Gram Staining Procedure, Principle, and Results

Gram staining is among the most important staining methods in microbiology. It was established in 1882 by the Danish bacteriologist Hans Christian Gram, primarily to identify germs causing pneumonia. Utilizing crystal violet or methylene blue as the dominant colour, gramme staining is frequently the first test administered. Gram-positive organisms are those that preserve their primary colour and appear purple-brown under a microscope. Gram-negative organisms are those that do not absorb primary stain and appear red under a microscope.

The initial staining of the slide with crystal violet dye is the first step in gramme staining. In the next stage, also known as fixing the dye, iodine is used to generate a crystal violet-iodine complex to prevent the dye from being easily removed. A decolorizer, often a mixture of ethanol and acetone, is then used to remove the dye. Gram staining is based on the capacity of the bacterial cell wall to retain the crystal violet dye after solvent treatment. Gram-positive microbes contain more peptidoglycans, whereas gram-negative organisms include more lipids.

Initially, all bacteria absorb crystal violet dye; however, the lipid layer of gram-negative organisms is dissolved when a solvent is applied. With the disintegration of the lipid layer, the main stain of gram-negative bacteria is lost. In contrast, the solvent dehydrates the gram-positive cell walls, inhibiting the diffusion of violet-iodine complex and leaving the bacteria marked. Critical to gramme staining is the duration of decolorization, as extended exposure to a decolorizing chemical can erase all stains from both types of bacteria.

The final stage in gramme staining is to apply a basic fuchsin stain on decolorized gram-negative bacteria to make them easier to identify. Also referred to as counterstain. Some laboratories use safranin as a counterstain, while basic fuchsin stains gram-negative organisms more strongly. Similarly, safranin stains weakly Hemophilus spp., Legionella app, and certain anaerobic bacteria.

History of Gram Staining

The Gram stain was first employed by Hans Christian Gram in 1884. (Gram,1884). Gram sought a technique that would permit viewing of cocci in tissue sections of the lungs of individuals who had died of pneumonia. For the visualisation of turbercle bacilli, Robert Koch had already developed a staining technique. Gram invented a technique employing Crystal Violet (Gentian Violet) as the principal stain, an iodine solution as the mordant, and ethanol as the decolorizer. This method did not dye the nuclei of eukaryotic cells in tissue samples, but it did stain the cocci found in the lungs of patients who had died from pneumonia. Gram discovered that his stain was effective for viewing a variety of disease-associated bacteria, such as “cocci of suppurative arthritis following scarlet fever.” When ethanol was added, however, Typhoid bacilli were easily decolored following treatment with crystal violet and iodine. Those organisms that stained blue/violet with Gram’s stain are now known to be grampositive bacteria, such as Streptococcus pneumoniae (found in the lungs of those with pneumonia) and Streptococcus pyogenes (from patients with Scarlet fever), whereas those that were decolorized are gramnegative bacteria, such as Salmonella Typhi, which causes Typhoid fever.

What is Gram Staining?

• Gram stain, the most common staining technique in bacteriology, is a sophisticated and differential staining technique.
• Cell wall composition is used to differentiate species in the Domain Bacteria through a series of staining and decolorizing procedures. Gram-positive bacteria include thick coatings of peptidoglycan in their cell walls (90% of cell wall). These discolour purple.
• Gram-negative bacteria have walls composed of 10 percent peptidoglycan and a high lipid content. These discolour pink.
• As both Archeae and Eukaryotes lack peptidoglycan, this staining method is not employed on them.
• Gram staining needs four fundamental steps: application of a primary stain (crystal violet) to a heat-fixed smear, addition of a mordant (Gram’s Iodine), quick decolorization using alcohol, acetone, or a mixture of the two, and counterstaining with safranin.
• In 1983, the chemical mechanism of the Gram stain was elucidated.

Purpose of Gram Staining

Gram stain is essential for determining the phenotype of bacteria. The staining process differentiates Bacteria-domain organisms based on cell wall structure. Gram-positive cells are stained blue to purple and have a thick peptidoglycan coating. Gram-negative cells are red to pink and contain a thin peptidoglycan coating.

Gram Staining Principle

• In gram staining, the bacteria were first treated with the primary stain known as crystal violet.
• Crystal violet is a positively charged dye that attracted to the bacterial cell’s net negative charge. 
• In an aqueous solution, Crystal violet (CV) dissociates into CV+ and Cl– ions. Both of these ions can penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells.
• The negatively charged components of bacterial cell interacts with the CV+ ions and stains the cells purple.
• In the second step it treated with mordant (mordant is a substance that helps bind the dye tightly to the cell wall ), the Gram’s iodine, it interacts with the CV+ of crystal violet, Making an insoluble complex(CV-I) and thus increasing dye retention.
• During decolorization step, when the bacteria treated with ethanol, the pores in peptidoglycan layer of gram-positive bacteria started to shrink, as a result, the peptidoglycan prevents the loss of crystal violet from the cell wall.
• Therefore, the dye-iodine complex is held during the decolorization step and the bacteria remain purple, indeed after the addition of a second dye.
• On the other hand, the gram-negative bacteria contain a very thin peptidoglycan layer, not highly crossed linked and the pores of peptidoglycan are larger as compared to the gram-positive bacteria. 
• Hence when t treated with alcohol may extract enough lipid from the outer membrane to increase the cell wall’s porosity further.
• As a result the alcohol more readily removes the crystal violet–iodine complex and decolorizing the cells.
• When the cells are treated with negative charge counterstain safranin or secondary stain, It easily stains the decolorized gram-negative cells, as a result of that, they appear red or pink.

Some bacteria, when stained with the Gram Stain, produce a pattern known as gram-variable, which consists of a mixture of pink and purple cells. The cell walls of Actinomyces, Arthrobacter, Corynebacterium, Mycobacterium, and Propionibacterium are more susceptible to rupture during cell division, causing these gram-positive cells to stain gram-negative. In Bacillus, Butyrivibrio, and Clostridium cultures, a decrease in peptidoglycan thickness coincides with an increase in gram-negative staining cells. In addition, the age of the culture may affect the staining results for all Gram-stained bacterial cultures.

Some bacteria do not exhibit the typical Gram staining. Gram-negative cocci of the species Acinetobacter, for instance, are resistant to the decolorization step of the Gram stain. Acinetobacter spp. frequently exhibit a gram-positive reaction to a properly prepared Gram stain. Despite the fact that Mycobacterium spp. are gramme positive, the waxy structure of the coat prevents the bacteria from readily staining with the dyes used in the Gram stain. The peculiar gram-positive cell wall structure of Gardnella bacteria leads them to stain gram-negative or gram-variable. Gram stain misinterpretation has led to incorrect or delayed diagnosis of infectious illness.

Reagents Required for Gram Staining

The Gram Stain technique has been modified by Hucker. Initially, Gentian Violet was utilised as the principal stain in the Gram stain. Today, crystal violet is commonly utilised. In Hucker’s approach, ammonium oxalate is added to prevent dye precipitation, and the counterstain is dissolved in alcohol. Burke modifies the Gram Stain by including sodium bicarbonate into the crystal violet solution. As iodine oxidises, sodium bicarbonate prevents the solution from becoming acidic, and an aqueous solution of Safranin is used as a counterstain. The indicated reagents can be manufactured or obtained from biological supply houses.

Gram Staining Requirements

• Sample bacterial colonies or suspension
• Gram Staining Kit (Reagents)
• Glass slide
• Inoculating loop
• Bunsen burner
• Staining rack 
• Wash bottle (or Tap water) 
• Microscope with 100X objective lens (compound microscope)

Reagent Preparation for Gram Staining

1. Primary Stain: Crystal Violet Staining Reagent

Solution A for crystal violet staining reagent

Crystal violet (certified 90% dye content)	2g
Ethanol, 95% (vol/vol)	20 ml

Solution B for crystal violet staining reagent

Ammonium oxalate	0.8 g
Distilled water	80 ml

Crystal Violet Staining Reagent Preparation

1. Mix A and B to obtain crystal violet staining reagent.
2. Store for 24 h and filter through paper prior to use.

2. Mordant: Gram’s Iodine

Iodine	1.0 g
Potassium iodide	2.0 g
Distilled water	300 ml

Gram’s Iodine Preparation

1. Grind the iodine and potassium iodide in a mortar and add water slowly with continuous grinding until the iodine is dissolved.
2. Store in amber bottles.

3. Decolorizing Agent

Ethanol, 95% (vol/vol)

*Alternate Decolorizing Agent

Some specialists prefer an acetone decolorizer, but others prefer a 1:1 mixture of acetone and ethanol. A number of combinations are available commercially, with the majority combining 25 to 50 percent acetone with ethanol. A few formulations contain a trace amount of isopropyl alcohol and/or methanol.

Acetone	50 ml
Ethanol (95%)	50 ml

4. Counterstain: Safranin

Stock solution:

Safranin O	2.5 g
95% Ethanol	100 ml

Working Solution:

Stock Solution	10 ml
Distilled water	90 ml

Preparation

• Combine 2.5 grammes of safranin-O with 100 millilitres of 95% ethanol.
• Prepare a working solution by combining 10 ml of the preceding solution with 90 ml of distilled water.

5. Carbol-fuchsin

1. Dissolve 3 grammes of basic fuchsin in 100 millilitres of 95% ethanol Mix 5 millilitres of liquid phenol with 95 millilitres of deionized water to create a 5% phenol solution.
2. Combine 10 ml of fuchsin solution with 100 ml of phenol solution at 5% concentration.
3. Allow the solution to settle at room temperature for 24 hours.
4. Keep in dark bottles

Gram Staining Procedure

Smear preparation

1. First, make slides grease-free slides by washing them with detergent (rube both sides of the slide with cotton and detergent) and dry it.

2. Use Bunsen burner to sterilize the inoculating loop, by holding it on flame.

3. After that, use to sterile loop to transfer a loopful of culture or the specimen on the grease-free slide. Then make a smear at the center (Smear should not be very thin or very thick.).

4. Now, dry the smear in air.

5. To fix the dry smear, pass it 3-4 times through the flame quickly with the smear side facing up.

Gram Staining

1. Cover the specimen slide with primary stain, the crystal violet, and leave for 1 min.

2. Now, gently wash off the stain by running tap water.

3. Flood the slide with mordant, the Gram’s iodine and leave for 1 minute.

4. Drain off the iodine, by washing the slide again in a gentle stream of tap water.

5. Flood the slide with decolorizing agent, the acid-alcohol, and wait for 20-30 seconds. 

6. Lightly rinse the slide under flowing tap water and drain completely.

7. Now, cover the slide with counterstain or secondary stain, the safranin, and wait for about 30 seconds to 1 minute.

8. Wash slide in a gentile and indirect stream of tap water until no color arises in the effluent and then absorb with absorbent paper.

9. Now, the slide is ready to observe under microscope.

Procedure of Microscopic Observation of Gram Stain

1. Place the microscope on a stable surface and turn on the light source, if applicable.
2. Make sure that the microscope is clean and free of any dirt or dust.
3. Position the microscope so that the stage (the platform on which the slide is placed) is at a comfortable height for you to view through the eyepieces.
4. Open the stage clips to hold the slide in place.
5. Place the slide on the stage with the specimen facing up. Adjust the stage so that the slide is centered under the objective lenses.
6. Look through the eyepieces and use the focusing knobs to bring the image into focus. You may need to adjust the eyepieces to accommodate your eyesight.
7. Use the objective lenses to magnify the image. The objective lenses are located on a rotating nosepiece, and you can switch between different magnification levels by turning the nosepiece.
8. Use the stage control knobs to move the slide around and view different areas of the specimen.
9. When you are finished observing the slide, turn off the light source and close the stage clips to protect the slide. Wipe the microscope clean and store it properly.

It’s important to handle the microscope with care and to follow the manufacturer’s instructions for use. Be sure to clean the lenses and the stage regularly to ensure the best possible image quality.

Change Magnification in Microscope

To change the magnification of a microscope from 10x to 100x, follow these steps:

1. Position yourself comfortably in front of the microscope and look through the eyepieces.
2. Use the focusing knobs to bring the image into focus at the current magnification level (10x).
3. Locate the objective lenses on a rotating nosepiece at the bottom of the microscope. The objective lenses are the small lenses that are closest to the slide.
4. Turn the nosepiece to the right or left to select the 100x objective lens. The nosepiece may have labels indicating the magnification level of each objective lens.
5. Look through the eyepieces again and use the focusing knobs to bring the image into focus at the new magnification level (100x).

Oil Immersion steps in microscope for 100x magnification

Oil immersion is a technique used to increase the magnification of a microscope beyond the highest objective lens (usually 100x or higher). It involves using a drop of oil between the objective lens and the specimen to reduce the refractive index and allow more light to pass through the lens. Here are the steps for using oil immersion with a microscope:

1. Make sure the microscope is on a stable surface and the light source is turned on.
2. Position yourself comfortably in front of the microscope and look through the eyepieces.
3. Place the slide on the stage with the specimen facing up. Adjust the stage so that the specimen is centered under the objective lenses.
4. Use the objective lenses to magnify the image to the highest level (100x or higher).
5. Add a drop of oil immersion oil to the center of the objective lens. You can use a pipette or a small dropper to do this.
6. Slowly lower the objective lens into the oil by turning the focusing knob. Be careful not to touch the oil or the slide with the lens.
7. Look through the eyepieces and use the focusing knobs to bring the image into focus at the new magnification level. You may need to adjust the eyepieces to accommodate your eyesight.
8. When you are finished observing the specimen, turn off the light source and remove the objective lens from the oil. Wipe the lens clean with a lens tissue or a soft cloth.

Gram Staining Result and Interpretation

After the staining the cell will appear in these following colors;

• Gram-Positive:  The gram-positive bacteria will appear in Dark purple color which is the color of Crystal violet.
• Gram-negative: The gram-negative bacteria will appear in Pale to dark red color which is the color of Safranin O.

Other Cells,

• Yeasts: Yeast cells will appear in Dark purple color.
• Epithelial cells: Epithelial cells will appear in Pale red color.

Examples of Gram-positive bacteria

Gram-positive bacteria are a group of bacteria that are characterized by their ability to retain the crystal violet stain during the Gram staining procedure. They have a thick peptidoglycan layer in their cell walls, which allows them to retain the stain and appear purple under the microscope. Here are a few examples of Gram-positive bacteria:

1. Staphylococcus aureus
2. Streptococcus pneumoniae
3. Bacillus anthracis
4. Clostridium tetani
5. Listeria monocytogenes
6. Corynebacterium diphtheriae
7. Streptococcus pyogenes
8. Enterococcus faecalis
9. Clostridium perfringens
10. Mycobacterium tuberculosis

Gram-positive bacteria are characterized by their ability to retain the crystal violet stain during the Gram staining procedure. They have a thick peptidoglycan layer in their cell walls, which allows them to retain the stain and appear purple under the microscope. Gram-positive bacteria can be found in a variety of environments, including soil, water, and the human body. They can cause a wide range of infections, from mild skin infections to more serious illnesses like pneumonia and sepsis.

Examples of Gram-negative bacteria

Here are 10 examples of Gram-negative bacteria:

1. Escherichia coli
2. Salmonella spp.
3. Klebsiella pneumoniae
4. Proteus mirabilis
5. Pseudomonas aeruginosa
6. Haemophilus influenzae
7. Neisseria gonorrhoeae
8. Shigella spp.
9. Vibrio cholerae
10. Francisella tularensis

Gram-negative bacteria are characterized by their inability to retain the crystal violet stain during the Gram staining procedure. They have a thin peptidoglycan layer in their cell walls and an outer membrane that contains lipopolysaccharides (LPS). These characteristics cause them to appear pink or red when stained with a counterstain like safranin. Gram-negative bacteria can be found in a variety of environments, including soil, water, and the human body. They can cause a wide range of infections, including respiratory infections, urinary tract infections, and gastrointestinal infections. Some Gram-negative bacteria are also responsible for more serious illnesses like sepsis and meningitis.

Risk of errors

False positive

• spread out too much (poor discoloration of bacteria in depth),
• There is a deposit of dye in the gentian violet bottle. To fix this, filter the dye.
• iodine solution that doesn’t drain well,
• Bleach didn’t have enough time,
• Use of fuchsin with certain germs: Neisseria and Acinetobacter, in particular, are “greedy” for fuchsin and concentrate it to a dark red that is hard to tell from violet.

False negative

• There are Gram- bacteria in every group of Gram+ bacteria, and sometimes there are a lot of them. These Gram- bacteria are dead Gram+ bacteria.
• low-quality or too-watered-down dyes,
• Lugol’s plan was put off for too long,
• bleach left for too long or not rinsed well enough.

Quality control

Spread a thin layer of stool on a slide, and then colour it. There should be a lot of bacteria that are both Gram positive and Gram negative.

Note: One or more tens of slides that have been dried, fixed, and individually wrapped in aluminium foil should be done with the same preparation of diluted stools. The temperature is +4°C. This gets rid of the smear thickness parameter and puts the focus on the quality of the reagents and the times for staining and removing the stain.

Comments and Tips

• The staining will be different depending on how thick the smear is. The bleaching step is the most important part of the staining process.
• Too much bleaching can make Gram positive smears look pink or red, which is a sign of a Gram negative result. Not enough bleaching can make Gram negative smears look blue to purple, which is a sign of a Gram positive result.
• The thickness of the smear determines how much colour change is needed.
• The ASM group says that cells should be prepared with a thin smear that doesn’t have any clumps or uneven spots. Thin smears should be stained for a short amount of time.
• Some people pour decolorizing agent over the slide for 15 seconds or less, while others say to add it drop by drop for 5 to 15 seconds, or until the colour of the decolorizing agent coming from the slide no longer shows any colour.
• It is best to use crops that are young and growing quickly. For an accurate to happen, the cell wall must be whole. Older cultures may have holes in the cell walls and often give different Gram results when a mix of cells is tested.
• Use brightfield microscopy to look at slides and adjust the brightness so that the colour of the sample can be seen.
• The KOH test can be used to confirm a Gram stain. If DNA forms a chain in 3% KOH, it means that the isolate is a Gram-negative organism. This is because 3% KOH breaks down the cell walls of Gram-negative organisms, which releases viscous chromosomal material that makes the suspension thick and stringy.
  1. a drop of 3% KOH on a slide for a microscope.
  2. Add a lot of bacteria that has been grown for 24–48 hours to the drop of KOH. Carefully stir
  3. In 30 seconds, the solution of Gram-negative bacteria will be thick and form a chain of mucus.

Gram-positive bacteria and Gram-negative bacteria

Gram positive cocci

Description of the Morphotype	Most Common Organisms
Pairs	Staphylococcus, Streptococcus, Enterococcus spp.
tetrads	Micrococcus, Staphylococcus, Peptostreptococcus spp
Groups	Staphylococcus, Peptostreptococcus, Stomatococcus spp.
Chains	Streptococcus, Peptostreptococcus spp.
Clusters, intracellular	Streptococcus spp. Microaerophilic, viridans streptococci, Staphylococcus spp.
Encapsulated	Streptococcus pneumoniae, Streptococcus pyogenes (rarely), Stomatococcus mucilaginosus
In the form of an ancestor	Streptococcus pneumoniae

Gram-negative cocci

	Neisseria spp., Moraxella catarrhalis.

Gram-positive bacilli

Description of the Morphotype	Most Common Organisms
Little	Listeria monocytogenes, Corynebacterium spp.
Medium	Lactobacillus, anaerobic bacilli
Big	Clostridium, Bacillus spp.
Diphtheroid	Corynebacterium, Propionibacterium, Rothia spp.
Pleomorphic, Gram variables	Gardnerella vaginalis
Pearl	Mycobacteria, lactobacilli affected by antibiotics and corynebacteria
Filamentous	Anaerobic morphotypes, cells affected by antibiotics
Filamentous, beaded, branched	Actinomycetes, Nocardia, Nocardiopsis, Streptomyces, Rothia spp.
Bifid or V shapes	Bifidobacterium spp., brevibacteria
Gram-negative coccobacilli	Bordetella, Haemophilus spp. (pleomorph)
Masses	Veillonella spp.
Chains	Prevotella, Veillonella spp.

Gram-negative bacilli

Description of the Morphotype	Most Common Organisms
Little	Haemophilus, Legionella (thin with filaments), Actinobacillus, Bordetella, Brucella, Francisella, Pasteurella, Capnocytophaga, Prevotella, Eikenella spp.
Bipolar	Klebsiella pneumoniae, Pasteurella spp., Bacteroides spp.
Medium	Enterics, pseudomonads
Big	Clostridia or devitalized bacilli
Curved	Vibro, Campylobacter spp.
Spiral	Campylobacter, Helicobacter, Gastrobacillum, Borrelia, Leptospira, Treponema spp
Fusiform	Fusobacterium nucleatum
Filaments	Fusobacterium necrophorum (pleomorph)

Why is gram staining important?

1. An essential test for the rapid presumptive diagnosis of infectious agent 
2. Gram staining is utilized to distinguish the bacteria as a Gram positive or Gram negative 
3. Used To examine the morphology of bacteria 
4. Used To examine the arrangement of bacteria 
5. Used To find out the evidence of capsule 
6. Used To find out the evidence of spore 
7. Used To find out the evidence of pus cells 
8. Used To find out the evidence of epithelial cells 
9. Used To find out the evidence of Yeast cells
10. Used to control initial therapy until definitive identification of microorganism concerned.
11. Morphology of stained bacteria can sometimes be diagnostic. For example Gram ve- intracellular diplococci in urethral pus provides a presumptive diagnosis of Gonorrhea.
12. Sometimes specimens may show organisms under a microscope but appear sterile in culture media. In these cases, Gram stain is the only clue to the nature , variety and relative proportion of infecting organism .
13. Aids in interpretation of culture reports.

Applications of Gram Staining

Gram staining is a widely used laboratory technique that is used to differentiate bacterial species based on the structure and composition of their cell walls. It was developed by Danish bacteriologist Hans Christian Gram in 1884, and it is still a valuable tool for microbiologists today. Here are some of the main applications of Gram staining:

1. Identification of bacterial species: Gram staining is often used as a preliminary step in the identification of bacterial species. By determining whether a bacterium is Gram-positive or Gram-negative, microbiologists can narrow down the possible species and perform further tests to confirm the identity of the bacterium.
2. Detection of bacterial infections: Gram staining is often used to detect bacterial infections in clinical samples, such as sputum, urine, and wound swabs. By identifying the presence of bacteria in the sample and determining whether they are Gram-positive or Gram-negative, healthcare providers can determine the appropriate treatment for the infection.
3. Antibiotic susceptibility testing: Gram staining can also be used to determine the susceptibility of bacteria to certain antibiotics. For example, Gram-negative bacteria are often resistant to certain types of antibiotics, while Gram-positive bacteria may be more susceptible. By performing a Gram stain, microbiologists can determine the best course of treatment for an infection.
4. Research and discovery: Gram staining is also used in research to study the characteristics and behavior of bacterial species. By analyzing the structures of bacterial cells and their ability to retain the crystal violet stain, scientists can learn more about the biology of these microorganisms and how they interact with their environment.

Overall, Gram staining is an important tool for microbiologists and healthcare providers for the identification and characterization of bacterial species, as well as for the detection and treatment of infections.

Advantages of Gram Staining

Gram staining is a widely used laboratory technique that is used to differentiate bacterial species based on the structure and composition of their cell walls. It has several advantages over other methods of bacterial identification, including:

1. Widely available and relatively easy to perform: Gram staining is a simple and inexpensive procedure that can be performed in most laboratories. It requires only basic equipment, such as a microscope, a heat source, and a few chemical reagents.
2. High degree of accuracy: Gram staining has a high degree of accuracy, especially when combined with other identification techniques. This makes it a reliable method for identifying bacterial species in clinical samples.
3. Can be performed on a variety of samples: Gram staining can be performed on a wide range of samples, including liquid cultures, solid media, and clinical specimens. This makes it a versatile technique for identifying bacteria in different types of samples.
4. Provides information about the structure of bacterial cells: In addition to identifying bacterial species, Gram staining can also provide information about the structure of bacterial cells. By examining the appearance of the cells under the microscope, microbiologists can learn more about the characteristics and behavior of different bacterial species.

Overall, Gram staining is an important tool for microbiologists and healthcare providers for the identification and characterization of bacterial species, as well as for the detection and treatment of infections.

Limitation of Gram Staining

1. This method does not work on acid-fast bacteria (Mycobacterium spp.) or other cell-wall-poor bacteria.
2. Incompatible with microscopic bacteria such as Ricktessia spp., Chlamydia spp., etc.
3. Several reagents are needed.
4. While under-decolorizing may lead to erroneous gram-positive results, over-decolorizing can lead to false gram-negative results.
5. Overly much primary stain may be retained by smears that are too thick or viscous, making it difficult to identify the correct Gram stain reactions. It’s possible that gram-negative bacteria don’t decolorize correctly.
6. When a culture is older than 16-18 hours, it will contain both living and dead cells. Deteriorating, dead cells will not properly hold the dye.
7. With time, the stain could crystallise. In order to get rid of extra crystals, filter the solution through a piece of gauze.
8. Patients who have been treated successfully with antibiotics or antimicrobial therapy may exhibit changed Gram stain reactivity in their swabs.
9. Pneumococci found in a direct smear of the lower respiratory tract may fail to develop in culture on rare occasions. Some types of the bacteria cannot survive in an oxygen-free environment.
10. In a purulent specimen, toxin-producing bacteria like Clostridia, Staphylococcus aureus, and Streptococcus pyogenes may kill off white blood cells.
11. It is possible to see Gram-negative bacteria like Campylobacter and Brucella, which only faintly stain, by employing a different counterstain (e.g., basic fuchsin).

What is the correct order of staining reagents in gram-staining?

The correct order of staining reagents in gram staining is as follows:

1. Crystal violet
2. Iodine
3. Alcohol or acetone
4. Safranin (counterstain)

Here is a brief description of each step:

1. Crystal violet: The sample is first treated with crystal violet, a violet-colored dye, which stains the bacterial cells.
2. Iodine: The sample is then rinsed and treated with an iodine solution, which serves as a mordant to help the crystal violet dye bind to the bacterial cell walls.
3. Alcohol or acetone: The sample is then rinsed again and treated with a decolorizing solution, which typically contains alcohol or acetone. This step helps to remove the excess crystal violet dye from the sample, resulting in the decolorization of the sample.
4. Safranin: Finally, the sample is treated with a counterstain called safranin, which stains the bacteria that were not retained by the crystal violet-iodine staining. This step allows for the differentiation between Gram-positive and Gram-negative bacteria based on their different responses to the crystal violet-iodine staining.

Animation of gram staining

Gram Staining Images

MCQ on Gram Staining

1. Which of the following is not a step in the gram staining process?

a) Applying crystal violet to the sample b) Applying a decolorizing solution c) Applying a mordant d) Applying a counterstain

2. Which of the following is not a characteristic of gram-positive bacteria?

a) They have a thick peptidoglycan layer in their cell walls b) They are resistant to decolorization c) They appear pink when stained with safranin d) They appear purple when stained with crystal violet

3. Which of the following is not a characteristic of gram-negative bacteria?

a) They have a thin peptidoglycan layer in their cell walls b) They are more susceptible to decolorization c) They appear purple when stained with crystal violet d) They appear pink when stained with safranin

4. Which of the following is not a reason for gram staining?

a) To differentiate between gram-positive and gram-negative bacteria b) To identify bacterial species c) To determine the susceptibility of bacteria to certain antibiotics d) To determine the presence of viruses in a sample

5. Which of the following solutions is not used in gram staining?

a) Crystal violet b) Iodine c) Decolorizing solution d) Ethanol

6. Which of the following is not a characteristic of a decolorizing solution?

a) It removes the crystal violet dye from gram-positive bacteria b) It removes the crystal violet dye from gram-negative bacteria c) It is often made with alcohol or acetone d) It is used after the crystal violet and iodine steps

7. What is the purpose of the counterstain in gram staining?

a) To make the sample more visible under the microscope b) To differentiate between gram-positive and gram-negative bacteria c) To identify bacterial species d) To determine the susceptibility of bacteria to certain antibiotics

8. Which of the following is not a common counterstain used in gram staining?

a) Safranin b) Eosin c) Thionine d) Methylene blue

9. Which of the following is not a characteristic of a negative gram stain result?

a) The cells are gram-negative b) The cells appear pink when stained with safranin c) The cells appear purple when stained with crystal violet d) The cells have a thin peptidoglycan layer in their cell walls

10. Which of the following is not a characteristic of a positive gram stain result?

a) The cells are gram-positive b) The cells appear pink when stained with safranin c) The cells appear purple when stained with crystal violet d) The cells have a thick peptidoglycan layer in their cell walls

Answer Key

1. c) Applying a mordant
2. c) They appear pink when stained with safranin
3. c) They appear purple when stained with crystal violet
4. d) To determine the presence of viruses in a sample
5. d) Ethanol
6. a) It removes the crystal violet dye from gram-positive bacteria
7. b) To differentiate between gram-positive and gram-negative bacteria
8. d) Methylene blue
9. b) The cells appear pink when stained with safranin
10. b) The cells appear pink when stained with safranin

FAQ

Reference

1. Tripathi N, Sapra A. Gram Staining. 2022 Aug 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan–. PMID: 32965827.
2. http://www.med-chem.com/pages/lab_procedures/pdf/gram_stain.pdf
3. https://www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-positive_Bacteria
4. https://www.austincc.edu/microbugz/handouts/Stain%20protocols.pdf
5. https://en.wikipedia.org/wiki/Gram_staining
6. https://basicmedicalkey.com/role-of-microscopy/
7. https://microbiologyinfo.com/gram-staining-principle-procedure-interpretation-examples-and-animation/
8. https://laboratoryinfo.com/gram-staining-principle-procedure-interpretation-and-animation/
9. https://www.labce.com/spg327545_gram_stain_principle.aspx
10. https://microbeonline.com/gram-staining-principle-procedure-results/
11. https://gpatindia.com/staining-microbiology-notes-of-gram-staining-and-acid-fast-staining/
12. https://www.slideshare.net/manojmahato9638/gram-stain-by-manoj-76506297