Gram stain: what it is, purpose, procedure and results (2023)


What is a Gram stain?

A Gram stain is a laboratory test that looks for bacteria at the site of a suspected infection or in certain body fluids. A medical laboratory scientist processes the Gram stain, which provides relatively quick results, allowing health care providers to know if bacteria are present, and if so, the general types. This can help guide further identification testing and treatment options.

Bacteria are a large group of protozoa. They can live in different places on your body and on your skin. While some types of bacteria are harmless or even beneficial, others can cause infection and disease. A Gram stain helps diagnose harmful bacteria.

Under a Gram stain, different types of bacteria change one of two color groups (pink to red or purple to blue) under a special set of stains and are classified as "gram negative" or "gram positive" as appropriate. Gram stain works by distinguishing bacteria based on the chemical and physical properties of their cell walls.

However, not all types of bacteria can be tested with the Gram stain method, and Gram stains alone are usually not diagnostic. Instead, they help determine the type of bacteria in general terms.

The Gram stain is an important staining technique in microbiology that has been used by scientists for hundreds of years. It is named after the Danish bacteriologist Hans Christian Gram, who first introduced it in 1882, primarily to identify pneumonia-causing organisms.

What are gram positive and gram negative bacteria?

When bacteria are present on a Gram-stained slide, a medical laboratory scientist classifies them as Gram-negative or Gram-positive based on the color they acquire from a variety of stains. There are also gram-variable bacteria, which means they are irregularly colored, and there is a mix of pink and purple bacteria.

It is important to note that gram negative does not mean that no bacteria are present, and 'positive' and 'negative' do not mean 'good' and 'bad' in this case.

gram positive organism

Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan, a substance that forms the cell walls of many bacteria. Peptidoglycan makes up about 90% of the cell wall of Gram-positive bacteria. As a result, they appear blue to violet under a Gram stain.

(Video) How to prepare the perfect Gram stain

Gram-positive organisms include:

  • staphSpecies.
  • streptococciSpecies.
  • CorynebacteriumSpecies.
  • ClostridiumSpecies.
  • ListeriaSpecies.

Gram-negative organism

Gram-negative bacteria have cell walls with thin layers of peptidoglycan (10% of the cell wall) and high levels of lipids (fatty acids). As a result, they appear red to pink under a Gram stain.

Gram-negative organisms include:

  • Neisseria gonorrhoeae and Neisseria meningitis.
  • MoraxellaSpecies.
  • Escherichia coli(E. coli).
  • PseudomonasSpecies.
  • protectionSpecies.
  • KlebsiellaSpecies.

What is the difference between a Gram stain and a bacterial culture?

Health care providers use a Gram stain and bacterial cultures if they suspect you have a bacterial infection. However, these tests provide different information.

Providers often order bacterial culture tests in addition to Gram stain tests. In a bacterial culture test, a medical laboratory scientist removes cells from the sample that will also be used for the Gram stain and places them in a special environment to encourage cell growth. Results are usually available in a few days, but some types of bacteria grow slowly, which can take several days or longer. Once the cells have multiplied and grown sufficiently, a medical laboratory scientist examines the cultured bacteria to determine their precise identity.

In a Gram stain, a sample is stained on glass slides and viewed under a microscope to determine if any bacteria are present. If bacteria are present, medical laboratory scientists can see if they are gram-negative or gram-positive based on whether the bacteria appear pink to red or purple to blue under the stain. The different types of bacteria are either gram negative or gram positive.

Although Gram stains provide faster results than bacterial cultures, in most cases, unlike a bacterial culture, a Gram stain cannot diagnose the type of bacteria. However, Gram stains can help point healthcare providers in the right direction for prescribing treatments.

What conditions do Gram stains help diagnose?

Health care providers order Gram stains to diagnose and treat certain bacterial infections, such as:Urinary tract infections (UTIs)and bacteriallung infection. Providers may not always order a Gram stain to help diagnose conditions caused by bacteria. For example, they do not usually change color due to food poisoning, but it is possible to do so through a stool sample (poop).

Common gram-negative bacteria and associated diseases

Examples of gram-negative bacteria (in italics) and the conditions they can cause include:

(Video) Gram Staining Procedure Animation Microbiology - Principle, Procedure, Interpretation

  • Escherichia coli: urinary tract infections (UTIs) andpoisoned food.
  • Legionella pneumoniae:legionelosis.
  • salmonella: food poisoning andtyphoid fever.
  • Klebsiella: Pneumonia, bloodstream infections, wound or wound infections andmeningitis bacterial.
  • Neisseria gonorrhoeae:hiker.
  • protection: HWI.
  • morganella morganii: HWI,Septicemia, pneumonia, wound infections, musculoskeletal infections, central nervous system infections andpericarditis.
  • Aeromonas: eye infections,Osteomyelitis, bacterial meningitis, respiratory infections after "near drowning" and joint infections.

Common gram-positive bacteria and their comorbidities

Examples of Gram-positive bacteria (in italics) and the conditions they can cause include:

  • steotococos neumonia: Neumonia bacterial.
  • staphylococcus aureus: Neumonia bacterial,Methicillin-resistant Staphylococcus aureus (MRSA)ytoxic shock syndrome.
  • group A strepspecies:sore throatinfections
  • Listeria: poisoned food.
  • Clostridium: poisoned food
  • Clostridium botulinumyClostridium butyricum:botulism.

When is a Gram test done?

Health care providers usually order a Gram stain along with a bacterial culture if they suspect you have a bacterial or sometimes fungal infection. It is one of the most common ways to quickly diagnose a bacterial infection in your body.

Who performs a Gram stain test?

Depending on the type of infection you have, many types of healthcare providers can collect a sample for a Gram stain test. For example, a gynecologist may take a swab for a Gram stain test for gonorrhea, and a pulmonologist may take a sample of your saliva or mucus to use in a Gram stain test for bacterial pneumonia.

Your provider then places the sample in a sterile container and sends it to a lab for testing. A medical laboratory scientist applies the sample to slides, applies a series of stains, and views the sample under a microscope. You compile a report and share it with your healthcare provider.

test details

How do I prepare for a Gram stain test?

You don't have to do anything special to prepare for a Gram stain test.

How is a gram stain test done?

A Gram stain test involves three general steps, including:

  • collecting the sample.
  • sample processing.
  • sample examination.

Collect Gram stain sample

For a Gram stain test, your doctor takes a sample from a suspected site of infection. Some of the ways a provider may collect Gram stain test samples are:

  • Brushing or scraping of tissue from the surface of a part of the body.
  • Collection of samples of fluids or waste from your body.
  • Use a fine needle aspiration to collect a sample of fluid from an area of ​​your body.

Stains picked up by a swab with brushes and scrapes include yours:

(Video) Gram Stain: introduction, procedure and result interpretation

  • Throat.
  • nostrils
  • Genitals.
  • skin wounds

Samples that you can collect directly into a sterile container include:

  • spit (sputum).
  • Urine (Cheeks).
  • Stool (poop).

Areas of your body that may require the use of fine needle aspiration include:

  • synovial fluid in your joints.
  • Fluid around your heart (pericardial fluid).
  • Fluid around the lungs (pleural fluid).
  • Cerebrospinal fluid (CSF) around the spinal cord.

Your provider then places the sample in a sterile container and sends it to a lab for testing.

Gram stain procedure

In the laboratory, a medical laboratory scientist smears or spreads the sample on glass slides. These slides are called swabs. They then apply a series of stains to the swab to perform a Gram stain.

The Gram staining process involves four basic steps, including:

  1. Application of a primary colorant (crystal violet).
  2. Addition of a mordant (Grams of Iodine).
  3. Quick bleaching with ethanol, acetone or a mixture of both.
  4. Contrast with safranin.

Gram stain test

The medical laboratory technician then classifies any bacteria that may be present by color and shape during microscopic evaluation:

  • color: Typically, Gram-positive bacteria appear purple to blue, and Gram-negative bacteria appear pink to red.
  • Form: The most common shapes include round (Cocina) or rod-shaped (bazillen).

The medical laboratory worker also looks for additional characteristics of the sample by looking at the clumps of bacteria on the slide. Examples include:

  • CocinaThey occur singly, in pairs, in groups of four, in clusters, or in chains.
  • bazillenwhich are thick, thin, short, long, or have enlarged spores at one end.
  • When bacteria are present in white blood cells.

The medical lab technician then creates a report and sends it to your doctor.

What are the risks of a Gram stain test?

There is no risk of having a Gram's spot involving a swab or fluid collection of sputum (spit), urine, or feces.

(Video) Microbiology: Gram Staining

There is a very small risk in blood tests. You may have a slight sensitivity or abruiseat the blood draw site, but this usually resolves quickly.

Possible risks of fine needle aspiration include:

  • discomfort or pain.
  • bleeding.
  • Infection.
  • Damage to neighboring buildings.

results and monitoring

What do the results of a Gram stain mean?

Gram stain test results show one of two categories: a negative Gram stain or a positive Gram stain. This is not to be confused with gram negative bacteria or gram positive bacteria.

Gram stain negative

If your test result shows a negative Gram stain or "no organisms seen," it usually means there are too few bacteria to see with the Gram stain method. Bacteria can still be detected by culture if the sample is cultured.

Gram stain positive

If your test result shows a positive Gram stain, it means that bacteria were present in your sample. If your result is positive, it will usually include information about what type of organism was present on the sample slide, including:

  • type of bacteria: Gram-positive or gram-negative.
  • form of bacteria: Around (Cocina) or smallpox (bazillen).
  • Other bacterial properties: Possibly size, relative quantity (number) and/or disposition of the bacteria.
  • other cells: If there are bacteria present in other cells (intracellular) and if there are red or white blood cells.
  • Mushrooms: Gram stains can check for the presence of fungi in the form of yeast or mold. You may need to do further testing to identify the specific type.

This information, along with signs and symptoms and other clinical findings, will help your healthcare provider determine which treatment might be most effective, sometimes before bacterial culture results are available.

When should I know the results of the test?

Gram stains generally give quick results. However, Gram stain results are generally considered preliminary, which means that they cannot always provide a diagnosis on their own. Results of a bacterial culture and/or other tests, e.g. B. Antigen and antibody tests for certain types of bacteria may be needed to confirm a diagnosis.

When do I have to call my doctor?

If you have been diagnosed with an infection and are having problems with your treatment or your infection is not improving, contact your doctor.

(Video) Gram Staining

A note from the Cleveland Clinic

Gram stains are a very common and often painless method of detecting the presence of a bacterial or fungal infection. While waiting for the results of a diagnostic test can be stressful, know that your medical team will support you no matter what the results. Don't be afraid to ask your doctor questions about your test or its results.


What is the purpose of the Gram stain procedure? ›

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 walls. The bacteria are differentiated through a series of staining and decolorization steps.

What is the principle procedure and results of Gram staining? ›

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.

What are the steps of the Gram stain procedure? ›

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 is the purpose of the Gram staining technique quizlet? ›

What is the purpose of Gram Stain? To be able to determine the composition of the cell wall. The advantage of this staining procedure is that those cells that decolorize can be differetiated from the cells that resist decolorization by alcohol.

What are the results of Gram stain? ›

A Gram stain is used, along with a culture of the sample from an infected site, to identify the cause of a bacterial infection. The Gram stain provides preliminary results on whether bacteria are present and the general type, such as the shape and whether they are Gram-positive or Gram-negative.

What is the conclusion of Gram staining? ›

CONCLUSION. Gram stain differentiates bacteria based on the differential staining properties of bacterial cell wall. Bacteria with thick peptidoglycan layer stains blue to purple and are treated as gram positive, while bacteria with thin peptidoglycan layer stains red to pink and are treated as gram negative.

What are the 4 steps of Gram staining quizlet? ›

Steps of gram staining technique:
  • Apply primary stain (crystal violet). All bacteria are stained purple by this basic dye.
  • Apply mordant (Gram's iodine). ...
  • Apply decolorizing agent (ethyl alcohol or ethyl alcohol acetone). ...
  • Apply secondary stain or counterstain (safranin).

How do you perform a Gram stain experiment? ›

There are six basic steps:
  1. Apply a smear of bacteria on to a slide. ...
  2. Add about 5 drops of Hucker's Crystal Violet to the culture. ...
  3. Add about 5 drops of iodine solution to the culture. ...
  4. Tilt slide and decolorize with solvent (acetone-alcohol solution) until purple color stops running. ...
  5. Add about 5 drops of Safranine O.

What is the most important step in the Gram stain process? ›

The thickness of the smear used in the Gram stain will affect the result of the stain. The step that is most crucial in effecting the outcome of the stain is the decolorizing step.

What is the purpose of a simple stain? ›

Simple staining allows one to observe the morphology and arrangement of the bacterial cells. Morphology refers to “form” or shape. We can use terms like spiral, bacilli (rod shaped), and cocci (spherical) to describe cell shapes.

What would be the purpose of doing a Gram stain in a real clinical lab? ›

Bacteria can be easily identified by light microscopy using appropriate staining methods. Various stains are used to visualize and differentiate microorganisms such as the gram staining technique. The gram stain is a common laboratory test that can quickly diagnose the presence of a bacterial infection.

What is the purpose for each gram staining reagent? ›

The first reagent is called the primary stain. Its function is to impart its color to all cells. The second stain is a mordant used to in- tensify the color of the primary stain. In order to es- tablish a color contrast, the third reagent used is the decolorizing agent.

What are the 4 stains used in Gram staining? ›

Reagents needed for Gram staining include:
  • Crystal violet (primary stain) [1]
  • Gram's iodine solution (the mordant) [1]
  • Acetone/ethanol (50:50 v:v) (the decolorizer) [1]
  • 0.1% basic fuchsin solution (the counterstain) [1]
  • Water.
Aug 8, 2022

What are the steps and times of a Gram stain? ›

Gram Staining Instructions
  • Heat fix the slide. ...
  • Stain with Crystal Violet for 1 minute by flooding the slide with stain. ...
  • Apply Iodine solution for 1 minute by flooding the slide with iodine. ...
  • CAREFULLY, decolorize for 3 seconds with Gram Stain Decolorizer by flooding the slide with decolorizer.

What is gram staining short note? ›

In microbiology and bacteriology, Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.

What is the procedure of simple staining? ›

Simple Stain. Simple stains are just that - add one stain to a fixed smear slide, let it sit, rinse it off, let it dry, and view. It is a quick procedure for determining the presence and morphology of bacteria in clinical samples such as stool and discharges.

What is meant by Gram positive? ›

Gram-positive bacteria are bacteria classified by the color they turn in the staining method. Hans Christian Gram developed the staining method in 1884. The staining method uses crystal violet dye, which is retained by the thick peptidoglycan cell wall found in gram-positive organisms.

What is the purpose of staining in microbiology? ›

Why Stain Cells? The most basic reason that cells are stained is to enhance visualization of the cell or certain cellular components under a microscope. Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample.

What is used to stain bacteria? ›

First cells are stained with crystal violet, followed by the addition of a setting agent for the stain (iodine). Then alcohol is applied, which selectively removes the stain from only the Gram negative cells. Finally, a secondary stain, safranin, is added, which counterstains the decolorized cells pink.

What are the 4 types of stain? ›

Simple Staining 2. Differential Staining 3. Gram Staining 4. Acid Fast Staining 5.

Why is the Gram stain so important in clinical microbiology quizlet? ›

The gram stain is useful in a clinical lab because it distinguishes between gram positive and gram negative cells, which aid in identifying a species or narrowing down the possibilities. It allows us to determine the cell arrangement, size, and morphology.

What is simple staining principle and procedure? ›

In simple staining, the bacterial cells are first fixed on a clean, oil-free slide and then flooded with stain (safranin, methylene blue, carbol fuchsin, Crystal violet, etc ). This stain will produce a distinctive contrast between the organism and its background so that we can easily distinguish them.

What is the principle of gram staining quizlet? ›

Gram Stain - principle. - differential stain in which a decolorization (95% alcohol solution) step occurs between the application of 2 basic stains. - Gram positive cell walls are rich in mucopolysaccharides - resist decolorization. - Gram-negative bacteria have a high concentration of lipids - dissolve in decolorizer.

What is the principle of staining techniques? ›

Principle: Staining technique involves electrostatic attraction between ions of opposite charge. Haemotoxylin is a complex with aluminium salt is cationic and acts as a basic dye. It is positively charged and reacts with negatively charged nucleic acids in the nucleus.

What is the purpose of staining? ›

Staining is used to highlight important features of the tissue as well as to enhance the tissue contrast. Hematoxylin is a basic dye that is commonly used in this process and stains the nuclei giving it a bluish color while eosin (another stain dye used in histology) stains the cell's nucleus giving it a pinkish stain.

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 introduction of gram staining? ›

In microbiology and bacteriology, Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.

What are the different types of Gram staining? ›

Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin.

What are the two main types of staining? ›

  • Types of staining techniques.
  • Simple staining.
  • Differential staining.
  • (Use of of single stain)
  • (Use of two contrasting stains)
  • Direct.
  • Indirect.
  • Separation.

Is gram staining a simple stain? ›

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.


1. Gram stain: Procedure and results
(Socially Distant Microbiology)
2. Reporting Staining Results
(Clinical Lab Science Videos)
3. Gram positive and gram negative bacteria (Gram Staining procedure explained)
(Henrik's Lab)
4. Gram's Staining
(Microbiology Mantra)
5. Gram stain results for gram positive bacteria (with examples)
6. Gram Staining: Principle, Procedure and Results
Top Articles
Latest Posts
Article information

Author: Domingo Moore

Last Updated: 01/09/2023

Views: 5912

Rating: 4.2 / 5 (53 voted)

Reviews: 92% of readers found this page helpful

Author information

Name: Domingo Moore

Birthday: 1997-05-20

Address: 6485 Kohler Route, Antonioton, VT 77375-0299

Phone: +3213869077934

Job: Sales Analyst

Hobby: Kayaking, Roller skating, Cabaret, Rugby, Homebrewing, Creative writing, amateur radio

Introduction: My name is Domingo Moore, I am a attractive, gorgeous, funny, jolly, spotless, nice, fantastic person who loves writing and wants to share my knowledge and understanding with you.