Staining

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 Bacteria have almost the same refractive index as water.

This means when you try to view them using a microscope

they appear as faint, gray shapes and are difficult to see. Staining cells makes them easier to see.

 Simple stains use only one dye that stains the cell wall of

bacteria much like dying eggs at Easter. Differential stains use two or more stains and categorize cells into groups. Both staining techniques allow the detection of cell morphology, or shape, but the differential stain provides additional information concerning the cell. The most common differential stain used in microbiology is the Gram stain.

Gram Stain
The Gram stain is a differential stain. Four different reagents are used and the results are based on differences in the cell wall of bacteria. Some bacteria have relatively thick cell walls composed primarily of acarbohydrate known as peptidoglycan. Other bacterial cells have thinner cell walls composed of peptidoglycan and lipopolysaccharides. Peptidoglycan is not soluble in non polar or organic solvents such as alcohol or acetone, but lipopolysaccharides are nonpolar and will dissolve in nonpolar organic solvents.

 Crystal violet acts as the primary stain. This stain can also be used as a simple stain because it colors the cell wall of any bacteria. Grams iodine acts as a mordant. This reagent reacts with the crystal violet to make a large crystal that is not easily washed out of the cell. At this point in the staining process all cells will be the same color. The difference in the cell wall structure is displayed by the use of the decolorizer. A solution of acetone and alcohol is used on the cells. The decolorizer does not affect those cell walls composed primarily of peptidoglycan but those with the lipid component will have large holes develop in the cell wall where the lipid is dissolved away by the acetone and alcohol.

These large holes will allow the crystal violet-iodine complex to be washed out of the cell leaving the cell colorless.

A counterstain, safranin, is applied to the cells which will dye the colorless cells

 The cells that retain the primary stain will appear blue or purple and are known as Gram positive. Cells that stain with the counter-stain will appear pink or red and are known as Gram negative. The lipopolysaccharide of the Gram negative cell not only accounts for the staining reaction of the cell but also acts as an endotoxin. This endotoxin is released when the cell dies and is responsible for the fever and general feeling of malaise that accompanies a Gram negative infection. When reporting a Gram stain you must indicate the stain used, the reaction, and the morphology of the cell. Round, purple (blue) cells would be reported as Gram positive cocci and rod-shaped, purple (blue) cells would be reported as Gram positive bacilli. There are standard abbreviations that may be used for these reports.

 Simple staining Methylene blue, Crystal violet, Safranin or carbolfuchsin act as simple stains 1. Cover the label on the slide with tape.

2. Place the slide on the staining rack and flood the slide with stain for 1 minute.
3. Rinse the slide with tap water, tilting the slide slightly to rinse all the stain from the slide. Tap the slide gently to remove excess water. 4. Place a piece of bibulous paper or paper towel on the lab table and put the slide on it. Fold the paper over the slide and gently blot the slide to remove the water. 5. Examine the stained smear with the microscope and record your results

 Simple stain techniques. Staining can be

performed with basic dyes such as crystal violet or methylene blue, positively charged dyes that are attracted to the negatively charged materials of the microbial cytoplasm. Such a procedure is the simple stain procedure.

Positive stains Dye binds to the specimen Negative stains Dye does not bind to the specimen, but rather around the specimen.

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 An alternative is to use a dye such as nigrosin or

Congo red, acidic, negatively charged dyes. They are repelled by the negatively charged cytoplasm and gather around the cells, leaving the cells clear and unstained. This technique is called the negative stain technique.

Stains consist of a positive and negative ion. In a basic dye, the chromophore is a cation. In an acidic dye, the chromophore is an anion. Staining the background instead of the cell is called negative staining.

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Differential stain techniques.

The differential stain technique distinguishes two

kinds of organisms. An example is the Gram stain technique. This differential technique separates bacteria into two groups, Gram-positive bacteria and Gram-negative bacteria.

1883 Hans Christian JoachimGram

Differential stain A differential stain uses two contrasting dyes to distinguish between different types of organisms. Examples include the Gram stain and the acid-fast stain. There are four terms generally used for multi-dye staining: primary stain, mordant, decolorizer, and counterstain. For example the Gram stain separates Gram positive (Gm+) from Gram negative (Gm-) bacteria. Nearly all bacteria can be classified as either Gm+ or Gm-. This is a fundamental type of stain used routinely in clinical situations to classify and differentiate microorganisms. Four reagents are used for Gram staining and are applied in the following order: CVI-E-S 1. Crystal Violet, the primary (purple) stain. 2. Iodine, the mordant which forms a complex with crystal violet. 3. 95% Ethanol, the decolorizing agent. 4. Safranin, a red counterstain.

POTENTIAL PROBLEMS
If the smear is too thick, the reagents will not penetrate

evenly (seen as a mix of red and purple). This is a very common problem when staining from a colony. Washing too vigorously can remove cells and/or dye safranin is particularly easy to remove, making Gram negative cells difficult or impossible to see. Decolorizing too much or little. Washing too much of the crystal violet off with water If the sample pH is acidic it must be neutralized with 1% NaHCO3 and rinsed prior to staining.

Differential Stains: Gram Stain

Color of Grampositive cells Primary stain: Crystal violet Mordant: Iodine Decolorizing agent: Alcohol-acetone Counterstain: Safranin Purple Color of Gram negative cells Purple

Purple
Purple Purple

Purple
Colorless Red

Gram positive cocci

Gram negative bacilli

Gram positive

Gram negative

Primary stain

Mordant

Decolorization

Counterstain
Positive Negative

Mechanism

Acid-fast technique.
Another differential stain technique is the acid-fast technique. This technique differentiates species of Mycobacterium from other bacteria. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed with a dilute acid-alcohol solution. Mycobacterium species resist the effect of the acidalcohol and retain the carbolfuchsin stain (bright red). Other bacteria lose the stain and take on the ubsequent methylene blue stain (blue). Thus, the acid-fast bacteria appear bright red, while the nonacid-fast bacteria appear blue when observed under oil-immersion microscopy.

procedure. A few species, particularly those in the genus Mycobacterium do not bind simple stains readily and must be stained by a harsher treatment: heating with a mixture of basic fuchsin and phenol (the Ziehl-Neelsen method). Once basic fuchsin has penetrated with the aid of heat and phenol, acid-fast cells are not easily decolorized by an acid-alcohol wash and hence remain red. This is due to the quite high lipid content of acid-fast cell walls; in particular, mycolic acida group of branched chain hydroxy lipidsappears responsible for acidfastness. Non-acid-fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain. This method is used to identify Mycobacterium tuberculosis and M. leprae ,the pathogens responsible for tuberculosis and leprosy, respectively

A few species of bacteria in the genera Mycobacterium and Nocardia, and the parasite Cryptosporidium do not readily stain with simple stains. However, these microorganisms can be stained by heating them with carbolfuchsin. The heat drives the stain into the cells. Once the microorganisms have taken up the carbolfuchsin, they are not easily decolorized by acid-alcohol, and hence are termed acid-fast. This acid-fastness is due to the high lipid content (mycolic acid) in the cell wall of these microorganisms.

1) Mycolic acid is a large fatty acid.

structure of mycolic acid

 The Ziehl-Neelsen acid-fast staining procedure (developed by Franz Ziehl, a German bacteriologist, and Friedrich Neelsen, a German pathologist, in the late 1800s) is a very useful differential staining technique that makes use of this difference in retention of carbolfuchsin.

Acid-fast microorganisms will retain this dye and appear red (figure 9.1a, b). Microorganisms that are not acid-fast, termed non-acid-fast, will appear blue or brown due to the counterstaining with methyleneblue after they have been decolorized by the acid-alcohol.

Differential Stains: Acid-Fast Stain

Cells that retain a basic stain

in the presence of acid-alcohol

are called acid-fast. Nonacid-fast cells lose the

basic stain when rinsed with

acid-alcohol, and are usually counterstained (with a

different color basic stain) to

see them.

Amodification of this procedure that employs a wetting agent (Tergitol No. 7) rather than heat to ensure stain penetration is known as the Kinyoun staining procedure (developed by Joseph Kinyoun, German bacteriologist, in the early 1900s).

Ziehl-Neelsen (Hot Stain) Procedure 1. Prepare a smear consisting of a mixture of E. coli and M. smegmatis. with a piece of paper towel. Soak the towel with the carbolfuchsin and heat, well above a Bunsen burner flame.) 2. Allow the smear to air dry and then heat-fix 3. Place the slide on a hot plate that is within a chemical hood (with the exhaust fan on), and cover the smear with a piece of paper toweling that has been cut to the same size as the microscope slide. Saturate the paper with Ziehls carbolfuchsin (figure 9.2a). Heat for 3 to 5 minutes. Do not allow the slide to dry out, and avoid excess flooding! Also, prevent boiling by adjusting the hot plate to a proper temperature. A boiling water bath with a staining rack or loop held 1 to 2 inches above the water surface also works well. (Instead of using a hot plate to heatdrive the carbolfuchsin into the bacteria, an alternate procedure is to cover the heat-fixed slide 4. Remove the slide, let it cool, and rinse with water for 30 seconds (figure 9.2b). 5. Decolorize by adding acid-alcohol drop by drop until the slide remains only slightly pink. This requires 10 to 30 seconds and must be done carefully (figure 9.2c). 6. Rinse with water for 5 seconds (figure 9.2d). 7. Counterstain with alkaline methylene blue for about 2 minutes (figure 9.2e). 8. Rinse with water for 30 seconds (figure 9.2f). 9. Blot dry with bibulous paper (figure 9.2g). 10. There is no need to place a coverslip on the stained smear. Examine the slide under oil immersion and record your results Acid-fast organisms stain red; the background and other organisms stain blue or brown. See figure 9.1 for an example of the Ziehl-Neelsen stain. 11. Examine the prepared slide of Mycobacterium tuberculosis.

Kinyoun (Cold Stain) Procedure

(This may be used instead of or in addition to the Ziehl-Neelsen procedure.) 1. Heat-fix the slide as previously directed. 2. Flood the slide for 5 minutes with carbolfuchsin prepared with Tergitol No. 7 (heat is not necessary). 3. Decolorize with acid-alcohol and wash with tap water. Repeat this step until no more color runs off the slide. 4. Counterstain with alkaline methylene blue for 2 minutes. Wash and blot dry. 5. Examine under oil. Acid-fast organisms stain red; the background and other organisms stain blue.