Are acid-fast bacteria Gram-positive or Gram-negative?

Are acid-fast bacteria Gram-positive or Gram-negative?

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Both procedures involve using a stain, decolorising agent, and then a second stain. I am tempted to conclude that acid-fast bacteria, which resist decolorisation (eg. by strong carbol fuchsin), also resist decolorisation in the Gram staining test (eg. by gentian violet) and are thus Gram-positive. After all, there are much fewer acid-fast bacteria than Gram-positive bacteria.

Is this line of thinking incorrect?

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Taxonomic Classification:

The most salient acid-fast bacteria are Mycobacterium spp., which are part of a phylum of gram-positive bacteria called Actinobacteria. This classification is based on 16S rRNA sequencing. However, based on genetic conservation, this paper makes the claim that they are more closely related to gram negative bacteria.

Gram Reaction:

Acid-fast bacteria are characterized by cell walls rich in mycolic acids. This characteristic causes both poor stain penetration and high stain retention which forms the basis of acid-fast stains. However, it also means that crystal violet will not penetrate the cell wall efficiently. Thus their gram reaction is variable and they generally stain light purple. The result is dependent on technique as well as mycolic acid content variation between genera (Nocardia spp, for example, are less acid-fast and stain gram positive). Needless to say, their cell walls don't fit well with the classic dichotomy of thin versus thick peptidoglycan which the gram stain differentiates between.

Example Gram Stains:

For comparison, the left image is of Bacillus subtilis, a gram positive, non-acid-fast bacilli. In the middle is M. phlei and on the right is M. smegmatis, both of which are acid-fast but show a weakly positive gram reaction. Images from Napa Valley College.

B. subtilis is again on the left for comparison. In the middle is M. fortuitum staining positive but more weakly than B. subtilis. On the right is N. astroides which is partially acid-fast but stains strongly gram positive. Images from Microbe Canvas.

Staining Procedures for Detecting Bacteria

When a single staining-reagent is used and all cells and their structures stain in the same manner, the procedure is called simple staining procedure.

This procedure is of two types – positive and negative (Fig. 17.5). In positive staining, the stain (e.g., methylene blue) is basic (cationic) having positive charge and attaches to the surface of object that is negatively charged.

In negative staining, the stain (e.g., India ink, nigrosin) is acidic (anionic) having negative charge and is repelled by the object that is negatively charged, and thus fills the spaces between the objects resulting in indirect staining of the object.

2. Differential Staining Procedure:

When more than one staining reagents are used and specific objects (e.g., specific microorganisms and/or particular structure of a microorganism) exhibit different staining reactions readily distinguishable, the procedure is called differential staining.

The most widely used differential staining in microbiology are Gram-staining and acid-fast staining. Acid-fast staining is especially useful in identifying Mycobacterium tuberculosis, the causative agent of tuberculosis.

A. Separation of Microbes into Groups:

A Danish scholar Christian Gram in 1884 devised a differential staining procedure which differentiates bacteria into gram-positive and gram-negative. This procedure is called Gram- staining technique. This technique has experienced numerous modifications from time to time and proves to be valuable for staining smears of pure cultures of bacteria.

The staining procedure (Fig. 17.6) is as follows:

I. A thin film of young culture (smear) is fixed on a clean slide.

II. The smear is stained for one minute with ammonium oxalate crystal violet. This stain sometimes yields over stained preparations in which certain gram-negative organisms (e.g. Gonococcus) also retain the stain. If this trouble is encountered, lesser amount of crystal violet should be used.

III. The slide is washed in tap water for not more than 2 seconds to remove excess stain.

IV. The slide is immersed for one minute in Lugol’s iodine solution. The bacteria become deeply stained and appear deep purple in colour due to crystal violet-iodine-complex formation.

V. The slide is washed in tap water and blot-dried.

VI. The slide is gently agitated for 30 seconds in 95% ethyl alcohol and blot-dried, gram-negative bacteria lose their stain in this step (i.e. decolourize). However, the gram- positive ones retain deep purple colour.

VII. The slide is now counterstained for 10 seconds in the safranin solution.

VIII. The slide is washed in tap water, dried and examined.

Gram-positive bacteria: deep purple (blue) gram-negative bacteria: pink (red).

Although different explanations have been given to answer why bacteria respond differently to the Gram-stain, it seems likely that the answer is related to the physical nature of their cell walls as when cell wall is removed from gram-positive bacteria, they become gram-negative. The peptidoglycan appears to act as a permeability barrier preventing loss of crystal violet-iodine-complex.

When gram-positive bacteria are treated with destaining agent (alcohol), the alcohol is thought to dehydrate the thick layer of peptidoglycan resulting in shrinkage of pores of peptidoglycan. Shrinkage of peptidoglycan pores prevents crystal violet-iodine-complex from escaping and the bacteria remain deep purple.

In contrast, peptidoglycan is very thin in gram-negative bacteria, not as highly crossed-linked as is in gram-positive ones, and has larger pores. Alcohol, therefore, readily penetrates the lipid-rich outer layer of the cell wall and extracts enough lipid thus increasing the porosity further.

For these reasons, alcohol more readily removes the deep purple crystal violet-complex from gram-negative bacteria and the latter become decolourized.

1. Fresh and young culture (less than 24 hours old) should always be used to avoid misleading results because old cultures of gram-positive bacteria tend to decolourize more rapidly and show gramnegativeness.

2. Smears should be thin and uniform to avoid over populated bacteria.

3. During heat-fixing, excessive heating should be avoided.

4. Over decolourization should be avoided.

(ii) Acid-Fast Staining:

The acid-fast stain is a differential stain developed first by Paul Ehrlich in 1882 and later on modified by Ziehl- Neelsen, and is in use even today by microbiologists. Majority of the bacteria are stained with simple stain and Gram-stain but certain bacteria do not do so because they have waxy components of the cell wall, hence their cell wall has limited permeability.

Such bacteria belong to genera like Mycobacterium and Nocardia and are stained by acid-fast stain the latter is used to identify Mycobacterium tuberculosis and M. leprae, the pathogens responsible for tuberculosis and laprosy, respectively.

The acid-fastness property of these bacteria is correlated with high lipid contents, which makes them difficult to stain. Hence for staining of these bacteria heating with strong dye is required. Once the acid-fast bacteria are stained, it is difficult to decolourize them even with acid and alcohol. Moreover, acid-fast staining serves also as good identification tool for a number of harmless saprophytic bacteria.

The reagents are prepared as given below:

Phenol (heat melted crystals) – 5.0 ml

The basic fuchsin is dissolved in ethanol and phenol is dissolved in water.

These two are mixed and kept for several days before filter and use:

Decolourising solvent (acid-alcohol):

Hydrochloric acid (conc.) – 3.0 ml

Methylene blue chloride – 0.3 g

The staining procedure (Fig. 17.7) is as follows:

I. A thin film of young culture (smear) is heat-fixed and air-dried on a clean slide.

II. The smear is now flooded with carbol fuchsin.

III. The slide is steamed over boiling water for 3-5 minutes More stain is added time to time to prevent smear from becoming dry.

IV. Slide is cooled and washed with distilled water until no colour appears from the smear.

V. Smear is decolourized with decolourising solvent (acid-alcohol) for 15-20 seconds. Some bacterial cells appear red (faint pink) in colour, while others decolourize. Slides arc washed with distilled water.

VI. Smears are now counterstained with methylene blue for 1-2 minutes and washed with distilled water.

VII. Slides are blot-dried with bibulous paper and examined directly under oil-immersion.

Acid-fast bacteria appear red.

Non-acid-fast bacteria appear blue.

Acid-fast staining helps classifying bacteria into two groups: acid-fast and non-acid-fast. Acid fast bacteria, particularly those in the genus Mycobacterium, do not bind simple stains but when stained by heating with a mixture of carbol fuchsin (basic fuchsin + phenol) they retain carbol fuchsin (the primary stain) after washing even with strong acid.

Once basic fuchsin penetrates with the aid of heat and phenol, cells of acid-fast bacteria are not easily decolourized by an aid-alcohol wash and hence remain red.

This is due to the quite high lipid content of cell walls of acid-fast bacteria in particular, mycolic acid (a group of branched chain hydroxy lipids) appears responsible for acid-fastness. The nonacid-fast bacteria get decolourized after washing with acid-alcohol they retain the counterstain methylene blue hence appear blue.

1. If necessary, more carbol fuchsin stain should be added to avoid its evaporation and dryness.

2. Carbol fuchsin stain should be prevented from heating to avoid “messy” preparations.

3. Over decolourization of the smear should be avoided.

B. Visualization of Various Structures:

(i) Endospore Staining:

Bacteria in the genera Bacillus and Clostridium form an exceptionally resistant structure capable of surviving for long periods in an unfavourable environment. This dormant structure is called an endospore since it develops within the cell. Endospore morphology and location vary with species and often are valuable in identification. Endospores are not easily stained well by most dyes.

Considerable amount of heating is required in order to make the stain penetrate the spore-coat, a thick wall primarily responsible for endospore resistance. But once stained, they strongly resist decolourization. This property is the basis of endospore staining techniques.

However, there are two staining procedures used by microbiologists to stain the endospores. These methods are the Schaeffer-Fulton method and Dorner method. For convenience, Schaeffer-Fulton method is given here.

The staining procedure of endospore (Fig. 17.8) by Schaeffer-Fulton method is as follows:

i. A thin film of young culture (smear) of endosporous bacteria is fixed on a clean slide.

ii. The smear is heat-fixed on to the slide by gentle warming.

iii. Smear is covered with the solution of malachite green which is a very strong stain that can penetrate the spore-coat of an endospore.

iv. The slide is kept on a suitable stand and heated with steam from below for 5 minutes. If the stain dries up during heating, more stain is added to the smear from time to time as per requirement.

v. The slide is washed gently under tap water.

vi. The slide is counter-stained with safrain for about 30 seconds.

vii. It is then washed with distilled water and dried with blotter.

viii. The smear is observed under oil immersion.

The endospore appears green while rest of the cell appears red.

Endospores are extremely resistant due to their thick wall, the spore coat. The spore coat does not take the stain easily. Malachite green, however, penetrates the spore coat of endospore after considerable heating. Once stained, the endospore does not decolourizes easily hence appears green even after washing. In contrast, the counter stain fails entering the endospore but stains rest of the cell content that appears red.

(ii) Flagella Staining:

Many bacteria are motile due to the presence of flagella that originate in the cytoplasm and project out from the cell wall. Bacterial flagella are fine, threadlike organelles that are so slender (about 10 to 30 nm in diameter) that they can only be observed directly using electron microscope.

To observe flagella with the light microscope, the thickness of flagella is increased by coating them with mordants like tannic acid and potassium alum, and they are stained with pararosaniline (Leifson’s method) or basic fuchsin (Gray’s method).

Flagella staining procedures provide taxonomically valuable informations (e.g., presence, distribution pattern, number) which are used in the identification and classification of bacteria.

However, Gray’s method of flagella staining is as follows:

The reagents are prepared as given below:

Tannic acid (20% aqueous solution) – 2.0 ml

Potassium alum (saturated aqueous solution) – 5.0 ml

Mercuric chloride (saturated aqueous solution) – 2.0 ml

Basic fuchsin (3%) in 95% alcohol – 0.4 ml

Solutions A and B are prepared by mixing their ingredients less than 24 hour before using and are stored in coloured bottles. Both solutions separately may be kept indefinitely.

I. Bacteria are grown on a suitable broth. If it is a solid medium, small quantity of 1% peptone is added in which the bacteria swim.

II. Medium is removed by centrifugation to obtain the pellet.

III. The pellet is suspended in 10% formaline to produce a light, faint turbidity, and for the prevention of flagella.

IV. The loopful of suspension is transferred on to a new greese-free slide and spread to prepare a thin film of young culture (smear).

V. The smear is flooded with freshly filtered mordant (solution A) and allowed to act 8-10 minutes.

VI. The smear is washed with a gentle stream of distilled water.

VII. The smear is flooded with freshly filtered basic fuchsine stain (solution B) and allowed to stand 5 minutes without heating.

VIII. The smear is gently washed with distilled water and air-dried.

IX. The slide is examined directly under oil-immersion.

Flagella stained red are observed.

1. Especially cleaned greese-free slides should be used.

2. Smears should not be heat-fixed.

3. Smears should not be blot-dried.

4. Reduced illumination should always be used in the microscope to observe flagella clearly.

Bacterial capsules are more easily confused with artifacts than any other structure pertaining to the organisms. Inasmuch as capsules sometimes show merely as unstained areas around the bacterial cells, there is a temptation to call any such surrounding area a capsule very often, however, they merely represent the tendency of a lightly stained surrounding medium to retract from bacterial cells on drying.

For this reason, the best way to demonstrate capsules is actually to stain them by some procedure which differentiates them from the bacterial cells itself. Though there are several methods of staining to accomplish this, the procedure of Anthony’s method is much simpler. E.E. Anthony devised this method for capsule staining in 1931.

However, Anthony’s method for capsule staining is the following:

The reagents used in the procedure are the following:

Crystal violet aqueous (85% dye content) – The stain

Copper sulfate aqueous (20% CuSO4.5H2O) – Decolourizing agent as well as counter stain Distilled water.

The staining procedure (Fig. 17.9) is as follows:

I. Two loopfuls of young culture of bacteria (e.g., 36-48 hour milk cultures of Klebsiella pneumoniae) is put on a clean glass slide.

II. A heavy smear is prepared and air-dried.

III. Smear is flooded with crystal violet stain for 2 minutes.

IV. The stain is washed off with copper sulfate (20%)

V. The stain is washed off with copper sulfate (20%).

VI. Copper sulfate is drained and the smear is gently blot-dried.

VII. The slide is examined directly under oil-immersion.

The bacterial cells appear dark blue, encircled by blue violet coloured capsule indicating that the bacteria are capsulated.

Crystal violet aqueous (85% dye content) is the primary stain, and on its application both the capsular material and the bacterial cell wall take the colour of the stain and appear dark blue. But the capsule being non-ionic fails to absorb the primary stain, while the cell wall being ionic absorbs it.

Copper sulfate aqueous (20%) acts both as a decolourizing agent as well as a counter stain. On application, the copper sulfate first decolourizes, the capsule by removing crystal violet and then imparts blue violet colour to it. As a result, the capsule finally appears blue violet in contrast to the dark blue colour of the bacterial cell.

1. Heavy smear should be prepared to counter the removal of bacterial cells during washing.

2. Smear should not be heat-fixed because the heating results in shinkage which may create a clear zone surrounding the cell that is an artifact and that can be mistaken for the capsule.

3. Washing off the smear with water should be avoided because the capsular materials are water soluble and may be dislodged and removed with water.

Acid-fast bacteria cause tuberculosis in humans and also in the animal. It is a zoonotic disease. It causes severe illness in poor people. It produces disease of the respiratory tract as it is an aerobic bacterium. Pathogenicity of this bacterium includes the presence of thick lipid layer around the cell wall that suppresses the immune system of the host. It is a slow-growing bacterium so the immune system didn’t recognize the division of bacteria. It forms the caseinous tubercle in the lungs. These tubercles burst and release bacteria in surroundings. This tubercle may travel from one place to another and produce tuberculosis of the bone.

They are fastidious bacteria. These bacteria can’t grow on simple media. For the culturing, LJ media is present to grow.

But rapid diagnostic tools are also present to diagnose this disease. Chest X-ray is used to detect the presence of tubercle formation in the lungs. PCR can be used to diagnose this disease.

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The Author is (currently) an autodidactic student of Industrial and Environmental microbiology, who is sick of people assuming all microbiology should be medical in nature, and who would really like to be allowed to go to graduate school one of these days now that he's finished his BS in Microbiology (with a bonus AS in Chemistry). He also enjoys exploring the Big Room (the one with the really high blue ceiling and big light that tracks from one side to the other every day) and looking at its contents from unusual mental angles. View all posts by Epicanis

Staining Techniques for Recognition of Bacteria

The following points highlight the top two staining techniques for recognition of bacteria. The techniques are: 1. Gram Stain Method 2. Ziehl Neelsen Method.

Staining Technique # 1. Gram Stain Method:

Before staining, the position of the smear on the slide should be confirmed by making a scratch with the help of a needle or sharp glass slide, NEVER with the fingernail which may become contaminated the slide should be placed in such as way that the smear is on the upper­most position on the staining rack or on glass rods levelled on the laboratory sink.

i. Cover the fixed smear with crystal violet or oxalated gentian violet stain which is allowed to act for 3 minutes

ii. Pour off excess of the stain wash away the remaining crystal violet stain with Gram’s iodine and allow the Gram’s iodine to act for 2 minutes

iii. Decolorize with alcohol for about 5 seconds drop by drop till the violet colour is removed from the film

v. Counterstain with basic fuchsin or safranin stain for 30 seconds

vi. Wash with tap water, blot with blotting paper, dry in air and examine the smear under oil immersion objective (Fig. 3.3).

On the basis of Gram stain, bacteria are mainly grouped into: (1) Gram-positive bacteria and Gram-negative bacteria. Gram-positive bacteria retain the gentian violet in spite of decolourisation by alcohol and stained violet, whereas Gram-negative bacteria are decolorized by alcohol, loose the violet stain completely and are stained pink by the counterstain.

The clinician can choose antibiotics effective against any of the groups (Table 3.1).

Principles of Gram Stain:

Gram-positive organisms retain the basic dyes at higher hydrogen ion concentration (pH 2-3) than the Gram-negative bacteria. The iodine treatment enhances the cytoplasmic acid character which retains strongly the basic dye. Hence iodine acts as mordant. A dye iodine complex or “lake” is formed within the cell, which is soluble in alcohol used as decolouriser. This complex diffuses out of Gram-negative bacilli.

Gram-reaction depends upon a difference in the permeability of the cell wall or the cytoplasmic membrane and the presence of a specific magnesium ribonucleate protein complex in the cell. Gram-positive staining colours the whole cell including the cell wall. If the magnesium ribonucleate is removed by autolysis or the cell wall is ruptured, the Gram-positive bacilli will become Gram-negative.

Staining Technique # 2. Ziehl Neelsen Method:

i. Flood or fully cover the slide with carbol fuchsin and heat gently with spirit lamp from below till steam rises. Allow the stain to act for 5 minutes

iii. Decolorize with 20% sulphuric acid for 1 minute till the yellowish brown or pink colour appears

v. Counterstain with methylene blue for 15 seconds ‘

vi. Wash, blot, dry and mount under oil immersion objective.

Acid fast bacilli are stained pink or red against the blue-back ground counterstain (Fig. 3.4).

Special Staining Methods:

Albert or Neisser method is used to demonstrate metachromatic granules whereas India Ink method and Leifson’s method are used to observe capsules and flagella of bacteria, respectively spores are stained by Gram’s method Or acid-fast stain, or malachite green stain.

Principles of Ziehl Neelsen Stain:

Some bacteria like tubercle bacilli cannot be easily stained by Gram stain because of the wax-like mycolic acid (fatty acid) in their cell wall they can be stained with a strong reagent (basic fuchsin in aqueous 5% phenol), applied with heat but subsequently they resist decolourisation by strong acid (20% sulphuric acid) and are stained pink or red.

Hence they are called “Acid fast bacilli” (AFB). Decolorized non-Acid fast organisms are counterstained with methylene blue.

Difference Between Gram Stain and Acid Fast


Gram stain refers to a staining technique for the preliminary identification of bacteria, in which a violet dye is applied, followed by a decolorizing agent and then a red dye while acid-fast stain refers to a differential stain used to identify acid-fast organisms such as members of the genus Mycobacterium. Thus, this is the main difference between Gram stain and acid fast stain.


Gram stain is a common technique used to characterize bacteria into two large groups while acid-fast stain is the technique used to differentiate Gram-positive bacteria.

Type of characterization

While gram stain characterizes bacteria with different types of cell walls, acid-fast stain characterizes bacteria with mycolic acid in the cell wall. Hence, this is another difference between Gram stain and acid fast stain.

Primary Stain

The primary stain used in Gram stain is crystal violet while the primary stain used in acid-fast stain is carbofuchsin.


Mordant is also a difference between Gram stain and acid fast stain. Iodine is used by the Gram stain as a mordant while no mordant is used in the acid-fast stain.


One other difference between Gram stain and acid fast stain is the counterstain. Safranin is the counterstain used in Gram stain while methylene blue is the counterstain used in the acid-fast stain.


Furthermore, Gram-staining differentiates bacteria as Gram-positive and Gram-negative while acid-fast stain differentiates Gram-positive bacteria as acid-fast and non-acid-fast bacteria. So, this is another difference between Gram stain and acid fast stain.


Moreover, in Gram stain, Gram-positive bacteria appear in blue color while Gram-negative bacteria appear in red color. In contrast, in acid-fast stain, acid-fast bacteria appear in red color while non-acid-fast bacteria appear in blue color.


Gram stain is one of the main bacteriological staining method used to distinguish between Gram-positive and Gram-negative bacteria. It is a differential staining technique which uses crystal violet as the primary stain and safranin as the counter stain. In comparison, the acid-fast stain is another differential staining method used to characterize Gram-positive bacteria, especially the members of the genus Mycobacterium. It uses carbofuchsin as the primary stain and methylene blue as the counter stain. However, the main difference between Gram stain and acid fast stain is the type of characterization.


1. “Staining Microscopic Specimens|Microbiology.” Lumen Learning, Lumen, Available Here.

Image Courtesy:

1. “Gram stain 01” By Y tambe – Y tambe’s file (CC BY-SA 3.0) via Commons Wikimedia
2. “Mycobacterium tuberculosis Ziehl-Neelsen stain 02” By CDC/Dr. George P. Kubica – CDC-PHIL ID #5789 (Public Domain) via Commons Wikimedia

About the Author: Lakna

Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things

8 Difference Between Gram Staining And Acid-fast Staining

Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet.

The Gram Staining Steps Include:

  • Add several drops of crystal violet to the smear and allow it to sit for 1 minute. Rinse the slide with water.
  • Add several drops of iodine to the smear and allow it to sit for 1 minute. Rinse the slide with water.
  • Add drops of ethanol one at a time until the runoff is clear. Rinse the slide with water.
  • Add several drops of safranin to the smear and allow it to sit for a minute.
  • Air dry, blot dry and observe under microscope.
  • Gram-negative cells will be stained pink by the safranin. This dye has no effect on Gram-positive cells, which remain purple.

What You Need To Know About Gram Staining

  • Gram staining is a technique for the preliminary identification of bacteria in which a violet dye is applied, followed by a decolorizing agent and then a red dye.
  • Gram staining characterizes bacteria with different types of cell walls.
  • The primary stain used in Gram stain is crystal violet.
  • Iodine is used by the gram stain as a mordant.
  • Safranin is the main counterstain used in gram staining.
  • Gram-staining differentiates bacteria into gram-negative and gram-positive bacteria.
  • In Gram stain, Gram-positive bacteria appear in blue color whereas Gram-negative bacteria appear in red color.

Difference Between Gram Positive and Gram Negative Bacteria

Christian Gram, a Danish Physician in 1884 developed a staining technique to distinguish two types of bacteria. The two categories of bacteria based on gram staining are Gram positive bacteria and Gram negative bacteria. Bacteria are first stained with crystal violet or gentian violet. All bacterial cells will stain blue or purple colour with crystal violet solution. Then the bacterial cells are treated with iodine solution (Lugol’s iodine) solution and washed with alcohol (de-staining solution). Those bacteria which retain the blue or purple colour of crystal violet are called Gram positive bacteria and those bacteria which loose the colour of crystal violet after washing with de-staining solution is called Gram Negative bacteria.

Gram negative bacteria are later stained with safranin or fuchsin for observation under microscope. Gram negative bacteria after safranin or fuchsin staining will appear red or pink colour. Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting the properties of peptidoglycan. Gram staining method is useful in differentiating majority of bacterial species into two broad categories. Even though all bacterial species cannot be differentiated based on gram staining technique, this method has immense application in clinical diagnostics and biological researches.

Similarities between Gram Positive and Gram Negative Bacteria

Ø Both are bacterial cells

Ø Both groups are prokaryotic

Ø Both lack membrane bounded organelles

Ø Both groups have covalently closed circular DNA as the genetic material

Ø Both groups contain extra-chromosomal genetic materials (plasmids)

Gram Positive (blue) and Gram Negative (Pink) Bacteria (source wikipedia)

Ø Both groups possess capsule

Ø In both groups, cell wall is made up of peptidoglycan

Ø In both groups, cytoplasm is surrounded by lipid bilayer with many membrane spanning proteins

Ø Both gram-positive and gram-negative bacteria commonly have a surface layer called an S-layer

Ø Both groups of bacteria undergo genetic recombination through transformation, transduction and conjugation

Ø Both groups undergo binary fission as a mode of asexual reproduction

Ø Both groups contain many flagellated and non-flagellated species

Ø Both gram positive and gram negative bacteria are inhibited by antibiotics (their sensitivity varies)

Ø Both groups includes flagellated (motile) and non-flagellated (non-motile) forms

Learn more…

Difference between Gram Positive Bacteria and Gram Negative Bacteria

Macrophage cytokine responses to commensal Gram-positive Lactobacillus salivarius strains are TLR2-independent and Myd88-dependent

The mechanisms through which cells of the host innate immune system distinguish commensal bacteria from pathogens are currently unclear. Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) expressed by host cells which recognize microbe-associated molecular patterns (MAMPs) common to both commensal and pathogenic bacteria. Of the different TLRs, TLR2/6 recognize bacterial lipopeptides and trigger cytokines responses, especially to Gram-positive and Gram-negative pathogens. We report here that TLR2 is dispensable for triggering macrophage cytokine responses to different strains of the Gram-positive commensal bacterial species Lactobacillus salivarius. The L. salivarius UCC118 strain strongly upregulated expression of the PRRs, Mincle (Clec4e), TLR1 and TLR2 in macrophages while downregulating other TLR pathways. Cytokine responses triggered by L. salivarius UCC118 were predominantly TLR2-independent but MyD88-dependent. However, macrophage cytokine responses triggered by another Gram-positive commensal bacteria, Bifidobacterium breve UCC2003 were predominantly TLR2-dependent. Thus, we report a differential requirement for TLR2-dependency in triggering macrophage cytokine responses to different commensal Gram-positive bacteria. Furthermore, TNF-α responses to the TLR2 ligand FSL-1 and L. salivarius UCC118 were partially Mincle-dependent suggesting that PRR pathways such as Mincle contribute to the recognition of MAMPs on distinct Gram-positive commensal bacteria. Ultimately, integration of signals from these different PRR pathways and other MyD88-dependent pathways may determine immune responses to commensal bacteria at the host-microbe interface.

Conflict of interest statement

The authors declare no competing interests.


Characterisation and validation of in…

Characterisation and validation of in vitro generated mouse BMDMs. BMDMs were stained with…

Clec4e is the most upregulated…

Clec4e is the most upregulated PRR in L. salivarius UCC118 co-cultured BMDMs. (…

TNF-α response triggered by L.…

TNF-α response triggered by L. salivarius UCC118 and TLR2 agonist (FSL-1) is partially…

Preparation and Staining for Other Microscopes

Samples for fluorescence and confocal microscopy are prepared similarly to samples for light microscopy, except that the dyes are fluorochromes. Stains are often diluted in liquid before applying to the slide. Some dyes attach to an antibody to stain specific proteins on specific types of cells (immunofluorescence) others may attach to DNA molecules in a process called fluorescence in situ hybridization (FISH), causing cells to be stained based on whether they have a specific DNA sequence.

Sample preparation for two-photon microscopy is similar to fluorescence microscopy, except for the use of infrared dyes. Specimens for STM need to be on a very clean and atomically smooth surface. They are often mica coated with Au(111). Toluene vapor is a common fixative.

Think about It

  • What is the main difference between preparing a sample for fluorescence microscopy versus light microscopy?

Clinical Focus: Nathan, Resolution

This example concludes Nathan’s story that started in The Properties of Light, Instruments of Microscopy, and above.

From the results of the Gram stain, the technician now knows that Nathan’s infection is caused by spherical, gram-positive bacteria that form grape-like clusters, which is typical of staphylococcal bacteria. After some additional testing, the technician determines that these bacteria are the medically important species known as Staphylococcus aureus, a common culprit in wound infections. Because some strains of S. aureus are resistant to many antibiotics, skin infections may spread to other areas of the body and become serious, sometimes even resulting in amputations or death if the correct antibiotics are not used.

After testing several antibiotics, the lab is able to identify one that is effective against this particular strain of S. aureus. Nathan’s doctor quickly prescribes the medication and emphasizes the importance of taking the entire course of antibiotics, even if the infection appears to clear up before the last scheduled dose. This reduces the risk that any especially resistant bacteria could survive, causing a second infection or spreading to another person.

Microscopy and Antibiotic Resistance

As the use of antibiotics has proliferated in medicine, as well as agriculture, microbes have evolved to become more resistant. Strains of bacteria such as methicillin-resistant S. aureus (MRSA), which has developed a high level of resistance to many antibiotics, are an increasingly worrying problem, so much so that research is underway to develop new and more diversified antibiotics.

Fluorescence microscopy can be useful in testing the effectiveness of new antibiotics against resistant strains like MRSA. In a test of one new antibiotic derived from a marine bacterium, MC21-A (bromophene), researchers used the fluorescent dye SYTOX Green to stain samples of MRSA. SYTOX Green is often used to distinguish dead cells from living cells, with fluorescence microscopy. Live cells will not absorb the dye, but cells killed by an antibiotic will absorb the dye, since the antibiotic has damaged the bacterial cell membrane. In this particular case, MRSA bacteria that had been exposed to MC21-A did, indeed, appear green under the fluorescence microscope, leading researchers to conclude that it is an effective antibiotic against MRSA.

Of course, some argue that developing new antibiotics will only lead to even more antibiotic-resistant microbes, so-called superbugs that could spawn epidemics before new treatments can be developed. For this reason, many health professionals are beginning to exercise more discretion in prescribing antibiotics. Whereas antibiotics were once routinely prescribed for common illnesses without a definite diagnosis, doctors and hospitals are much more likely to conduct additional testing to determine whether an antibiotic is necessary and appropriate before prescribing.

A sick patient might reasonably object to this stingy approach to prescribing antibiotics. To the patient who simply wants to feel better as quickly as possible, the potential benefits of taking an antibiotic may seem to outweigh any immediate health risks that might occur if the antibiotic is ineffective. But at what point do the risks of widespread antibiotic use supersede the desire to use them in individual cases?

Key Concepts and Summary

  • Samples must be properly prepared for microscopy. This may involve staining, fixation, and/or cutting thin sections.
  • A variety of staining techniques can be used with light microscopy, including Gram staining, acid-fast staining, capsule staining, endospore staining, and flagella staining.
  • Samples for TEM require very thin sections, whereas samples for SEM require sputter-coating.
  • Preparation for fluorescence microscopy is similar to that for light microscopy, except that fluorochromes are used.

Multiple Choice

What mordant is used in Gram staining?

What is one difference between specimen preparation for a transmission electron microscope (TEM) and preparation for a scanning electron microscope (SEM)?

  1. Only the TEM specimen requires sputter coating.
  2. Only the SEM specimen requires sputter-coating.
  3. Only the TEM specimen must be dehydrated.
  4. Only the SEM specimen must be dehydrated.

Fill in the Blank

Ziehl-Neelsen staining, a type of _______ staining, is diagnostic for Mycobacterium tuberculosis.

The _______ is used to differentiate bacterial cells based on the components of their cell walls.


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