
over-decolorized cells
may be mistaken for
gram-negative
coccobacilli.
Acinetobacter spp.
Gram-negative
coccobacilli
Gram-negative cocci;
gram-variable staining
is common
May be mistaken
for Neisseria spp. and
reported as gram-
negative cocci; search
the smear to find some
organisms that
demonstrate elongated
forms, which are not
seen in Neisseria.
Yeast,
especially Cryptococc
us neoformans
Gram-positive round or
oval cells with budding
Gram-variable cells
May be mistaken for
artifacts; size and
shape distinguish them
from bacteria