Microscopy and Staining
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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. -
Infection Control in Dentistry: How to Asepsis Photographic Mirrors?
Infection control in dentistry: how to asepsis photographic mirrors? Amanda Osório Ayres de Freitas* Mariana Marquezan* Giselle Naback Lemes Vilani* Rodrigo César Santiago* Luiz Felipe de Miranda Costa* Sandra Regina Torres** Abstract: The aim of this study was to evaluate the efficacy of six different methods of disinfection and sterilization of intraoral photographic mirrors through microbiological testing and to analysis their potential harm to mirrors’ surface. Fourteen occlusal mirrors were divided into seven groups. Group 1 comprised two mirrors as received from manufacturer. The other six groups comprised mirrors disinfected/sterilized by autoclave, immersion in enzymatic detergent, and friction with chlorhexidine detergent, chlorhexidine wipes, common detergent and 70% ethylic alcohol. Microbiological and quality surface analyses were performed. Sterilization in autoclave was microbiologic effective, but caused damage to the mirror surface. Chlorhexidine (in wipes or detergent) and liquid soap were effective disinfectant agents for photographic mirrors decontamination, without harmful effect on its surface. Enzymatic detergent immersion and friction with 70% ethylic alcohol were not effective as disinfectant agents for photographic mirrors decontamination. According to the results, the more effective and safe methods for photographic mirrors disinfection were friction with chlorhexidine wipes or detergent, as well as liquid soap. Results, the most efficacious methods for photographic mirrors disinfection were friction with chlorhexidine wipes and detergent, as well as common detergent. Descriptors: Dental Instruments; Decontamination; Microbiology; Surface Properties. *Doutoranda em Odontologia na Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brasil **Pósdoutora em odontologia pela University of Washington (UW), Seattle, WA, Estados Unidos ISSN 22365843 │ 93 Introduction Dental photography is an important tool for diagnostic and treatment planning, and it’s also a registration of the patient’s condition before and after treatment. -
A Study of Rawitz's 'Inversion Staining' by ALEKSANDRA PRZEL^CKA
231 A Study of Rawitz's 'Inversion Staining' By ALEKSANDRA PRZEL^CKA {From the Cytological Laboratory, Department of Zoology, University Museum, Oxford, and the Nencki Institute, 3 Pasteur St., Warsaw 22; present address, Nencki Institute) SUMMAHY The Rawitz method involves mordanting with tannic acid and potassium antimony tartrate, and staining with basic fuchsine. The mordanting causes basic fuchsine to act as though it were an acid dye ('inversion staining'). A modification of the method is described in the present paper. This modification makes it possible to obtain the same results in a shorter time. The chief substances stained by Rawitz's method are phospholipids, certain pro- teins, and certain polysaccharides. Although the method cannot be regarded as a cytochemical test in the strict sense, yet it gives useful indications of chemical composition and in addition is valuable to the morphological cytologist as a technique for showing certain cytoplasmic inclusions (mitotic spindle, acrosome, mitochondria, 'Golgi apparatus' of certain cells). INTRODUCTION T is well known that the so-called 'Golgi apparatus' is extremely difficult to I reveal by any staining method. Baker, in the course of his investigation on this organelle in the epididymis of the mouse, found that it can be stained by basic fuchsin after a special mordanting process (1957). The method was taken from Rawitz (1895), who found that basic fuchsin, if mordanted with tannic acid and potassium antimony tartrate, loses the character of a dye for chro- matin and colours the cytoplasm instead. Rawitz called this effect 'inversion staining'. Since this technique, when applied to various kinds of cytological material, gave good selectivity in visualizing certain delicate cell structures, it seemed interesting to investigate the nature of the chemical compounds which are responsible for positive Rawitz staining. -
Eosin Staining
Science of H & E Andrew Lisowski, M.S., HTL (A.S.C.P.) 1 Hematoxylin and Eosin Staining “The desired end result of a tissue stained with hematoxylin and eosin is based upon what seems to be almost infinite factors. Pathologists have individual preferences for section thickness, intensities, and shades. The choice of which reagents to use must take into consideration: cost, method of staining, option of purchasing commercially-prepared or technician-prepared reagents, safety, administration policies, convenience, availability, quality, technical limitations, as well as personal preference.” Guidelines for Hematoxylin and Eosin Staining National Society for Histotechnology 2 Why Do We Stain? In order to deliver a medical diagnosis, tissues must be examined under a microscope. Once a tissue specimen has been processed by a histology lab and transferred onto a glass slide, it needs to be appropriately stained for microscopic evaluation. This is because unstained tissue lacks contrast: when viewed under the microscope, everything appears in uniform dull grey color. Unstained tissue H&E stained tissue 3 What Does "Staining" Do? . Contrasts different cells . Highlights particular features of interest . Illustrates different cell structures . Detects infiltrations or deposits in the tissue . Detect pathogens Superbly contrasted GI cells Placenta’s large blood H&E stain showing extensive vessels iron deposits There are different staining techniques to reveal different structures of the cell 4 What is H&E Staining? As its name suggests, H&E stain makes use of a combination of two dyes – hematoxylin and eosin. It is often termed as “routine staining” as it is the most common way of coloring otherwise transparent tissue specimen. -
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. -
Electron Microscopy and the Investigation of New Infectious Diseases
Review Electron microscopy and the investigation of new infectious diseases Alan Curry@) Objectives: To review and assess the role of electron microscopy in the investigation of new infectious diseases. Design: To design a screening strategy to maximize the likelihood of detecting new or emerging pathogens in clinical samples. Results: Electron microscopy remains a useful method of investigating some viral infections (infantile gastroenteritis, virus-induced outbreaks of gastroenteritis and skin lesions) using the negative staining technique. In addition, it remains an essential technique for the investigation of new and emerging parasitic protozoan infections in the immunocompromised patients from resin-embedded tissue biopsies. Electron microscopy can also have a useful role in the investigation of certain bacterial infections. Conclusions: Electron microscopy still has much to contribute to the investigation of new and emerging pathogens, and should be perceived as capable of producing different, but equally relevant, information compared to other investigative techniques. It is the application of a combined investigative approach using several different techniques that will further our understanding of new infectious diseases. Int J Infect Dis 2003; 7: 251-258 INTRODUCTION at individually by a skilled microscopist have con- The electron microscope was developed just before tributed to the decline of electron microscopy. Against World War II in several countries, but particularly in this background, the inevitable question must be Germany.l The dramatic increase in resolution available asked-does electron microscopy still have a useful in comparison with light microscopy promised to role to play in the investigation of emerging or new revolutionize many aspects of cell biology, virology, infectious diseases? bacteriology, mycology and protozoan parasitology. -
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. -
Negative Stain Grid Preparation
Negative Stain Grid Preparation Generic Protocol for Single Particle – Page 2 Example For Bacteria – Page 5 Example For Viruses – Page 6 Example For Purified Bacterial Flagella – Page 7 Drafted by E. Montemayor, 6/15/2020. Revisions by EJM, JCS, ERW, 1/6/2021 1 Generic protocol for negative stain grid preparation Think of this as a “try this first” pipeline for new samples. Sample concentration and type of stain tend to be the most important variables, then additives like detergents or crosslinking should be tried next. Overview at a glance: Step 1: Prepare serial four-fold dilutions of your sample, covering at least two order of magnitude in sample concentration. Typical ceiling for sample concentration is ~ 1 mg/mL. Step 2: Prepare negative stain solution. Uranyl stains are most popular as a first pass, especially for single particle work. Consider filtering (0.1 µm or 0.2 µm syringe filters) and centrifuging (5-15 min, 15,000 rpm, and at room temperature (RT)) your stain to reduce crystals and debris. Step 3: Glow discharge grids. Standard thickness carbon on a 200 copper mesh is a popular support. Step 4: Place grid in self closing tweezers. Use a microscope or magnifying lens to help you touch only the edge of the grid with the tweezers. Step 5: Set up 2x 20 µL drops of buffer and 3x 20 µL drops of stain on a strip of parafilm. Apply 3 µL of sample to carbon side of grid and incubate for 1 minute. Step 6: Side blot to remove sample but do not let grid dry out. -
Second Harmonic Imaging Microscopy
170 Microsc Microanal 9(Suppl 2), 2003 DOI: 10.1017/S143192760344066X Copyright 2003 Microscopy Society of America Second Harmonic Imaging Microscopy Leslie M. Loew,* Andrew C. Millard,* Paul J. Campagnola,* William A. Mohler,* and Aaron Lewis‡ * Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06030-1507 USA ‡ Division of Applied Physics, Hebrew University of Jerusalem, Jerusalem 91904, Israel Second Harmonic Generation (SHG) has been developed in our laboratories as a high- resolution non-linear optical imaging microscopy (“SHIM”) for cellular membranes and intact tissues. SHG is a non-linear process that produces a frequency doubling of the intense laser field impinging on a material with a high second order susceptibility. It shares many of the advantageous features for microscopy of another more established non-linear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce 3D images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It also does not arise via photon emission from molecular excited states, as do both 1- and 2-photon excited fluorescence. SHG of intrinsic highly ordered biological structures such as collagen has been known for some time but only recently has the full potential of high resolution 3D SHIM been demonstrated on live cells and tissues. For example, Figure 1 shows SHIM from microtubules in a living organism, C. elegans. The images were obtained from a transgenic nematode that expresses a ß-tubulin-green fluorescent protein fusion and Figure 1 also shows the TPEF image from this molecule for comparison. -
Applications of Microscopy in Bacteriology
Microscopy Research, 2016, 4, 1-9 Published Online January 2016 in SciRes. http://www.scirp.org/journal/mr http://dx.doi.org/10.4236/mr.2016.41001 Applications of Microscopy in Bacteriology Mini Mishra1, Pratima Chauhan2* 1Centre of Environmental Studies, Department of Botany, University of Allahabad, Allahabad, India 2Department of Physics, University of Allahabad, Allahabad, India Received 28 September 2015; accepted 2 January 2016; published 5 January 2016 Copyright © 2016 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Abstract Bacteria are smallest primitive, simple, unicellular, prokaryotic and microscopic organisms. But these organisms cannot be studied with naked eyes because of their minute structure. Therefore in search for the information about the structure and composition of bacterial cells, cell biologist used light microscopes with a numerical aperture of 1.4 and using wavelength of 0.4 µm separa- tion. But there are still certain cellular structures that cannot be seen through naked eyes, and for them electron microscope is used. There are certain improved types of light microscope which can be incorporated to improve their resolving power. Hence microscopy is playing a crucial role in the field of bacteriology. Keywords AFM, SEM, TEM, Microscopy, Bacteriology 1. Introduction To get acquainted with the world of bacteria like small organisms, very effective and advanced technique is re- quired. The size of bacteria ranges between 0.5 - 5.0 micrometer in length; the smallest of them are members of mycoplasma which measures 0.3 micrometers [1]. -
Electron Microscopy of Negatively Stained and Unstained Fibrinogen (Scanning Transmission Electron Microscopy) LEONARD F
Proc. Natl. Acad. Sci. USA Vol. 77, No. 6, pp. 3139-3143, June 1980 Biochemistry Electron microscopy of negatively stained and unstained fibrinogen (scanning transmission electron microscopy) LEONARD F. ESTIS* AND RUDY H. HASCHEMEYER Department of Biochemistry, Cornell University Medical College, 1300 York Avenue, New York, New York 10021 Communicated by Alton Meister, December 26,1979 ABSTRACT Electron microscopic images of negatively are always met: (i) The great majority of images of "apparent stained fibrinogen are predominantly asymmetric rods 450 A particles" in any field are of fibrinogen molecules. (Ui) The in length and about 60 A in width. The molecules appear to have considerable flexibility, and mass distribution along the major number of identifiable fibrinogen molecules is defined by fi- axis is not uniquely distinguished despite apparent beading in brinogen concentration and application time and not by the some particles. Scanning transmission electron microscopy of buffer, pH, or staining conditions. (Mii) Large-field views of unstained fibrinogen again demonstrates that a majority of fibrinogen contain predominantly well-separated molecules molecules are rodlike. The results differ from those obtained with clearly defined morphology and dimensions. by negative staining in that a substantial fraction of images are Conditions trinodular with striking resemblance to those obtained by C. required to achieve these goals are primarily E. Hall and H. S. Slayter IJ Biophys. Bioehem. Cytol. (1959) 5, related to attachment of fibrinogen to the substrate film. Once 11-161 using the mica replictecaique. The above results were this problem was solved, images of the unstained molecule were obtained on glow-discharged carbon substrate films by a simple also obtained by high-resolution scanning transmission electron low-concentration, lon&-attachment-time modification of microscopy (STEM). -
Spontaneous Generation & Origin of Life Concepts from Antiquity to The
SIMB News News magazine of the Society for Industrial Microbiology and Biotechnology April/May/June 2019 V.69 N.2 • www.simbhq.org Spontaneous Generation & Origin of Life Concepts from Antiquity to the Present :ŽƵƌŶĂůŽĨ/ŶĚƵƐƚƌŝĂůDŝĐƌŽďŝŽůŽŐLJΘŝŽƚĞĐŚŶŽůŽŐLJ Impact Factor 3.103 The Journal of Industrial Microbiology and Biotechnology is an international journal which publishes papers in metabolic engineering & synthetic biology; biocatalysis; fermentation & cell culture; natural products discovery & biosynthesis; bioenergy/biofuels/biochemicals; environmental microbiology; biotechnology methods; applied genomics & systems biotechnology; and food biotechnology & probiotics Editor-in-Chief Ramon Gonzalez, University of South Florida, Tampa FL, USA Editors Special Issue ^LJŶƚŚĞƚŝĐŝŽůŽŐLJ; July 2018 S. Bagley, Michigan Tech, Houghton, MI, USA R. H. Baltz, CognoGen Biotech. Consult., Sarasota, FL, USA Impact Factor 3.500 T. W. Jeffries, University of Wisconsin, Madison, WI, USA 3.000 T. D. Leathers, USDA ARS, Peoria, IL, USA 2.500 M. J. López López, University of Almeria, Almeria, Spain C. D. Maranas, Pennsylvania State Univ., Univ. Park, PA, USA 2.000 2.505 2.439 2.745 2.810 3.103 S. Park, UNIST, Ulsan, Korea 1.500 J. L. Revuelta, University of Salamanca, Salamanca, Spain 1.000 B. Shen, Scripps Research Institute, Jupiter, FL, USA 500 D. K. Solaiman, USDA ARS, Wyndmoor, PA, USA Y. Tang, University of California, Los Angeles, CA, USA E. J. Vandamme, Ghent University, Ghent, Belgium H. Zhao, University of Illinois, Urbana, IL, USA 10 Most Cited Articles Published in 2016 (Data from Web of Science: October 15, 2018) Senior Author(s) Title Citations L. Katz, R. Baltz Natural product discovery: past, present, and future 103 Genetic manipulation of secondary metabolite biosynthesis for improved production in Streptomyces and R.