The gram-stain tutorial
The Gram-stain is a differential stain that requires the use of at least three chemical reagents applied sequentially to a heat-fixed smear. The first reagent (Crystal Violet) is the primary stain. It gives color to all cells. This is followed by a second reagent (Gram's Iodine), which acts as a mordant and helps the color to stick to the cell wall. In order to establish a color contrast, this is followed by the addition of an acid/alcohol decolorizing reagent (ethyl alcohol, 95%), which may or may not remove the primary stain from the entire cell or just from some of the cell structures. This is a protein-dehydrating agent and a lipid solvent. This is the most critical step. Finally, the counterstain is applied (Safranin), which contrasts in color to the primary stain. After decolorization, if the primary stain is removed, the decolorized cell will accept and assume the counterstain contrasting color (pink-red). If the primary stain is not removed or washed out during decolorization, the counterstain cannot be absorbed and the cell will retain the color of the primary stain (purple-blue).
Thanks to the Gram-stain, the most important differential stain used in Bacteriology, cell types or structures and their arrangements can be distinguished from each other on the basis of the stain that has been retained. Named after Dr. Christian Gram, it separates the bacterial cells into two major groups: Gram-positive and Gram-negative, making it a critical tool in the classification and differentiation of microbes.
Since the best Gram-stained preparations are those made with fresh cultures (<24 hours), as cultures age the organisms tend to lose their ability to retain the primary stain, especially Gram-positive organisms. Therefore, Gram-stains made from cultures that are >24 hours old may appear to be Gram-variable, in that some cells will appear purple, while others will appear red.
Thanks to the Gram-stain, the most important differential stain used in Bacteriology, cell types or structures and their arrangements can be distinguished from each other on the basis of the stain that has been retained. Named after Dr. Christian Gram, it separates the bacterial cells into two major groups: Gram-positive and Gram-negative, making it a critical tool in the classification and differentiation of microbes.
Since the best Gram-stained preparations are those made with fresh cultures (<24 hours), as cultures age the organisms tend to lose their ability to retain the primary stain, especially Gram-positive organisms. Therefore, Gram-stains made from cultures that are >24 hours old may appear to be Gram-variable, in that some cells will appear purple, while others will appear red.
STEP 1: prepare a smear
Use a plastic loop or dropper to smear some specimen on a clean slide. Mix with 1-2 drops of sterile or deionized (DI) water. This method enables cells to adhere to the slide so that they are not washed off during the staining procedure. It is important to insure that shrinking of cells does not occur during the staining process, because artifacts and cell distortion can result. Prepare thin smears so you can visualize the individual cells and their arrangement. It only takes a very small amount of bacterial material to make a good smear. The smear should air dry completely prior to heat-fixing and staining.
Step 2: heat-fix or methanol fix your smear slide
Allow your slide to fully air dry, then fix your slide. For heat fixation, place the slide on a slide warmer for at least 1 minute. You can also place the slide in front of an incinerator for up to 60 seconds or flame your slide through the blue portion of the flame of a Bunsen burner 3x. You can methanol fix a slide by flooding the slide with methanol for 1 minute.
step 3: gram stain procedure
Initially, both Gram-positive AND Gram-negative cells are stained by the Primary Stain, the Crystal Violet. In the second step of the procedure, Gram's Iodine is added to the smear as a mordant to complex with the crystal violet and forms an insoluble complex in Gram-positive cells. At this point, the cell types will both appear purple. The dye-mordant complex will not be removed from Gram-positive bacteria but is leached from Gram-negative cells during the alcohol or acetone (95% ethyl alcohol) in the Decolorization step. Decolorization is the most important step! Decolorization serves as a protein-dehydrating reagent and a lipid solvent. The alcohol increases the porosity of the cell wall by dissolving the lipids in the outer layers of the bacterium. The crystal violet complex can then be more easily removed from the thinner area and less cross-linked area of the peptidoglycan layer. After decolorization, Gram-positive cells still remain purple, but Gram-negative cells are colorless. In the final step, a counterstain, safranin or carbol fuschin, is added to counterstain the Gram-negative cells.
Good stains
A mix of Gram-positive (purple) and Gram-negative (red/pink) organisms is seen in the images here
results of overdecolorization
Overdecolorization results if you leave the decolorizer on for too long. This will result in loss of the primary stain, causing Gram-positive organisms to appear Gram-negative. Here are some examples:
underdecolorization: |
If you rinse the decolorizer off too early, a Gram-negative bacterium might retain some of the crystal violet-iodine complex, giving it a purple, Gram-positive appearance, as seen in the image below of E. coli, a normally Gram-negative bacilli appearing Gram-positive, purple, here.
The best Gram stained preparations are made with fresh cultures <24 hours old. As cultures age, particularly with Gram-positive organisms, they tend to lose their ability to retain the primary stain and may appear to be Gram-variable, so some will appear purple, and others will appear red. Additionally, patients on antibiotics may also have cells displaying Gram-variability, because the antibiotics may change the properties of the cell wall, changing the way that the cells retain stain.
Other situations that cause gram positive bacteria to appear gram negative:
- Antibiotic therapy has been started, such as Penicillin, which damages the cell wall, making it difficult for the cell wall to retain the Crystal Violet stain
- Cell wall has become damaged by lysozyme, a potent enzyme and cytotoxin that "pokes holes" in the cell wall, causing its contents to leak out, making it difficult for the cell wall to retain stain
- Cultures >18-24 hours old, because the cell wall thins, loses its strength, and becomes damaged as the bacteria age and cultures age
- Action of autolytic enzymes, which cause "autodigestion" of the cell and its contents
- Bacteria that have been phagocytosed by phagocytic bacteria (neutrophils, macrophages)
- When only the ends stain and the vacuole in the middle does not hold the stain as well (gives it a "safety-pin" appearance
Precipitate, debris and stain crystallization:
Stain precipitate, cellular debris, and stain crystallization are quite common and need to be recognized. In order to help prevent this, ensure that your slide is completely dried and fixed before staining, and completely dried before applying oil and observing microscopically under the 100x objective. Make sure your reagents stay closed between use. Slightly mix the bottle of reagent before use (stains). Some stains may need to be filtered occasionally to help prevent this from occurring. Also, the thinner the specimen on the slide, the better the stain will be. Sometimes it is helpful to take a spreader slide with a thick specimen to create a thinner smear on your slide.
importance and applications of initial gram stain:
- Rapid, presumptive diagnosis (ex: bacterial meningitis, sepsis, bacteremia)
- Prophylactic or empirical antibiotic therapy may begin earlier in the meantime while cultures are growing out
- Appropriate and/or additional media may be added based on initial Gram stain findings
- Screening of the quality of the specimen submitted
- Quantitation of bacteria and cells seen
- Bacterial morphology, which may aid in diagnosis
Gram-positive (purple) organisms:
Gram-positive cocci in chains: STREPTOCOCCI (Image on the Left)
Gram-positive cocci in pairs: DIPLOCOCCI (Image on the Middle)
Gram-positive organisms stain PURPLE-BLUE in color because they retain the Crystal Violet stain in their thick cell walls. Cocci are SPHERICAL (ROUND) in shape to sightly ovoid or slightly elongated ("lancet").
Streptococcus spp in chains include the following: S. agalactiae (Group B), S. pyogenes (Group A), S. salivarius, S. mutans, S. mitis, S. equi, S. viridans...
Streptococci spp in pairs (diplococci) with elongated cocci, pointed at the ends ("lancet-shape"), are seen in S. pneumoniae, causative agent of bacterial pneumonia.
Enterococcus faecalis (Group D Streptococci) (image on the right), is a Gram-positive cocci seen in pairs and short chains.
Gram-positive cocci in pairs: DIPLOCOCCI (Image on the Middle)
Gram-positive organisms stain PURPLE-BLUE in color because they retain the Crystal Violet stain in their thick cell walls. Cocci are SPHERICAL (ROUND) in shape to sightly ovoid or slightly elongated ("lancet").
Streptococcus spp in chains include the following: S. agalactiae (Group B), S. pyogenes (Group A), S. salivarius, S. mutans, S. mitis, S. equi, S. viridans...
Streptococci spp in pairs (diplococci) with elongated cocci, pointed at the ends ("lancet-shape"), are seen in S. pneumoniae, causative agent of bacterial pneumonia.
Enterococcus faecalis (Group D Streptococci) (image on the right), is a Gram-positive cocci seen in pairs and short chains.
Gram-positive cocci in clusters: STAPHYLOCOCCI (*NOTE: Oftentimes, as seen in the images above, staphylococci are seen not only as grape-like clusters, but also as tetrads (packets of 4), sarcinae (packets of 8), and short chains, singles or pairs that broke off while dividing.)
Gram-positive organisms stain PURPLE-BLUE in color because they retain the Crystal Violet in their thick cell walls. The cocci found in staphylococci are spherical/round and appear in grape-like clusters, or when dividing, found in tetrads (packets of four), sarcinae (packets of 8, 3D).
There are many species of Staphylococcus, including S. aureus, MRSA, S. epidermidis, S. saprophyticus
Gram-positive organisms stain PURPLE-BLUE in color because they retain the Crystal Violet in their thick cell walls. The cocci found in staphylococci are spherical/round and appear in grape-like clusters, or when dividing, found in tetrads (packets of four), sarcinae (packets of 8, 3D).
There are many species of Staphylococcus, including S. aureus, MRSA, S. epidermidis, S. saprophyticus
Gram-positive bacilli (rods)
Gram-positive organisms stain PURPLE-BLUE in color because they retain the Crystal Violet in their thick cell walls. Gram-positive bacilli are shaped like RODS and may be thick, thin, short or long rods. The bacilli may either be spore-forming (ex: Clostridium spp), or non-spore-forming, as seen in the image above. Bacilli (rods) may be observed microscopically as single rods, in pairs, or in chains (streptobacilli).
Gram-positive organisms stain PURPLE-BLUE in color because they retain the Crystal Violet in their thick cell walls. Gram-positive bacilli are shaped like RODS and may be thick, thin, short or long rods. The bacilli may either be spore-forming (ex: Clostridium spp), or non-spore-forming, as seen in the image above. Bacilli (rods) may be observed microscopically as single rods, in pairs, or in chains (streptobacilli).
Gram-positive bacilli (rods)
Gram-positive organisms stain PURPLE-BLUE in color because they retain the Crystal Violet in their thick cell walls. Gram-positive bacilli are shaped like RODS and may be thick, thin, short or long rods. The bacilli may either be spore-forming (ex: Clostridium botulinum, as seen in the image above), or non-spore-forming. C. botulinum is a spore-forming Gram-positive bacilli (rod) that excretes the toxin responsible for a deadly type of food poisoning, botulism.
Gram-positive organisms stain PURPLE-BLUE in color because they retain the Crystal Violet in their thick cell walls. Gram-positive bacilli are shaped like RODS and may be thick, thin, short or long rods. The bacilli may either be spore-forming (ex: Clostridium botulinum, as seen in the image above), or non-spore-forming. C. botulinum is a spore-forming Gram-positive bacilli (rod) that excretes the toxin responsible for a deadly type of food poisoning, botulism.
Gram-positive bacilli (rods): BRANCHING
Branching, Gram-positive bacilli (rods) are indicative of the fungus-like bacteria Nocardia spp or Actinomyces/Actinomycetes spp. as seen in the image on the left, a light, Gram-positive staining organism seen with light microscopy. Propionibacterium spp, Eubacterium spp, Brevibacterium spp are all branching bacteria as well.
Branching, Gram-positive bacilli (rods) are indicative of the fungus-like bacteria Nocardia spp or Actinomyces/Actinomycetes spp. as seen in the image on the left, a light, Gram-positive staining organism seen with light microscopy. Propionibacterium spp, Eubacterium spp, Brevibacterium spp are all branching bacteria as well.
Gram-negative cocci
Gram-negative cocci are spherical (round) bacteria that stain a pink-red color because they retain the Safranin dye in their thin cell walls. Gram-negative cocci in pairs are referred to as DIPLOCOCCI and are found in the Neisseria spp and Moraxella spp. Veillonella spp are anaerobic Gram-negative cocci. |
Gram-negative bacilli (rods)
Gram-negative bacilli (rods) may be observed singly, in pairs or in chains, may be curved (as in Vibrio spp), or "seagull wing-shaped" (as in Campylobacter spp), or pointed at the ends (as in Fusobacterium). They may be short or long, and stain as pink-to-red when observed by Gram-stain microscopically. The image above is that of Escherichia coli. NOTE: There are times when Gram-negative bacilli (rods) will take on a "safety pin-like" appearance, because sometimes cellular structures in the middle of rod do not stain very well.
Gram-negative bacilli (rods) may be observed singly, in pairs or in chains, may be curved (as in Vibrio spp), or "seagull wing-shaped" (as in Campylobacter spp), or pointed at the ends (as in Fusobacterium). They may be short or long, and stain as pink-to-red when observed by Gram-stain microscopically. The image above is that of Escherichia coli. NOTE: There are times when Gram-negative bacilli (rods) will take on a "safety pin-like" appearance, because sometimes cellular structures in the middle of rod do not stain very well.
Gram-negative very short, egg-shaped bacilli: COCCOBACILLI
Gram-negative rods that are very short, oval, egg-shaped bacilli are referred to as coccobacilli. The image above is a Gram-stain of Haemophilus influenzae, a causative agent of bacterial respiratory infection, meningitis, ear infection, and other types of infections. Most Haemophilus spp and some Moraxella spp are coccobacilli.
Gram-negative rods that are very short, oval, egg-shaped bacilli are referred to as coccobacilli. The image above is a Gram-stain of Haemophilus influenzae, a causative agent of bacterial respiratory infection, meningitis, ear infection, and other types of infections. Most Haemophilus spp and some Moraxella spp are coccobacilli.
candida albicans (yeast) in gram stain:
YEAST
Yeast stains purple-blue, or Gram-positive, when observed microscopically as seen in this image of Candida albicans above. Yeast stands out as a large, oval-shaped microbe that may or may not be budding, and that may or may not exhibit pseudohyphae. Other species of Candida and other yeasts stain like this as well.
Yeast stains purple-blue, or Gram-positive, when observed microscopically as seen in this image of Candida albicans above. Yeast stands out as a large, oval-shaped microbe that may or may not be budding, and that may or may not exhibit pseudohyphae. Other species of Candida and other yeasts stain like this as well.
Cryptococcus (type of yeast) in gram stains:
red blood cells:
white blood cells: neutrophils
white blood cells: eosinophils
white blood cells: basophils
white blood cells: monocytes and lymphocytes
Making blood smears for malaria testing:
An example of the end result of the thin smear can be seen on the left side of the slide, with a nice feathered edge. On the right side, a thick smear is seen. For the thick smear, use a pipette to drop 2-3 drops of blood in a circular area on the slide. Allow to fully air dry, and do not fix prior to staining with Quick-Diff. Air dry for 4-24 hours.
plasmodium vivax
plasmodium falciparum
plasmodium ovale
plasmodium malariae
babesia spp
The Stains
Bacteria are slightly negatively charged, which produces an attraction between cationic chromophores and an organism.
BASIC DYES: Methylene Blue (cationic chromophores), Malachite Green, Crystal Violet, Safranin
-Carry a positive charge
-Attracted to the opposite charge (negative) on the bacterial cell wall
-Basic dye molecules form ionic bonds to the negative charges on the surface of the bacterial cell wall
ACIDIC DYES: Eosin (anionic chromophores), Nigrosin, India Ink, Congo Red,
-Carry a negative charge
-Repelled by a like charge (example: repelled by the negative charge on the bacterial cell wall)
-They color the background surrounding the cell
-The cell is seen in light outline against a dark background
Crystal violet stain, in aqueous solutions, dissociates, or breaks apart into CV+ and Cl- ions. The CV+ ions are attracted to and interact with the negative ions in the bacterial cell wall, staining it purple.
Iodine, I-, interacts with CV+ and forms a large complex, helping the stain to color the cell wall.
Decolorizer interacts with the lipids in the cell membrane, eating away at the Gram-negative lipopolysaccharide layer, and leaving the peptidoglycan layer exposed to receive the counterstain.
Counterstain Safranin or Carbol Fuschin is positively charged,
Pleomorphism: Irregularity of form, demonstrating several different shapes/irregular, and variable staining patterns (mix of pink and purple): Examples: Corynebacterium, Actinomyces, Proprionibacterium are just a few examples of bacteria that can stain Gram-variable because they have fragile cell walls that break easily during cell division resulting in Gram-variable staining. In culture, Bacillus and Clostridium spp may have some cells that appear to stain Gram-negative, even though they are really Gram-positive, because during growth, there is a reduction of the peptidoglycan layer in the cell wall thickness. In all cultures >24 hours, any bacterium type may begin to stain Gram-variable due to age.
Metachromic granules: distinct reddish-purple granules that appear within cells stained with methylene blue (masses of volutin, a polymetaphosphate)
Palisade arrangement: Side-by-side, parallel arrangement of rod-shaped bacilli (coryneform)
Vacuoles: spaces in the cell where nutrients (sugars), gases, pigments, enzymes are stored
BASIC DYES: Methylene Blue (cationic chromophores), Malachite Green, Crystal Violet, Safranin
-Carry a positive charge
-Attracted to the opposite charge (negative) on the bacterial cell wall
-Basic dye molecules form ionic bonds to the negative charges on the surface of the bacterial cell wall
ACIDIC DYES: Eosin (anionic chromophores), Nigrosin, India Ink, Congo Red,
-Carry a negative charge
-Repelled by a like charge (example: repelled by the negative charge on the bacterial cell wall)
-They color the background surrounding the cell
-The cell is seen in light outline against a dark background
Crystal violet stain, in aqueous solutions, dissociates, or breaks apart into CV+ and Cl- ions. The CV+ ions are attracted to and interact with the negative ions in the bacterial cell wall, staining it purple.
Iodine, I-, interacts with CV+ and forms a large complex, helping the stain to color the cell wall.
Decolorizer interacts with the lipids in the cell membrane, eating away at the Gram-negative lipopolysaccharide layer, and leaving the peptidoglycan layer exposed to receive the counterstain.
Counterstain Safranin or Carbol Fuschin is positively charged,
Pleomorphism: Irregularity of form, demonstrating several different shapes/irregular, and variable staining patterns (mix of pink and purple): Examples: Corynebacterium, Actinomyces, Proprionibacterium are just a few examples of bacteria that can stain Gram-variable because they have fragile cell walls that break easily during cell division resulting in Gram-variable staining. In culture, Bacillus and Clostridium spp may have some cells that appear to stain Gram-negative, even though they are really Gram-positive, because during growth, there is a reduction of the peptidoglycan layer in the cell wall thickness. In all cultures >24 hours, any bacterium type may begin to stain Gram-variable due to age.
Metachromic granules: distinct reddish-purple granules that appear within cells stained with methylene blue (masses of volutin, a polymetaphosphate)
Palisade arrangement: Side-by-side, parallel arrangement of rod-shaped bacilli (coryneform)
Vacuoles: spaces in the cell where nutrients (sugars), gases, pigments, enzymes are stored
Bacterial structure and cell wall:
Gram-positive organisms:
The cell wall of Gram-positive bacteria is THICK and contains a thick, mesh-like layer of peptidoglycan. This layer of peptidoglycan (murein) accounts for about 50-60% of the bacterial cell envelope, or cell wall and surrounds the cell membrane. As a result of this thick layer of peptidoglycan, the initial stain in the Gram-stain, crystal violet, is retained (trapped). The second stain, iodine, helps keep the crystal violet stain trapped in the thick cell because it serves as a mordant and complexes with the crystal violet. The thick layer of peptidoglycan and the mesh-like structure can be difficult for some antibiotics to penetrate, making some infections with Gram-positive organisms difficult to treat or eradicate.
Gram-negative organisms:
The cell wall of Gram-negative bacteria contains only a THIN layer of peptidoglycan, accounting for simply about 10% of the cell envelope, or cell wall. Because of this, they do not retain the primary crystal violet stain and are counter-stained a pink-red color by safranin or carbol-fuschin. The rapid decolorization with ethanol or acetone-alcohol rinses the crystal violet out of the cell completely, and it is counter-stained with safranin or carbol-fuschin. It also contains lipopolysaccharide (LPS) sugars, which contain the potent O antigen, which upon release when the cell bursts or is destroyed by antibiotics or the immune system, is responsible or many of the drastic and severe symptoms patients may experience when they have an infection with a Gram-negative bacterium. Symptoms may include such things as fever, shock, drop of blood pressure, and sepsis.