Bacterial Staining Procedures

Created by pmrayfield 

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The components of a stain

They are solutions consisting of a solvent (usually water or ethanol) and a chromogen.


The portion of the stain that is the colored molecule (often a benzene derivative).


The portion of the chromogen that gives it its color. A chromogen may have multiple chromophores, with each adding intensity to the color.


the charged portion of the chromogen that allows it to act as a dye through ionic or covalent bonds between the chromogen and the cell.

Basic Stains

The term basic here means alkaline. In these stains the auxochrome becomes positively charged as a result of picking up a hydrogen ion or losing a hydroxide ion. This makes it attracted to the negative charges on the surface of most bacterial cells. Thus, the cell becomes colored. Common basic stains include methylene blue, crystal violet and safranin.


Basic stains are applied to bacterial smears that have been heat-fixed. Heat-fixing kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible. It also distorts the cells to some extent.

Simple Stain Preparation

1. Begin with a heat-fixed emulsion
2.Cover the smear with stain (60 sec)
3. Rinse the slide with water
4. Gently blot dry with bibulous paper - do not rub
5. Observe under oil immersion

What are the consequences of overstaining

Overstaining the bacterial smear may cause the cell wall disruption or totally destroy the cell wall which results in the loss of true morphological characteristics of the bacterial cell

What are the consequences of under staining

The cells may lose the stain when washed with alcohol or water which causes a problem in identifying the cell.
In Gram staining if it is not stained properly with the Methylene Blue then it will lose the stain when washed and will counter stain with Safranin and cause false Gram negatives.

What are the components of a negative stain

It uses a dye solution in which the chromogen is acidic and carries a negative charge. It gets its negative charge by giving up a hydrogen ion. The negative charge on the bacterial surface repels the negatively charged chromogen, so the cell remains unstained against a colored background.

What is the name of an acidic stain used for negative staining?


Why would you use negative staining?

to determine morphology and cellular arrangement in bacteria that are too delicate to withstand heat-fixing. Also, where determining the accurate size is crucial, a negative stain can be used because it produces minimal cell shrinkage.

Negative Stain Preparation

1. Begin with a drop of acidic stain at one end of a clean slide
2. Aseptically add organisms and emulsify with a loop. Do not over-inoculate and avoid spattering the mixture
3. Take a second clean slide, place it on the surface of the first slide, and draw it back into the drop
4. Do it until the drop flows across the width of the spreader slide
5. Then push the spreader slide to the other end and dispose of the spreader slide
6. Air dry and observe under the microscope

Differential Stains

Allow a microbiologist to detect differences between organisms or differences between parts of the same organism.

Why would you use a another differential stain besides the Gram stain?

There are some organisms that are not distinguishable by the Gram stain, and for those that have other important cellular attributes such as acid-fastness, a capsule, spores or flagella.

Primary Stain

The first stain applied in many differential staining techniques; usually subjected to a decolorization step that forms the basis for the differential stain.


A substance added to a staining solution to make it stain more intensely.


Most critical step in Gram staining. Removes the color from Gram negative cells and allows them to be counterstained to differentiate between the cells.


Stain applied after decolorization to provide contrast between cells that were decolorized and those that were not.

Gram Stain

A differential stain in which a decolorization step occurs between the application of 2 basic stains. The most important and widely used differential stain. It typically is the first differential test run on a specimen brought into the lab for identification. In some cases, a rapid, presumptive ID or elimination of a particular organism is possible.

Crystal violet-Iodine Complex

Forms when the mordant, iodine, is added to the primary stain, crystal violet, to enhance crystal violet staining

Resisting Decolorization

Based on the different wall constructions of G+ and Gram- cells. G- cell walls have a higher lipid content (because of the outer membrane) and a thinner peptidoglycan layer than G+ cell walls. The alcohol/acetone in the decolorizer extracts the lipid, making the G- wall more porous and incapable of retaining the CV-IC, thereby decolorizing it. The thicker peptidoglycan and greater degree of cross-linking trap the CV-IC more effectively, making the G+ wall less susceptible to decolorization

Gram Stain Preparation

1. Begin with a heat-fixed emulsion
2. Cover the slide with crystal violet stain for 30 sec. Rinse
3. Cover the smear with iodine stain for 1 min. Rinse
4. Decolorize with ethanol by running it over the edge of the slide 3x. Immediately wash off with water
5. Counterstain with Safranin stain for 1 min. Rinse
6. Gently blot dry with bibulous paper. Observe under oil immersion

Acid-Fast Stain

A differential stain used to detect cells capable of retaining a primary stain when treated with an acid alcohol. It is important in ID'ing bacteria in the genus Mycobacterium, some of which are pathogens. Because so few organisms are Acid-fast, the Acid-fast stain is run only when infection by an acid-fast organism is suspected. The presence of mycolic acids in the cell walls of acid-fast organisms is the cytological basis for the acid-fast differential stain.

Mycolic Acid

A waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution. This waxy wall repels typical aqueous stains - as a result, most acid-fast positive organisms are only weakly G+.

Two variations in the acid-fast staining technique

1. Ziehl-Neelsen (ZN) Method - uses heat as part of the staining process.
2. Kinyoun (K) Method - a "cold" stain
*In both protocols the bacterial smear may be prepared in a drop of serum to help the "slippery" acid-fast cells adhere to the slide.


A phenolic compound that is the primary stain used in acid-fast staining. It is used because it is lipid-soluble and penetrates the waxy cell wall.

Acid-fast decolorization

Acid alcohol is used to decolorize nonacid-fast cells; acid-fast cells resist this decolorization.

ZN Method

Acid-fast staining method that uses a slightly less soluble and less concentrated carbolfuchsin stain than the K method. To enhance uptake of the carbolfuchsin, the preparation is stream-heated for several minutes. The ZN method is slightly more sensitive than the K method.

Characteristic arrangement of acid-fast cells

the acid-fast organisms show characteristic clumping of the cells.

Clinical use for acid-fast staining

1. bacteria in the genus Mycobacterium, some are pathogens
2. Useful in identifying acid-fast bacilli (AFB)
3. rapid, preliminary diagnosis of tuberculosis and leprosy
4. Performed on patient samples to track the progress of antibiotic therapy
5. Performed on patient samples to determine the degree of contagiousness - a prescribed number of microscopic fields is examined and the number of AFB is determined and reported using a standard scoring system.

Acid-Fast Stain (K Method) Preparation

1. Begin with a heat-fixed emulsion - emulsion can be prepared in a drop of sheep serum
2. Apply Kinyoun carbolfuchsin stain for 5 min - perform with adequate ventilation. Rinse
3. Decolorize with acid-alcohol. Rinse
4. Counterstain with Brilliant Green stain for 1 min. Rinse
5. Gently blot dry with bibulous paper. Observe under oil immersion

How does heating the bacterial smear during a ZN stain promote entry of carbolfuchsin into the acid-fast cell wall?

Heating melts the mycolic acid and allows the stain to penetrate the cell walls.

Are acid-fast negative cells stained by carbolfuchsin? If so, how can this be a differential stain?

It uses acid alcohol as a decolorizing agent which extracts the carbolfuchsin from the nonacid-fast cells while ineffective on the acid-fast positive cells. The nonacid-cells are then counterstained with brilliant green to show the difference.


Composed of mucoid polysaccharides or polypeptides that repel most stains. Capsule production increases virulence in some microbes (such as anthrax bacillus and the pneumococcus Streptococcus pneumoniae) by making them less vulnerable to phagocytosis.

The stains of the Capsule staining technique

Typically an acidic stain such as Congo red or nigrosin, which stains the background, and a basic stain that colorizes the cell proper are used.This technique begins as a negative stain; cells are spread in a film with an acidic stain and are not heat-fixed.

Why do you not heat-fix cells with capsules?

Heat-fixing causes the cells to shrink, leaving an artificial white halo around them that might be interpreted as a capsule. In the place of heat-fixing, cells may be emulsified in a drop of serum to promote their adhering to the glass slide

Capsule Stain

A differential stain used to detect cells capable of producing an extracellular capsule. The acidic stain colorizes the background while the basic stain colorizes the cell, leaving the capsules as unstained, white clearings around the cells. Under a microscope you can see that they lack uniform capsule size and even the absence of a capsule in some cells.

Capsule Stain Preparation

1. Begin with a drop of Congo red or nigrosin stain at one end of a clean slide - add a drop of serum
2. Aseptically add organisms and emulsify with a loop
3. Take a 2nd clean slide, place it on the surface of the 1st slide, and draw back into the drop
4. Do it until the drop flows across the width of the spreader slide
5.Then push the spreader slide to the other end and dispose it
6. Air dry and do NOT heat-fix
7. Flood the slide with Maneval's Stain for 1 min. Rinse
8. Blot dry with bibulous paper. Observe under oil immersion

What is the purpose of emulsifying the bacteria in serum in capsule staining?

To promote the cells adhering to the glass slide since they can't be heat-fixed

Some oral bacteria produce an extracellular capsule. Of what benefit is a capsule to these cells?

Prevents the cells from being damaged by enzymes and other body materials


A dormant form of bacterium that allows it to survive poor environmental conditions.


A protein that makes up the tough outer covering of a spore and makes it resistant to heat and chemicals. The keratin also resists staining, so extreme measures must be taken to stain the spore.

Endospore Stain

A differential stain used to detect the presence and location of spores in bacterial cells.

Shaeffer-Fulton Method

A primary stain of malachite green is forced into the spore by steaming the bacterial emulsion. Alternatively, malachite green can be left on the slide for 15 min or more to stain the spores. Safranin is then used to counterstain vegetative and spore mother cells.

Malachite Green

Stain used in Endospore staining that is water-soluble and has a low affinity for cellular material, so vegetative cells and spore mother cells can be decolorized with water and counterstained with Safranin.

Classification of Endospores

1. The location of the spore within the spore mother
2. Shape: spherical or elliptical (oval)
3.Size relative to the cell - whether they cause the cell to look swollen or not

The spore is located in the middle of the cell


The spore is located at the end of the cell


The spore is located between the end and middle of the cell



Heterotrophs that digest dead organic matter, a decomposer

Endospore producing organisms

1. Bacillus - most are soil, freshwater or marine saprophytes and a few are pathogens (ie B. anthracis, the causal agent of anthrax)
2. Clostridium - most are soil or aquatic saprophytes or inhabitants of human intestines, but 4 pathogens are fairly well known - C. tetani (tetanus), C. botulinum (botulism), C. perfringens (gas gangrene), and C. difficile (pseudo-membranous colitis).

Endospore Stain (Schaeffer-Fulton Method) Preparation

1. Begin with a heat-fixed emulsion
2. Cover the smear with a strip of bibulous paper. Apply Malachite Green stain. Steam for 7-10 min.Keep the paper moist with stain.
3. Remove the paper and dispose of it. Rinse the slide
4. Counterstain with Safranin stain for 1 min. Rinse
5. Blot dry with bibulous paper. Observe under oil immersion.

Why does the Endospore Stain exercise call for an older (5-day) culture of Bacillus?

Older bacteria are under greater nutritional stress so there will be more endospores present and able to visualize

What is the main reason you would perform a wet mount or hanging drop technique?

To observe and determine motility

False Motility in the Wet Mount Technique

Viewing must be done quickly because of drying of the preparation. As the water recedes, bacteria will appear to be herded across the field. This is not motility. You should look for independent darting motion of the cells.

What are the benefits to using the Hanging Drop technique instead of the wet mount?

Allows longer observation of the specimen because it doesn't dry out as quickly. Can observe cell size, shape, binary fission, and motility

False motility in the Hanging Drop technique

Observer must be careful to distinguish between true motility and the Brownian Motion created by collisions with water molecules. In the latter, cells will appear to vibrate in place. With true motility, cells will exhibit independent movement over greater distances.

What do simple wet mounts and the hanging drop technique better than other techniques allow that other techniques do not?

Most bacterial preparations result in death of the cells as a result of heat-fixing and staining.These 2 techniques allow observation of living cells to determine motility. They are also used to see natural cell size, arrangement, and shape. All of which are useful in ID of a microbe

Wet Mount Preparation

1. Place a loop-full of water on a clean glass side
2. Add bacteria to the drop. Don't over innoculate. Flame the loop after transfer
3. Gently lower a cover glass with your loop supporting one side over the drop of water. Avoid trapping air bubbles
4. Observe under 40x or oil immersion

Hanging Drop Preparation

1. Apply a light ring of petroleum jelly around the well of a depression slide
2. Apply a drop of water to a cover glass, if using a broth culture then omit the water
3. Aseptically add a drop of bacteria to the water. Flame loop
4. Invert the depression slide so the drop is centered in the well. Gently press until the petroleum jelly has created a seal between the slide and cover glass.
5. The drop should not be in contact with the depression slide. Observe under 40x or oil immersion.

In the wet mount technique, what should you do in order to have the best focus under the microscope?

Because of the thickness of the water, cells show up in many different focal planes and are mostly out of focus. To get the best possible image, adjust the condenser height and reduce the light intensity with the iris diaphragm.

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