Diagnostic Stains & Laboratory Reagents
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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. -
Clinically Pertinent Cytological Diff-Quick and Gram Stain
Clinically Pertinent Cytological Diff-Quick and Gram Stain Evaluation for the Reptilian Practitioner Kendal E Harr, DVM, MS, Dipl ACVP (Clinical Pathology), April Romagnano, PhD, DVM, DABVP (Avian) Session #214 Affiliation: URIKA, LLC, Mukilteo, WA 98275, USA (Harr), Avian and Exotic Clinic of Palm City, Palm City, Florida and the Animal Health Clinic, Jupiter, FL 33458, USA (Romagnano). Abstract: The goals of this work were to: 1) improve knowledge of preanalytic sampling techniques including blood smears, fine needle aspirates, imprints, smears, and fluid preparation including oral, dermal and cloacal swabs, cystic and solid mass sampling, joint fluids and effusions, and fecal smears and floats; and 2) enable the reptilian practitioner to better identify basic cells, classify disease processes, as well as infectious agents such as bacteria, fungi, and other structures. Discussion of diagnoses and treatment will follow. Generalized disease processes cross species and classes. The most important rule for cytologic interpretation is to not overinterpret the cytologic findings. Cytology helps guide therapeutic decision making by classification of disease process as neoplasia, fungal infection, etc but may not provide a definitive diagnosis. One should only interpret to the correct level of diagnosis and know when to refer the cytology and biopsy the lesion. Preanalytical Blood collection Collect less than < 1% of a reptile’s body weight. Use heparinized, size appropriate pediatric microtainers or Capijects®. Use the jugular vein in species where possible as the large bore vein decreases the likelihood of lymph dilution common in samples from the caudal vein. The right jugular may be larger in some species of lizard and tortoise but the size difference is not as dramatic as in avian species. -
Comparative Evaluation of Ziehl-Neelsen and Kinyoun Staining in the Diagnosis of Clinically Suspected Cases of Pulmonary Tuberculosis
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Asian Pacific Journal of Health Sciences Asian Pac. J. Health Sci., 2019; 6(4):37-42 e-ISSN: 2349-0659, p-ISSN: 2350-0964 ____________________________________________________________________________________________________________________________________________ Document heading doi: 10.21276/apjhs.2019.6.4.8 Original Research Article Comparative Evaluation of Ziehl-Neelsen and Kinyoun Staining in the Diagnosis of Clinically Suspected Cases of Pulmonary Tuberculosis Sachin Kumar Mishra1, Sonali Mishra2* 1Assistant Professor, Department of Microbiology, Atal Bihari Vajpayee Government Medical College, NH-86, In-front of Khel Parisar, Sanchi Rd, Vidisha, 464001, Madhya Pradesh, India 2Assistant Professor, Department of Biochemistry, Atal Bihari Vajpayee Government Medical College, NH-86, In-front of Khel Parisar, Sanchi Rd, Vidisha, 464001, Madhya Pradesh, India Received: 26-08-2019 / Revised: 17-11-2019 / Accepted: 26-11-2019 ABSTRACT Background: Bacteriological examination of sputum is the cornerstone in diagnosis of pulmonary tuberculosis in developing world, which is usually done using a Ziehl-Nelseen (ZN) method. However, due to limited laboratory facilities that can satisfy the procedure, applicability of this procedure appears to be adversely affected in field conditions and at peripheral health institutions. Hence, it has become necessary to look for a procedure which can be used as alternative in such conditions. Material and Methods: This was a cross sectional study conducted in the Department of Microbiology, Index Medical College Hospital & Research Centre, Indore, Madhya Pradesh in conjunction with the Chest TB Clinic of Index Hospital, New Delhi for a period of 1year [from February 2018 to January 2019]. -
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. -
Carbol Fuchsin Acc. to Ziehl-Neelsen
Rev.07– 27/10/2011 Carbol Fuchsin acc. to Ziehl-Neelsen Manufacturer: Diapath Via Savoldini,71- 24057 MARTINENGO- BG- PH+39.0363.986.411 [email protected] CODE PACKAGING C0421 125 ml C0422 500 ml C0423 1000 ml Description Carbol fuchsin solution is used for the staining of acid resistant bacteria in the Ziehl-Neelsen method (see, also special stain kit code 010201). This staining is suitable to highlight mycobacteria, Nocardia and parasites on histological sections, smears, excreta and cultures. The protocol is based on typical structure of acid resistant bacteria, which acquire and keep dyes so that following decolorizing treatments are possible. Dark blue background is obtained with Methylene blue. Composition Phenol CAS No. 108-95-2 EC No. 203-6327 Basic fuchsine CAS No. 58969-01-0 EC No. 221-816-5 C.I. 42510 Ethanol CAS No.64-17-5 EC No.20-578-6 Staining protocol Histological sections (Ziehl-Neelsen staining) 1. Dewax sections and hydrate to distilled water 2. Carbol fuchsin solution for 30 minutes 3. Wash very well in running cold water 4. Acid alcohol* till sections are pale pink 5. Water for 5 minutes 6. Methylene blue solution for 30 seconds 7. Distilled water 8. Dehydrate very quickly, clarify and mount *Acid alcohol: 100 ml of ethyl alcohol 70° + 3 ml hydrochloric acid Cytological specimens (Ziehl-Neelsen staining) 1. Carbol fuchsin for 30 minutes 2. Wash in distilled water 3. Acid alcohol* 10 seconds 4. Wash in distilled water 5. Methylene blue for 30 seconds 6. Distilled water 7. Dehydrate very quickly, clarify and mount NOTE: Methylene blue could cover the possible presence of acid resistant bacteria in the 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. -
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. -
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. -
Wright's Stain
WRIGHT’S STAIN - For in vitro use only - Catalogue No. SW80 Our Wright’s Stain can be used to stain blood Interpretation of Results smears in the detection of blood parasites. Wright’s Stain is named for James Homer If malaria parasites are present, the Wright, who devised the stain in 1902 based on a cytoplasm stains pale blue and the nuclear modification of the Romanowsky stain. The stain material stains red. Schüffner’s dots and other distinguishes easily between blood cells and RBC inclusions usually do not stain or stain became widely used for performing differential very pale with Wright’s stain. Nuclear and white blood cell counts, which are routinely cytoplasmic colors that are seen in the malarial ordered when infections are expected. The stain parasites will also be seen in the trypanosomes contains a fixative, methanol, and the stain in one and any intracellular leishmaniae that are solution. Thin films of blood are fixed with present. methanol to preserve the red cell morphology so Refer to an appropriate text for a detailed that the relationship between parasites to the red description of characteristic morphological cells can be seen clearly. structures of different parasitic organisms and human cell types. Formula per Litre • Make sure all slides are clean prior to Wright’s Stain .............................................. 1.8 g making the blood smear to ensure that the Methanol .................................................. 1000 mL stain absorbs properly • Tap water is unacceptable for the rinsing Recommended Procedure solution as the chlorine may bleach the stain 1. Dip slide for a few seconds in methanol as a fixative step and allow slide to air dry • Finding no parasites in one set of blood completely. -
Appendix A: Standardization of Staining Methods
Appendix A: Standardization of Staining Methods H. Lyon, D. Wittekind, E. Schulte No detailed descriptions of staining methods are provided in this book. The reader is referred to one or more of the excellent texts covering this field (cf. Preface). Nevertheless, we have feIt it appropriate to inc1ude this appendix which gives some of our views on the technical aspects of procedures which should be given particular emphasis. It is our opinion that the need for standardization and quan titative methods in daily work is pressing. This appendix sets out the appropriate general considerations followed by a few selected methods in order to cover this area. A.l General Considerations According to Boon and Wittekind (1986) the principle aim of standardizing staining methods is to render their application reproducible and therefore reliable. This is of the utmost importance when dyes and stains are used for automated cell pattern recognition (Wittekind, 1985; Wittekind and Schulte, 1987). The theoretical background for standardization of cell and tissue preparation sterns from the fact that any preparatory step - from cell sampling to mounting of the stained slide - will somehow affect the structure of the cell and ultimately lead to the production of a particular staining pattern which, in strict tenns, is an artifact. What we eventually observe by microscopy is, from the perspective of a cell, the product of a rather violent procedure: In cytological preparations the cells have been isolated from their tissue, spread out on the surface of a glass slide and immersed in a liquid poison which abruptly arrests and - sensu stricto - "fixes" the cell in the very last moment of its life. -
Cold Acid Fast Staining Method: Efficacy in Diagnosis of Mycobacterium Tuberculosis
African Journal of Microbiology Research Vol. 3(9) pp. 546-549, September, 2009 Available online http://www.academicjournals.org/ajmr ISSN 1996-0808 ©2009 Academic Journals Full Length Research Paper Cold acid fast staining method: Efficacy in diagnosis of Mycobacterium tuberculosis Anita Pandey*, Molly Madan, Ashish K. Asthana, Ritu Kansal, Anupam Das Department of Microbiology, Subharti Medical College, Swami Vivekanand Subharti University, Meerut, Uttar Pradesh, India. Accepted 16 July, 2009 Ziehl Neelson, a carbol fuschin based stain, using the 3 component hot process is a standard staining technique for staining sputum smears for detection of Mycobacterium tuberculosis. We evaluated a 2 step cold staining method for detection of acid fast bacilli in sputum smears. 1836 sputum samples from patients of pulmonary tuberculosis were studied. Smears were prepared from each sample and slides were simultaneously stained by both methods and examined. The 2 step cold stain method was found to be equally sensitive as the ZN method detecting 20% of cases, when the primary stain was kept for a period of 20 min. The CS method also has the advantage of being simple, economical and less cumbersome and in the procedure of staining, heating, a much too precise step in ZN method for large scale application, is eliminated. The CS method may therefore prove as a good alternative for diagnosis of M.tuberculosis, particularly in smaller laboratories and peripheral centers where there is limitation of the funds and equipments. Key words: Cold stain, acid fast bacilli, Mycobacterium tuberculosis. INTRODUCTION Demonstration of acid fast bacilli in the sputum is the methylene blue as decolouriser and counter-stain has been easiest, quickest and a reliable tool for the diagnosis of advocated as an alternative staining technique by various pulmonary tuberculosis (WHO, 1974). -
Isolation of an Acid-Fast Organism from the Aqueous Humor in a Case of Sarcoidosis
Henry Ford Hosp Med Journal Vol 27, No 2, 1979 Isolation of an Acid-Fast Organism from the Aqueous Humor in a Case of Sarcoidosis C. L. Barth, PhD,* M. S. Judge, MS,** L. H. Mattman, PhD,** and P. C. Hessburg, MD * * * The anterior chamber fluid from the eye of this patient with Introduction sarcoidosis was found to contain microcolonies of a cell- wall-deficient organism that was propagated and identified as acid-fast by the Intensified Acid-Fast stain. The colonies Anterior uveitis is frequently a local manifestation of were inoculated into mice and retrieved from the dead or systemic Boeck's sarcoidosis.^-^ In fact, an early name for sacrificed animals. This report suggests that the acid-fast sarcoidosis was uveoparotid fever. In such cases the aque microbe in the aqueous humor of this case of uveitis- ous humor appears to be sterile by routine culture methods. sarcoidosis may be the same organism as that found in the Similarly, in the case described here no classical microorga blood in sarcoidosis. Thus, it may be associated not only nisms appeared in a battery of media for aerobes, an with the primary disease, but also with the complications of aerobes. Mycoplasma, Mycobacteria, and fungi. However, Boeck's sarcoidosis. special media and staining detected an organism which resembles the isolates from the blood of nine other cases of Boeck's sarcoidosis evaluated in a parallel study.^ Data regarding four ofthese patients have been published.^ This acid-fast organism remains to be identified, but wfth im proved staining and culture methods diagnosis of sar- coidosis-and its complications may be facilitated.