DOCSLIB.ORG

Gram's Serendipitous Stain

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Gram's Serendipitous Stain Gram’s Serendipitous Stain In spite of his “defective” method, his name lives on … hile working with which acted as a mordant to fix infected lung tissue the dye. Then he poured ethanol Wfrom cadavers, a over the slide to wash away the Danish pathologist stumbled dye. Certain bacteria upon a remarkable technique that (pneumococci) retained the color that is still in common usage (gram-positive), while other over 100 years later. How did species became bleached or de- this come about? colorized by the alcohol (gram- negative). His initial work with Hans Christian Gram (born in this staining process was Jay Hardy, CLS, SM (ASCP) 1853) studied botany at the performed on Streptococcus University of Copenhagen in pneumoniae and Klebsiella Denmark. His study of plants pneumoniae. introduced him to the basis of Jay Hardy is the founder and pharmacology and the use of the CEO of Hardy Diagnostics. microscope. He began his career in microbiology as a Medical In 1883, he graduated from Technologist in Santa medical school and after Barbara, California. traveling throughout Europe, he In 1980, he began settled in Berlin. His initial work manufacturing culture media concerned the study of human for the local hospitals. red blood cells. He was among Today, Hardy Diagnostics is the first to recognize that the third largest media macrocytes were characteristic manufacturer in the US. of pernicious anemia. To ensure rapid and reliable In 1884, while examining lung turn around time, Hardy tissue from patients who had Diagnostics maintains six distribution centers, and died of pneumonia, Gram had produces over 2,700 discovered that certain stains products used in clinical and were preferentially industrial microbiology taken up and “I am aware that as yet it is laboratories throughout the retained by world. bacterial cells. In very defective and imperfect” the first step, he Hans Christian Gram dried a fluid smear on a glass slide over a burner flame and poured Gentian Gram was a modest man, and in his initial publication he (crystal) violet solution over it. remarked, "I have therefore After rinsing with water, he added Lugol’s solution - published the method, although I potassium triiodide solution - am aware that as yet it is very defective and imperfect; but it is classification in medical hoped that also in the hands of microbiology today. other investigators it will turn out to be useful." The Gram Stain Gram did not use a counterstain Mechanism in his procedure. It was a few years later, that the German What makes some cells retain pathologist Carl Weigert (1845- crystal violet (Gram positive) 1904) from Frankfurt, added a while others readily release the final step of staining with stain when alcohol is added safranin. Gram himself never (Gram negative)? used the red counterstaining in order to visualize the gram- Gram-positive bacteria have a Crystal violet (CV) dissociates in negative bacteria. thick mesh-like cell wall made of aqueous solutions into CV+ and peptidoglycan (50-90% of cell chloride (Cl – ) ions. These ions In 1891, Gram became a lecturer wall), which stain purple and penetrate through the cell wall in pharmacology, and later that Gram-negative bacteria have a and cell membrane of both year was appointed professor at thinner layer (10% of cell wall), Gram-positive and Gram- the University of Copenhagen. In which stain pink. Gram-negative negative cells. The CV+ ion 1900 he resigned his Chair in bacteria also have an additional interacts with negatively charged Pharmacology to become outer membrane which contains components of bacterial cells Professor of Medicine. The king lipids, and is separated from the and stains the cells purple. of Denmark awarded him the cell wall by the periplasmic Iodine (I – or I3 – ) interacts Dannebrog Commander’s Cross, space. with CV+ and forms large first-class in 1912, and the complexes of crystal violet and Golden Medal of Merit in 1924. iodine (CV – I) within the inner He retired in 1923 and died in and outer layers of the cell. 1938. When a decolorizer such as The staining technique he alcohol or acetone is added, it developed is still the most interacts with the lipids of the important method for cell membrane. A Gram- distinguishing between two negative cell will lose its outer major classes of bacteria. His membrane and the peptidoglycan discovery in 1884 occurred layer is left exposed. The CV – I during the golden years of complexes are washed from the clinical microbiology. This is Gram-negative cell along with when Petri plates were invented the outer membrane. In contrast, (1887), Agar was discovered a Gram-positive cell becomes (1881), and Pasteur and Koch There are four basic steps of the dehydrated from an ethanol were at their peak of Gram stain, which include treatment. The large CV – I performance, discovering the applying a primary stain (crystal complexes become trapped etiologic agents of many of the violet) to a heat-fixed smear of a within the Gram-positive cell vexing diseases of that day. bacterial culture, followed by the due to the multilayered nature of addition of a mordant (Gram's its peptidoglycan. In spite of his modesty and iodine), rapid decolorization doubts of success, this technique, with alcohol or acetone, and The decolorization step is critical the Gram Stain, continues to be a counterstaining with safranin or and must be timed correctly; the standard procedure for bacterial basic fuchsin. crystal violet stain will be removed from both Gram- positive and negative cells if the decolorizing agent is left on too long (a matter of seconds). After decolorization, the Gram- positive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively- charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color. Bacillus spp showing ambiguous staining results. Older cells readily decolorize and will appear gram negative, while the younger cells retain their gram positive nature. An “Imperfect” procedure, and caution must be Method exercised. Some bacteria, after staining In addition, for all bacteria with the Gram stain, yield a stained using the Gram stain, the Gram-variable pattern: a mix of age of the culture may influence pink and purple cells are seen. the results of the stain as seen in the photo above. The genera Actinomyces, Arthobacter, Corynebacterium, Using improved formulations, Mycobacterium, and such as Gram Stain Advanced Propionibacterium have cell will help in overcoming some of walls particularly sensitive to these problems. breakage during cell division, resulting in Gram-negative Jay Hardy, CLS, SM (ASCP) staining of these Gram-positive Santa Maria, CA cells. The staining of these organisms result in an uneven or granular appearance. In cultures of the Gram-positive Bacillus, Butyrivibrio, and Clostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative. For this reason, these organisms are very prone to over- decolorization in the acetone- alcohol rinse step of the .
Load more

Recommended publications
  • Gst Gram Staining Learning Objectives the Student Will  Use Aseptic Techniques in the Safe Inoculation of Various Forms of Media
    GSt Gram Staining Learning Objectives The student will Use aseptic techniques in the safe inoculation of various forms of media. Follow oral and written instructions and manage time in the lab efficiently. Use the bright field light microscope to view microbes under oil immersion, make accurate observations and appropriate interpretations and store the microscope according to lab procedures. Properly prepare a bacterial smear for accurate staining and describe the chemical basis for simple staining and negative staining. Background/Theory Differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms may appear to be different colors. Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining. This link to the OpenStax Microbiology text provides more detail on these differential staining techniques. (OpenStax CNX, 2018) The Gram stain is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria containing the two most common types of cell walls. (OpenStax CNX, 2018) One type consists of an inner plasma membrane and a thick outer layer of peptidoglycan. The other type consists of a double phospholipid Figure 1 Simplified structures of Gram negative cells (left) and Gram positive bilayer with a thin layer of cells (right) peptidoglycan between the two. The Gram Staining technique remains one of the most frequently used staining techniques. The steps of the Gram stain procedure are listed below and illustrated in Figure. (OpenStax CNX, 2018) 1.
    [Show full text]
  • Laboratory Exercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation
    City University of New York (CUNY) CUNY Academic Works Open Educational Resources Queensborough Community College 2016 Laboratory Exercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation Joan Petersen CUNY Queensborough Community College Susan McLaughlin CUNY Queensborough Community College How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/qb_oers/16 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] Laboratory Exercises in Microbiology: Discovering the Unseen World through Hands-On Investigation By Dr. Susan McLaughlin & Dr. Joan Petersen Queensborough Community College Laboratory Exercises in Microbiology: Discovering the Unseen World through Hands-On Investigation Table of Contents Preface………………………………………………………………………………………i Acknowledgments…………………………………………………………………………..ii Microbiology Lab Safety Instructions…………………………………………………...... iii Lab 1. Introduction to Microscopy and Diversity of Cell Types……………………......... 1 Lab 2. Introduction to Aseptic Techniques and Growth Media………………………...... 19 Lab 3. Preparation of Bacterial Smears and Introduction to Staining…………………...... 37 Lab 4. Acid fast and Endospore Staining……………………………………………......... 49 Lab 5. Metabolic Activities of Bacteria…………………………………………….…....... 59 Lab 6. Dichotomous Keys……………………………………………………………......... 77 Lab 7. The Effect of Physical Factors on Microbial Growth……………………………... 85 Lab 8. Chemical Control of Microbial Growth—Disinfectants and Antibiotics…………. 99 Lab 9. The Microbiology of Milk and Food………………………………………………. 111 Lab 10. The Eukaryotes………………………………………………………………........ 123 Lab 11. Clinical Microbiology I; Anaerobic pathogens; Vectors of Infectious Disease….. 141 Lab 12. Clinical Microbiology II—Immunology and the Biolog System………………… 153 Lab 13. Putting it all Together: Case Studies in Microbiology…………………………… 163 Appendix I.
    [Show full text]
  • Francisella Tularensis 6/06 Tularemia Is a Commonly Acquired Laboratory Colony Morphology Infection; All Work on Suspect F
    Francisella tularensis 6/06 Tularemia is a commonly acquired laboratory Colony Morphology infection; all work on suspect F. tularensis cultures .Aerobic, fastidious, requires cysteine for growth should be performed at minimum under BSL2 .Grows poorly on Blood Agar (BA) conditions with BSL3 practices. .Chocolate Agar (CA): tiny, grey-white, opaque A colonies, 1-2 mm ≥48hr B .Cysteine Heart Agar (CHA): greenish-blue colonies, 2-4 mm ≥48h .Colonies are butyrous and smooth Gram Stain .Tiny, 0.2–0.7 μm pleomorphic, poorly stained gram-negative coccobacilli .Mostly single cells Growth on BA (A) 48 h, (B) 72 h Biochemical/Test Reactions .Oxidase: Negative A B .Catalase: Weak positive .Urease: Negative Additional Information .Can be misidentified as: Haemophilus influenzae, Actinobacillus spp. by automated ID systems .Infective Dose: 10 colony forming units Biosafety Level 3 agent (once Francisella tularensis is . Growth on CA (A) 48 h, (B) 72 h suspected, work should only be done in a certified Class II Biosafety Cabinet) .Transmission: Inhalation, insect bite, contact with tissues or bodily fluids of infected animals .Contagious: No Acceptable Specimen Types .Tissue biopsy .Whole blood: 5-10 ml blood in EDTA, and/or Inoculated blood culture bottle Swab of lesion in transport media . Gram stain Sentinel Laboratory Rule-Out of Francisella tularensis Oxidase Little to no growth on BA >48 h Small, grey-white opaque colonies on CA after ≥48 h at 35/37ºC Positive Weak Negative Positive Catalase Tiny, pleomorphic, faintly stained, gram-negative coccobacilli (red, round, and random) Perform all additional work in a certified Class II Positive Biosafety Cabinet Weak Negative Positive *Oxidase: Negative Urease *Catalase: Weak positive *Urease: Negative *Oxidase, Catalase, and Urease: Appearances of test results are not agent-specific.
    [Show full text]
  • Biofire Blood Culture Identification System (BCID) Fact Sheet
    BioFire Blood Culture Identification System (BCID) Fact Sheet What is BioFire BioFire BCID is a multiplex polymerase chain reaction (PCR) test designed to BCID? identify 24 different microorganism targets and three antibiotic resistance genes from positive blood culture bottles. What is the purpose The purpose of BCID is to rapidly identify common microorganisms and of BCID? antibiotic resistance genes from positive blood cultures so that antimicrobial therapy can be quickly optimized by the physician and the antibiotic stewardship pharmacist. It is anticipated that this will result in improved patient outcomes, decreased length of stay, improved antibiotic stewardship, and decreased costs. When will BCID be BCID is performed on all initially positive blood cultures after the gram stain is routinely performed and reported. performed? When will BCID not For blood cultures on the same patient that subsequently become positive with be routinely a microorganism showing the same morphology as the initial positive blood performed? culture, BCID will not be performed. BCID will not be performed on positive blood cultures with gram positive bacilli unless Listeria is suspected. BCID will not be performed on blood culture bottles > 8 hours after becoming positive. BCID will not be performed between 10PM-7AM on weekdays and 2PM-7AM on weekends. BCID will not be performed for clinics that have specifically opted out of testing. How soon will BCID After the blood culture becomes positive and the gram stain is performed and results be available? reported, the bottle will be sent to the core Microbiology lab by routine courier. BCID testing will then be performed. It is anticipated that total turnaround time will generally be 2-3 hours after the gram stain is reported.
    [Show full text]
  • The Use of Matrix-Assisted Laser Desorption Ionization
    Session: OS202 MALDI-TOF - diagnostics for the micro lab in the 21st century Category: 4b. Diagnostic bacteriology – non-culture based, including molecular and MALDI-TOF 25 April 2017, 14:30 - 14:40 OS1031 The use of matrix-assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF MS) for rapid bacterial identification in patients with smear- positive bacterial meningitis - a study of diagnostic accuracy Boaz Bishop*1, Yuval Geffen2, Ola Kassis3, Adi Kamar4, Mical Paul5, Ami Neuberger6 1Rambam Medical Center; Department of Internal Medicine B 2Rambam Health Care Campus; Microbiology Laboratory 3Technion 4Rambam Medical Center; Microbiology Laboratory 5Rambam Health Care Center; Division of Infectious Diseases 6Rambam Medical Center; Unit of Infectious Diseases Background: Bacterial meningitis is a rapidly occurring fatal disease, thus immediate diagnosis and adequate therapy are crucial. Diagnosis is obtained by cerebral spinal fluid (CSF); cultures results are the gold standard for diagnosis, but are available only within 24-72 hours. MALDI-TOF-MS identifies a wide range of bacteria by examining protein profiles. Little data exists regarding the use of MALDI- TOF-MS for examining CSF samples. We aimed at assessing the accuracy of MALDI-TOF-MS in rapid identification of CSF pathogens in patients with meningitis. Material/methods: We conducted a prospective study examining samples with positive CSF Gram stains with MALDI-TOF-MS. The results were compared with the CSF culture; identification was considered accurate when identical to the CSF culture results (species and genus level). Lab workers preforming the MALDI-TOF MS and ones interpreting its results were blinded to the result of the direct smear, besides the fact of it being positive.
    [Show full text]
  • GRAM STAIN REAGENTS - for in Vitro Use Only - Catalogue No
    GRAM STAIN REAGENTS - For in vitro use only - Catalogue No. SG51-55 Our Gram-Stain Reagents are intended to be The last step is the application of a counterstain. The used as a differential stain for the microscopic most common counterstain is safranin, which colors examination of bacterial cultures and laboratory decolorized cells pink. An alternate counterstain is basic specimens. fuchsin, which gives the decolorized cells more of a Gram staining is the single most useful test in bright pink or fuchsia coloration. The basic fuchsin the microbiology laboratory given its simplicity and counterstain works particularly well for anaerobic ability to differentiate bacteria into two main bacteria, but poorly for Legionella and Bordetella groups: gram-positive organisms and gram negative species. organisms. Hans Christian Joachim Gram first devised the original procedure in the late 19 th century, and although modifications have since been Formulation per Litre made, the basic principles and results remain the same. Our formulation is often referred to as the SG51 Gram Crystal Violet Hucker Modification. The staining spectrum includes almost all Crystal Violet ................................................ 20.0 g bacteria, many fungi, and parasites such as Ammonium Oxalate ....................................... 8.0 g Trichomonas , Strongyloides , and protozoan cysts. Methanol ................................................. 200.0 mL The notable exceptions include intracellular pathogens such as Chlamydia and Rickettsia , and SG52 Gram Iodine those organisms lacking a true cell wall such as Mycobacterium , Mycoplasma , and Ureaplasma . Iodine Crystals .............................................. 3.33 g The differential properties of the staining Potassium Iodide ........................................... 6.67 g process are attributed to the differences in composition between gram-positive and gram- SG53 Gram Decolorizer negative cell walls.
    [Show full text]
  • LAB 2: Staining and Streaking Protocols for Simple Stain, Gram Stain, Streak Plate Technique and Culture Maintenance
    LAB 2: Staining and Streaking Protocols for Simple stain, Gram Stain, Streak Plate Technique and Culture Maintenance Lab 2a: INTRODUCTION To Staining Live specimens are difficult to see with the bright field microscope. The contrast between a cell, which is primarily water, and the background, which is water, is poor. Staining is used to increase contrast and can be employed to provide information about the chemistry of a specimen. Stains, or dyes, are salts in which one of the ions is colored. In a basic stain, the color is in the positively charged ion. In an acidic stain the color is in the negatively charged ion. Bacterial surfaces have a slight negative charge. Thus, there is an affinity between a positively charged color ion and the negatively charged bacterial cell. In the Direct or Positive Staining Procedure a cell takes up a positively charged dye and becomes stained. Methylene blue, crystal violet, and safranin, are all basic dyes. In the Indirect or Negative Staining Procedure, a cell is immersed in a negatively charged dye. As the cell will repel the dye, the cell appears clear in a background of color. Nigrosine is an example of an acidic stain. Staining procedures that use one dye to increase contrast between specimen and background are simple staining procedures. Complex staining procedures employ a series of stains and chemical reagents to increase contrast and reveal information about the specimen. Any staining procedure that allows differentiation of one type of bacterium from another is a differential staining procedure. For all staining procedures, it is first required that cells be fixed to the slide.
    [Show full text]
  • Use of the Diagnostic Bacteriology Laboratory: a Practical Review for the Clinician
    148 Postgrad Med J 2001;77:148–156 REVIEWS Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from Use of the diagnostic bacteriology laboratory: a practical review for the clinician W J Steinbach, A K Shetty Lucile Salter Packard Children’s Hospital at EVective utilisation and understanding of the Stanford, Stanford Box 1: Gram stain technique University School of clinical bacteriology laboratory can greatly aid Medicine, 725 Welch in the diagnosis of infectious diseases. Al- (1) Air dry specimen and fix with Road, Palo Alto, though described more than a century ago, the methanol or heat. California, USA 94304, Gram stain remains the most frequently used (2) Add crystal violet stain. USA rapid diagnostic test, and in conjunction with W J Steinbach various biochemical tests is the cornerstone of (3) Rinse with water to wash unbound A K Shetty the clinical laboratory. First described by Dan- dye, add mordant (for example, iodine: 12 potassium iodide). Correspondence to: ish pathologist Christian Gram in 1884 and Dr Steinbach later slightly modified, the Gram stain easily (4) After waiting 30–60 seconds, rinse with [email protected] divides bacteria into two groups, Gram positive water. Submitted 27 March 2000 and Gram negative, on the basis of their cell (5) Add decolorising solvent (ethanol or Accepted 5 June 2000 wall and cell membrane permeability to acetone) to remove unbound dye. Growth on artificial medium Obligate intracellular (6) Counterstain with safranin. Chlamydia Legionella Gram positive bacteria stain blue Coxiella Ehrlichia Rickettsia (retained crystal violet).
    [Show full text]
  • Gram Stain Workshop for the Laboratory Generalist
    Gram Stain Workshop for the Laboratory Generalist Karen Stiles, SM(ASCP)CM State Training Coordinator Assistant Chemical Terrorism Coordinator Nebraska Public Health Laboratory 402-559-3590 [email protected] 1 GRAM STAIN OBJECTIVES: Upon completion, the participant will be able to: 1. Explain the principle of the Gram stain procedure, including what elements can affect staining results 2. Correlate the most common pathogens with positive Gram stains from blood cultures and direct specimen sterile body fluid smears 3. Perform and interpret Grams stains 2 Purpose of Gram Stain Classify bacteria based on form, size, cellular morphology, Gram reaction Assess quality of specimen Identify specific infectious agent from morphology and Gram reaction Correlation with culture growth Correlation with culture-independent methodologist Guide presumptive antibiotic therapy 3 Principle of Gram Stain Cell wall composition Gram positive – think peptidoglycan layer with teichoic acid Gram negative – high in lipid content Basic premise Crystal Violet – all cells take up primary stain Gram’s iodine – mordant to form complex Decolorizer – mixture of acetone and alcohol Dehydrate lipids in Gram negative cell walls, wash out complex Gram positive cells resistant, retain stain complex Safranin - counterstain 4 Gram negative cells take up counterstain Preparation of Samples Specimen Type Preparation CSF/sterile body fluids Cyto/Centrifuge Blood Culture Broth Drop to slide Tissue Touch prep Tissue homogenate Drop to slide Swabbed material Roll
    [Show full text]
  • Gram Stain Introduction
    Gram Stain Introduction The Gram stain is a differential staining procedure used to categorize bacteria as Gram positive or Gram negative based on the chemical and physical properties of their cell’s walls. The bacteria are differentiated through a series of staining and decolorization steps. Gram positive cells will stain purple and Gram-negative cells will stain red to pink. Supplies and Reagents 1. Personal protective equipment (PPE) 3. 2. Slide rack 1. 3. Timer 4. Absorbent paper, such as bibulous paper 5. Water (tap water or deionized) 6. Crystal violet 7. Gram’s iodine 8. Decolorizer 9. Safranin (or carbol fuchsin) 5. 7. 10. Brightfield microscope with 100X objective 11. Immersion oil Instructions 1. Use appropriate PPE according to your laboratory’s procedures and safety manual. 2. Place the prepared fixed smear on a slide rack then flood the slide with crystal violet. 3. Wait at least 15 seconds* then rinse the slide with 9. 11. water. 4. Flood the slide with Gram’s iodine. 5. After 15 seconds* rinse the slide with water. 6. Apply the decolorizer to the slide. 7. Rinse the slide immediately with water. 8. Flood the slide with counterstain. 9. Wait at least 15 seconds* then rinse the slide with water. 10. Blot the slide with absorbent paper. Be careful not to wipe the cells off the slide. 11. Allow the newly stained slide to air dry completely. 12. View the slide under oil using the oil immersion objective for a total magnification of 1000X. 13. Record results based on your laboratory’s criteria.
    [Show full text]
  • GRAM STAIN Item Number: 6250, 6251, 6252, 6253, 6254, 6255, 6256
    GRAM STAIN Item Number: 6250, 6251, 6252, 6253, 6254, 6255, 6256 GRAM STAIN SET For staining bacteria from cultures or patient specimens by the differential Gram stain method SUMMARY This procedure is based upon the Hucker modification of the original Gram stain method. Gram-positive and Gram-negative bacteria are both stained by the crystal violet stain. Addition of the iodine leads to the formation of a crystal violet-iodine complex within the cell wall. The decolorizer extracts lipids from the cell wall of Gram-negative bacteria, thereby increasing the porosity of the cell wall and allowing the crystal violet-iodine complex to diffuse from the cell. At the same time, Gram-positive bacteria are dehydrated, causing a decrease in cell wall porosity and trapping the crystal violet-iodine complex within the cell. Due to the increase in porosity, the safranin counterstain is able to permeate the cell wall of the Gram-negative bacteria. STORAGE CONDITIONS Store at controlled room temperature (15-30°C) with bottles tightly sealed. INDICATIONS OF DETERIORATION If precipitate forms in the Crystal Violet Solution, warm gently to 37°C and shake well to re-dissolve. SPECIMEN COLLECTION AND PREPARATION Liquid Media: Spread the bacteria suspension on a clean glass slide using a sterile loop and allow to air dry. Heat-fix the smear by allowing smear to dry on a hot plate or in oven at ~42°C. Solid Media: Place a loopful of deionized water on a clean glass slide. Touch a sterilized wire loop to a colony and suspend the cells in the water droplet; spread the suspension on the slide and allow to air dry.
    [Show full text]
  • The Gram Stain
    Gram Stain Pagina 1 di 3 http://www-micro.msb.le.ac.uk/index.html: Microbiology Video Library: Gram Stain Search The Gram Stain In 1884, Hans Christian Gram, a Danish doctor working in Berlin, accidentally stumbled on a method which still forms the basis for the identification of bacteria. While examining lung tissue from patients who had died of pneumonia, he discovered that certain stains were preferentially taken up and retained by bacterial cells. Over the course of the next few years, Gram developed a staining procedure which divided almost all bacteria into two large groups - the Gram stain. Individual bacterial cells are hard to see, partly because they are small, but also because they are almost transparent. In addition to magnification under a microscope, optical tricks must also be used to be able to see them: Phase contrast microscopy Staining Either of these methods can make bacterial cells visible under the microscope. Other staining methods are described elsewhere in these documents, e.g. the Ziehl-Neelsen acid- fast staining procedure, but the Gram stain procedure is as follows: 1. Place a slide with a bacterial smear on a staining rack. 2. STAIN the slide with crystal violet for 1-2 min. 3. Pour off the stain. Note: fingers stain Gram-positive - use forceps! 4. Flood slide with Gram's iodine for 1-2 min. 5. Pour off the iodine. 6. Decolourize by washing the slide briefly with acetone (2-3 seconds). 7. Wash slide thoroughly with water to remove the acetone - do not delay with this step. 8.
    [Show full text]