The basic principle of the gram staining technique involves the ability of the cell wall to retain the primary stain. The cell wall structure will determine whether the organism is gram-positive or gram-negative. Gram-positive bacteria have higher peptidoglycan content, whereas gram-negative bacteria have more lipid content in the cell wall.
What is the purpose of the primary stain in Gram staining?
The Gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. The primary stain ( crystal violet) binds to peptidoglycan, coloring cells purple. Both gram-positive and gram-negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet.
What are the steps in Gram staining?
Gram Staining - Step 1. Flood heat-fixed emulsion with Crystal Violet, let stand for 1 minute, rinse with water. Gram Staining - Step 2. Add iodine solution, let stand for 1 minute, rinse with water. Gram Staining - Step 3. Decolorize with alcohol solution holding slide on slight angle, add until run off is clear (20-30 sec), rinse with water.
What is the important procedure in Gram staining?
The procedure/steps of Gram Stain
- Prepare and fix the specimen to the microscope slide before staining.
- Cover the smear with crystal violet, the primary stain, for 20 seconds.
- Gently rinse off the stain with water.
- Cover the smear with Gram’s iodine, the mordant, for 1 minute.
- Pour off the excess Gram’s iodine.
Which bacteria will not stain using the Gram staining method?
This waxy barrier also prevents stains from penetrating the cell, which is why the Gram stain does not work with mycobacteria such as Mycobacterium, which are pathogens of humans and animals. For these bacteria, the acid–fast staining technique is used. Figure 6. Acid-fast bacilli in sputum
What is the principle behind gram staining and acid fast staining?
Principle of acid fast staining (in mycobacteria): Heat softens the wax in the cell wall and allows the stain (basic fuchsin) to enter. The fuchsin dye is more soluble in phenol than in water or alcohol. Phenol in turn is more soluble in lipids or waxes, thus the dye-phenol mixture enters the cell.
What is the principle behind simple staining?
Simple staining involves directly staining the bacterial cell with a positively charged dye in order to see bacterial detail, in contrast to negative staining where the bacteria remain unstained against a dark background.
What are the 4 steps of gram staining?
The performance of the Gram Stain on any sample requires 4 basic steps that include applying a primary stain (crystal violet) to a heat-fixed smear, followed by the addition of a mordant (Gram's Iodine), rapid decolorization with alcohol, acetone, or a mixture of alcohol and acetone and lastly, counterstaining with ...
What's the purpose of gram staining?
A Gram stain is most often used to find out if you have a bacterial infection. If you do, the test will show if your infection is Gram-positive or Gram-negative. A Gram stain may also be used to diagnose fungal infections.
What is the difference between simple staining and gram staining?
The Gram stain is a differential stain, as opposed to the simple stain which uses 1 dye. As a result of the use of 2 dyes, making this procedure a differential stain, bacteria will either become purple/blue or pink during the procedure.
How is Gram staining used to identify bacteria?
The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with.
Why Safranin is used in Gram staining?
The safranin is also used as a counter-stain in Gram's staining. In Gram's staining, the safranin directly stains the bacteria that has been decolorized. With safranin staining, gram-negative bacteria can be easily distinguished from gram-positive bacteria.
Why crystal violet is used in Gram staining?
The gram stain utilizes crystal violet as the primary stain. This basic dye is positively charged and, therefore, adheres to the cell membranes of both gram negative and positive cells. After applying crystal violet and waiting 60 seconds the excess stain is rinsed off with water.
What is the purpose of simple staining in microbiology?
Simple staining can be used for all types of bacterial cells to give contrast to the other- wise colorless cell in order to determine cell morphology, size, and cell grouping. This technique is simple because only one dye is used and direct because the actual cell is stained.
What are the two purpose of simple staining?
The simple stain can be used to determine cell shape, size, and arrangement. True to its name, the simple stain is a very simple staining procedure involving only one stain. You may choose from methylene blue, Gram safranin, and Gram crystal violet.
What is the purpose of simple staining in microbiology quizlet?
The purpose of a simple stain is to allow visualization of bacteria by examination of the shape and arrangement. List 3 other dyes besides Methylene blue that can be used for simple staining.
What is the difference in principle between a simple and a differential stain?
Simple staining uses only one staining reagent and is used to determine shape, dimensions, and arrangement of microbial cells. Differential staining uses more than one staining reagent (dye) to differentiate cellular structures.
When was Gram staining first used?
Last updated on May 30th, 2021. Gram staining method, the most important procedure in Microbiology, was developed by Danish physician Hans Christian Gram in 1884. Gram staining is still the cornerstone of bacterial identification and taxonomic division. This differential staining procedure separates most bacteria into two groups on the basis ...
What color does a gram positive cell lose?
After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.
What color are Gram negative bacteria?
The gram-negative bacteria appear colorless and gram-positive bacteria remain blue. Application of counterstain (safranin): The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue. Find information and process for the Preparation of Gram Staining Regent.
What is the difference between Gram positive and Gram negative bacteria?
The differences in cell wall composition of Gram-positive and Gram-negative bacteria account for the Gram staining differences. Gram-positive cell wall contains a thick layer of peptidoglycan with numerous teichoic acid cross-linking which resists the decolorization.
What is the decolorizing agent in bacteria?
The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative bacteria.
How long to flood cells with crystal violet stain?
Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results.
What is the best way to check for staining reactions?
Always check new batches of stain and reagents for correct staining reactions using a smear containing known Gram-positive and Gram-negative organisms.
What is Gram staining?
The Gram staining is one of the most crucial staining techniques in microbiology. It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882, mainly to identify organisms causing pneumonia.[1] .
When was Gram staining first used?
Last Update: February 23, 2021. Introduction. The Gram staining is one of the most crucial staining techniques in microbiology. It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882, mainly to identify organisms causing pneumonia.[1] . Often the first test performed, ...
What is the primary color of gram stain?
Often the first test performed, gram staining involves the use of crystal violet or methylene blue as the primary color.[2] . The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms.
What is the term for organisms that retain the primary color and appear purple-brown under a microscope?
The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms. The organisms that do not take up primary stain appear red under a microscope and are Gram-negative organisms. The Gram staining is one of the most crucial staining techniques in microbiology.
How long to counterstain a smear?
The smear is counterstained with basic fuchsin solution for 40 to 60 seconds. The fuchsin solution is washed off with water, and excess water is blotted with the bibulous paper. The slide can also be air-dried after shaking off excess water. 3.
How long does it take for crystal violet to wash off?
Crystal violet stain is added over the fixed culture. After 10 to 60 seconds, the stain is poured off, and the excess stain is rinsed with water. The goal is to wash off the stain without losing the fixed culture. Iodine solution is used to cover the smear for 10 to 60 seconds.
Which cocci are Gram positive?
Gram-positive cocci in clusters: Usually characteristic of Staphy lococcusspecies such as S. aureus.
What is Gram staining?
The Gram staining is one of the most crucial staining techniques in microbiology. It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882, mainly to identify organisms causing pneumonia. Often the first test performed, gram staining involves the use of crystal violet or methylene blue as the primary color. The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms. The organisms that do not take up primary stain appear red under a microscope and are Gram-negative organisms.
What is the first step in gram staining?
The first step in gram staining is the use of crystal violet dye for the slide's initial staining. The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine complex to prevent easy removal of dye. Subsequently, a decolorizer, often solvent of ethanol and acetone, is used to remove the dye. The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment. Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content.
Do Gram negatives lose their primary stain?
Initially, all bacteria take up crystal violet dye; however, with the use of solvent, the lipid layer from gram-negative organisms is dissolved. With the dissolution of the lipid layer, gram negatives lose the primary stain. In contrast, solvent dehydrates the gram-positive cell walls with the closure of pores preventing diffusion of violet-iodine complex, and thus, bacteria remain stained. The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria.
Why does a Gram negative organism get decolorized?
Since Gram negative organism have thin peptidoglycan layer(1-2 layers)and have additional lipopolysaccharide layerwhich gets dissolved due to the addition of alcohol, so gram negative organism fails to retain the complex and gets decolorized as the complex is washed away.
What color does a Gram positive cell lose after decolorization?
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. Requirements and preparation of reagents.
What determines whether an organism is gram psitive or negative?
The structure of the organism’s cell wall determines whether the organism is gram psitive or negative. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary stain by resisting declorization while others get decolorized by a decolorizer.
What is the color of gram positive?
Observe under microscope. Interpretation of Gram staining. The staining results of gram stain are as follows : Gram Positive: Dark purple.
What is the role of iodine in the cell membrane?
Iodine (I), used as mordant interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. When a decolorizer such as alcohol or acetoneis added, it interacts with the lipids of the cell membrane.
How long to leave crystal violet stain on a smear?
Cover the smear with crystal violet stain and leave for 1 minute.
Why is a Gram positive cell dehydrated?
In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. This closes the pores in the cell wall and prevents the stain from exiting the cell. The large CV–I complexes become trapped within the Gram-positive cell also due to the thick and multilayered (40 layers)nature of its peptidoglycan.
How Does Gram Staining Work?
Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin. Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. The process involves three steps:
Why do Gram positive bacteria stain violet?
Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process.
How to stain a cell?
How To- Staining Protocol and Concerns: 1 Make a slide of cell sample to be stained. Heat fix the sample to the slide by carefully passing the slide with a drop or small piece of sample on it through a Bunsen burner three times. 2 Add the primary stain (crystal violet) to the sample/slide and incubate for 1 minute. Rinse slide with a gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet. 3 Add Gram's iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall. 4 Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a gentle stream of water. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells. 5 Add the secondary stain, safranin, to the slide and incubate for 1 minute. Wash with a gentle stream of water for a maximum of 5 seconds. If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope. If the bacteria is Gram negative, it will lose the primary stain and take the secondary stain, causing it to appear red when viewed under a microscope.
What is the process of staining cells with crystal violet?
The process involves three steps: Cells are stained with crystal violet dye. Next, a Gram's iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water.
Does alcohol decolorize a cell?
However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells. Add the secondary stain, safranin, to the slide and incubate for 1 minute.
What is the objective of gram stain?
This test differentiates the bacteria into Gram-Positive and Gram-Negative Bacteria, which helps in the classification and differentiation of microorganisms. The Gram stain separates bacteria into two groups: (1) Gram-positive microorganisms that retain the primary dye (Crystal violet) and ...
What is Gram Staining?
Gram staining is a differential bacterial staining technique used to differentiate bacteria into Gram Positive and Gram Negative types according to their cell wall composition.
How to stain a specimen with gram stain?
The procedure/steps of Gram Stain. Prepare and fix the specimen to the microscope slide before staining. Cover the smear with crystal violet, the primary stain, for 20 seconds. Gently rinse off the stain with water. Cover the smear with Gram’s iodine, the mordant, for 1 minute. Pour off the excess Gram’s iodine.
How to do Gram stain?
The procedure/steps of Gram Stain 1 Prepare and fix the specimen to the microscope slide before staining. 2 Cover the smear with crystal violet, the primary stain, for 20 seconds. 3 Gently rinse off the stain with water. 4 Cover the smear with Gram’s iodine, the mordant, for 1 minute. 5 Pour off the excess Gram’s iodine. 6 Run the acid-alcohol decolorizer over the smear until the solution appears clear. 7 Gently rinse with water. 8 Cover the smear with safranin, the secondary or counterstain, for 20 seconds. 9 Gently rinse the stain with water. 10 Blot dry with bibulous paper.
What is the primary color of Gram stain?
Gram-positive organisms contain a highly cross-linked layer of peptidoglycan that retains the primary dye, crystal violet (CV), following the application of the mordant, iodine (I). The iodine and crystal violet form ...
What stain is used to make gram negative cells pink?
The cells appear colorless. To make the colorless cells visible, a secondary stain, safranin, is applied, leaving the gram-negative cells pink. Created with BioRender.com.
How long to cover a smear with iodine?
Cover the smear with Gram’s iodine, the mordant, for 1 minute.
What is the decolorising agent used in Gram positive cells?
It acts as a lipid solvent and as a protein- dehydrating agent. In gram-positive cells, the decolourising agent acts initially as a lipid solvent , dissolving out the little quantity of lipid present in the cell wall, thereby forming minute pores in it. Subsequently, it dehydrates the cell wall proteins (peptides), which close the pores.
Why do Gram positive cells lose their violet color?
The reasons are as follows: (a) Over-decolourisation may occur, due to which the gram-positive cells also lose their violet colour. (b) Over-fixation may occur, due to which the gram-positive cells lose their ability to resist decolourisation.
How to make a smear of bacteria?
A smear of bacteria is prepared at the center of the slide in two methods as follows. (a) If a bacterium grown on agar plate or agar slant is to be observed, a drop of water is put at the center of the slide and a loop of bacteria from the plate or slant is transferred to it by a loop sterilized over flame. Then, by slow rotation of the loop in the drop, a bacteria suspension is made and it is spread till a smear is obtained. (b) If a bacterium grown in liquid broth is to be observed, a drop of the bacteria suspension is directly placed at the center of the slide by a flame-sterilized loop and a smear is made by spreading.
What is the solvent that extracts the blue dye complex from the lipid-rich, thin-walled gram?
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-positive bacteria. The gram-negative bacteria appear colorless and gram-positive bacteria remain blue.
What is the color of the primary stain?
The cells finally retain the purple-blue colour of the primary stain. On the other hand, the gram-negative cell wall contains large quantity of lipid (LPS), which is dissolved out by the decolourising agent, resulting in the formation of large pores. These pores do not close appreciably on dehydration of cell wall proteins.
How is a smear fixed?
The smear is fixed by heating. Heating results in coagulation of the cellular proteins, due to which the cells stick to the slide surface and do not get washed away during staining, Heat- fixation is done by quickly passing the slide high above a flame 2-3 times, with the smear surface facing upward, so that the smear does not get heated up.
How long does a slide stay on a stain tray?
The slide is kept on a staining tray and flooded with the primary stain, crystal violet, for 1 minute.
Steps of Gram Staining
Principle of Gram Stain
- 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 peptidoglycanwith numerous teichoic acid cross-linking, which resists decolorization. In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that penetrate through Gram-positive and Gram-negativ…
Procedure of Gram Staining
- Smear Preparation
Fix material on a slide with methanol or heat. If the slide is heat fixed, allow it to cool to the touch before applying the stain. - Gram Staining Procedure
After performing a gram stain, thetechnicianshould 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.
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 105organisms per milliliter of centrifuged fluid. Gram staining technique is not recommended fo…
Quality Control
- 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. 1. Gram-positive bacteria may lose their ability to retain crystal violet and stain Gram negatively for the following reasons: 1.1. cell wall damage of bacteria due to antibiotic therapy or excessive heat fixation of the smear. 1.2. over- decolorizatio…
Historical Background
Principle of Gram Staining Technique
- The basic principle of the gram staining technique involves the ability of the cell wall to retain the primary stain. The cell wall structure will determine whether the organism is gram-positive or gram-negative. Gram-positive bacteria have higher peptidoglycan content, whereas gram-negative bacteria have more lipid content in the cell wall. 1. Cry...
Specimen
- Various specimens can be used in this technique. Some of the commonly used specimens are following 1. Urine 2. Ascitic fluid 3. Sputum 4. Cerebrospinal fluid (CSF) 5. Blood 6. Swabs from throat, rectum, wound, nostrils, cervix, etc 7. Synovial fluid 8. Pleural fluid
Reagents
- Reagents used in this technique are following 1. Crystal-violet stain 2. Gram’s Iodine 3. Decolorizer (ethanol) 4. Safranin 5. Water
Clinical Uses of Gram-Staining
- Provides preliminary clue in anaerobic culture.
- Identifies Candida and Cryptococcus Spp.
- Classifies the bacteria into two groups, gram-positive and gram-negative bacteria.
- Determines the cell morphology, size, and arrangement.
Steps in Gram-Staining Technique
- Take a grease-free dry slide. And wipe the bottom of the biofilm slide clean.
- Clean top edges of the slide about 2mm
- Build up a ridge of petroleum jelly on the top and bottom of a cover slide
- Cover slip with petroleum jelly
- Put the biofilm on slide with cover slip
- Take a grease-free dry slide. And wipe the bottom of the biofilm slide clean.
- Clean top edges of the slide about 2mm
- Build up a ridge of petroleum jelly on the top and bottom of a cover slide
- Cover slip with petroleum jelly
- Put the biofilm on slide with cover slip
- Stain with primary crystal violet stain and incubate for almost 1 minute.
Errors During The Gram-Staining Procedure
- Prolonged decolourisation
- Excessive counterstaining
- Excessive heat while fixation
- Excessive washing between steps
Artifacts in Gram-Staining
- Dirty glass slides
- Gram-staining reagents contamination
- Contaminated water used to rinse slides
Requirements and Preparation of Reagents
- Primary Stain : Crystal violet
Solution A : 1. Crystal violet = 2 gm 2. Ethyl alcohol= 20 ml Solution B : 1. Ammonium oxalate = 0.8 gm 2. Distilled water = 80 ml Mix solution A and B. Keep for 24 hours and filter. Store in an amber-colored bottle. - Mordant : Gram’s Iodine
1. Iodine = 1 gm 2. Potassium iodide = 2 gm 3. Distilled water = to 100 ml Mix and Store in an amber-colored bottle.
Interpretation of Gram Staining
- The staining results of gram stain are as follows: 1. Gram-positive will be dark-purple color 2. Gram-negative will be pale to dark red color 3. Yeasts will be dark purple color 4. Epithelial cells will be pale red color