Gram Stain Introduction

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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. * Be sure to check manufacturer’s instructions for the timing of each step. This job aid is a component of the free, on-demand CDC training course “Biochemicals and Gram Negative Organism ID” Find the course at https://www.cdc.gov/labtraining. .

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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.
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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.
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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).
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