Fundamentals of Microbiology

Lesson 6. MICROBIOLOGICAL STAINING METHODS

Module 2. Microscopy

Lesson 6

MICROBIOLOGICAL STAINING METHODS

6.1 Introduction

Sometimes it is convenient to determine overall bacterial morphology without the use of harsh staining or heat-fixing techniques that change the shape of cells. This might be the case when the bacterium does not stain well or when it is desirable to confirm observations made on the shape and size of bacteria observed in either a wet-mount or hanging drop slide. Staining of specimens is carried out to increase visibility of specimen, accentuate specific morphological features and preserves specimens. Staining of specimen makes internal and external structures of cell more visible by increasing contrast with background.

6.2 Preparing a Smear

A properly prepared smear accomplishes two things. One, it causes bacteria to adhere to a slide so that they can be stained and observed and second, it also kills them, rendering pathogenic bacteria safe to handle. An objective in preparing smears is to learn to recognize the correct density of bacteria to place on the slide. If there are too many cells, they will overlap each other giving false positives or crowding effect and if they are too low in number, they cannot be located on the slide. A smear is also required to undergo fixation, a process by which organism is killed and firmly attached to microscope slide and internal and external structures are preserved and fixed in position. Fixation can be achieved either by heat fixation (commonly used in light microscopy) which preserves overall morphology but not internal structures or chemical fixation using chemical fixatives (used in light and electron microscopy) which protects fine cellular substructure and morphology of larger, more delicate organisms.

6.2.1 Procedure

• A circle should be marked on the under side of a slide with a glass etching tool. Several circles can be located on the same slide.

• The slide must be grease-free. A good way to clean a slide is to repeatedly breathe on it, followed by rubbing vigorously with a soft tissue or paper towel to remove the fog. When the slide de-fogs immediately after breathing on it, it is sufficiently clean.

• To prepare a smear from a dry culture, a very small drop of distilled water should be placed over the circled area. After aseptically removing material from a culture it is them mixed with the drop or placed directly on the slide if it is a dilute broth culture. It takes very little material to produce a successful smear.

• The drop is air-dried completely, which takes a short time if a small drop is prepared.

• While holding the slide with a clothes pin it is quickly passed it through a flame. Three quick passes are usually sufficient to kill the bacteria and cause them to adhere.

• After cooling the slide, the staining procedure is conducted.

6.3 Staining Methods

Staining methods can be broadly categorized in to two types, simple and differential and include including negative staining, Gram staining, endospore staining, capsule staining, flagellar staining, acid fast staining etc.

6.3.1 Simple staining

In this type of staining bacterial cells are stained with a single reagent. Positively charged dyes such as methylene blue, crystal violet etc. is used for this purpose. These stains are taken up by the cells and bind to negatively charged cell components (cell wall, nucleic acids). Staining can be positive or negative (Fig. 6.1) depending up on the type of dyes (cationic or anionic)

Fig. 6.1 Types of staining

6.3.1.1 Procedure

• Prepare a thin smear of bacterial culture by placing a loopful of culture on a clean slide and spreading it slowly in circular motion on the surface of slide. Allow the smear to get dried.
• Heat fix the smear by passing it quickly on the bunsen flame.
• Flood the smear with methylene blue/crystal violet for 2 or 1 min. respectively.
• Wash the smear with tap water and dry it.
• Put a drop of immersion oil and observe under 100X lens of light microscope.
• Cells will appear blue (methylene blue) or purple (crystal violet). Look for various cell shapes.

6.3.2 Negative staining

In this stain the bacterial cells do not take up the cell but the background gets stained and the cell appears as unstained transparent entity. Acidic dyes such as nigrosin are used. Negative staining is advantageous because the bacteria which do not take up the stain can be observed in this way. Another advantage of this procedure is that it does not require heat fixing of smear so cell distortion due to heat do not take place.

6.3.2.1 Procedure (Fig. 6.2)

• Place a drop of nigrosin close to one end of a clean slide.
• Mix a loopful of culture in it.
• Prepare a smear of this mixture using the edge of another slide.
• Air-dry it and observe under oil immersion lens.

Unstained bacterial cells will appear in contrast to a dark background.

Fig. 6.2 Procedure for negative staining

6.4 Differential Staining

Simple staining depends on the fact that bacteria differ chemically from their surroundings and thus can be stained to contrast with their environment. Bacteria also differ from one another chemically and physically and may react differently to a given staining procedure. This is the principle of differential staining. Differential staining can distinguish between types of bacteria.

6.4.1 Gram staining

The Gram stain (named after Christian Gram, Danish scientist and physician, 1853–1938) is the most useful and widely employed differential stain in bacteriology. It divides bacteria into two groups, Gram negative and Gram positive. The first step in the procedure involves staining with the basic dye crystal violet. This is the primary stain. It is followed by treatment with an iodine solution, which functions as a mordant; that is, it increases the interaction between the bacterial cell and the dye so that the dye is more tightly bound or the cell is more strongly stained. The smear is then decolorized by washing with an agent such as 95% ethanol or isopropanol-acetone. Gram positive bacteria retain the crystal violet-iodine complex when washed with the decolorizer, whereas Gram negative bacteria lose their crystal violet-iodine complex and become colorless. Finally, the smear is counterstained with a basic dye, different in color than crystal violet. This counter stain is usually safranin. The safranin will stain the colorless, Gram negative bacteria pink but does not alter the dark purple color of the Gram positive bacteria. The end result is that Gram positive bacteria are deep purple in color and Gram negative bacteria are pinkish to red in color (Fig. 6.3). The Gram stain does not always yield clear results. Species will differ from one another in regard to the ease with which the crystal violet-iodine complex is removed by ethanol. Gram positive cultures may often turn Gram negative if they get too old. Thus, it is always best to Gram stain young, vigorous cultures rather than older ones. Furthermore, some bacterial species are Gram variable. That is, some cells in the same culture will be Gram positive and some, Gram negative. Therefore, one should always ensure to run Gram stains on several cultures under carefully controlled conditions in order to make certain that a given bacterial ‘strain’ is truly Gram positive or Gram negative. Indistinct Gram-stain results can be confirmed by a simple test using KOH. Place a drop of 10% KOH on a clean glass slide and mix with a loopful of bacterial paste. Wait for 30 seconds and then pull the loop slowly through the suspension and up and away from the slide. A Gram negative organism will produce a mucoid string; a Gram positive organism remains fluid.

Fig. 6.3 Gram staining method

6.4.2 Acid-fast staining (Ziehl-Neelsen and Kinyoun)

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. 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 (Fig. 6.4). Microorganisms that are not acid-fast, termed non-acid-fast, will appear blue or brown due to the counterstaining with methylene blue after they have been decolorized by the acid-alcohol. A modification 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).

Fig. 6.4 Acid fast staining method

6.4.3 Endospore staining

Bacteria in genera such as Bacillus and Clostridium produce quite a resistant structure capable of surviving for long periods in an unfavorable environment and then giving rise to a new bacterial cell. This structure is called an endospore since it develops within the bacterial cell. Endospores are spherical to elliptical in shape and may be either smaller or larger than the parent bacterial cell. Endospore position within the cell is characteristic and may be central, subterminal, or terminal. Endospores do not stain easily, but, once stained, they strongly resist decolorization. This property is the basis of the Schaeffer-Fulton (Alice B. Schaeffer and MacDonald Fulton were microbiologists at Middlebury College, Vermont, in the 1930s) or Wirtz-Conklin method (Robert Wirtz and Marie E. Conklin were bacteriologists in the early 1900s) of staining endospores. The endospores are stained with malachite green. Heat is used to provide stain penetration. The rest of the cell is then decolorized and counterstained a light red with safranin.

6.4.3.1 Procedure (Fig. 6.5)

• With a wax pencil, place the names of the respective bacteria on the edge of four clean glass slides.
• Aseptically transfer one species of bacterium with an inoculating loop to each of the respective slides, air dry (or use a slide warmer), and heat-fix.
• Place the slide to be stained on a hot plate or boiling water bath equipped with a staining loop or rack. Cover the smear with paper toweling that has been cut the same size as the microscope slide.
• Soak the paper with the malachite green staining solution. Gently heat on the hot plate (just until the stain steams) for 5 to 6 min after the malachite green solution begins to steam. Replace the malachite green solution as it evaporates so that the paper remains saturated during heating. Do not allow the slide to become dry.
• Remove the paper using forceps, allow the slide to cool, and rinse the slide with water for 30 sec.
• Counterstain with safranin for 60 to 90 sec.
• Rinse the slide with water for 30 sec.

Fig. 6.5 Endospore staining