Staining of Microorganisms

There are two types of scanning probe microscopy. The first is called scanning tunneling microscopy or STM and the second is atomic force microscopy or AFM. The STM technique passes a probe above the specimen, creating an electric current between the specimen and the probe. The intensity of the current is measured. The AFM also passes a probe across the specimen and it moves up and down because of the different forces between the atoms and the probe. The amount of deflection is measured and an image is constructed.

STAINING OF MICROORGANISMS

Without staining, it would be difficult to see most specimens because they do not have a lot of contrast between the different structures. For this reason, staining is used in many microscopy settings in order to see the structures better.

In preparing the specimen, there are wet mounts and dry mounts available. Wet mounts involve suspending the specimen in a liquid medium. Water and sometimes stains are used after which a coverslip is applied to the glass slide. Fixation actually involves attaching the cells to the slide. It involves heat fixation or chemical treatment to kill the specimen. The organisms do not move and can easily be stained. Chemical fixating agents include acetic acid, methanol, ethanol, glutaraldehyde, and formaldehyde.

Stains are also added to cause coloration of the specimen. All stains are salts that have both a negative and positive ion. One ion will be the chromophore and the other will be uncolored. If the chromophore belongs to the positive ion, it will be called a basic dye. If it belongs to the negative ion, it will be called an acidic dye. There will be a positive stain that is absorbed by what you want to see and a negative stain, which is absorbed by the background.

Most positive chromophores are basic dyes that stick to bacterial cell walls. These include crystal violent, basic fuchsin, malachite green, safranin, and methylene blue. Negatively charged chromophores made up of acidic dyes are not taken up by the cell wall; these include rose Bengal, eosin, and acid fuchsin.

Simple staining involves a single dye, while differential staining uses multiple dyes in order to show different structures as different colors. Gram staining, endospore

staining, acid-fast staining, and capsule staining are all techniques that require differential staining.

You will likely be involved in Gram staining, which was invented by Hans Christian Gram in 1884. It is a differential stain that will tell the difference between certain types of bacteria based on their cell wall structure. There are several steps to this that you should know about:

• The sample is fixed with heat. • Crystal violet is used to give all cells a dark purple color. • Gram’s iodine is a mordant used to trap the crystal violent within the peptidoglycan cell wall. • Ethanol or acetone and ethanol are used to decolorize cells with a thinner cell wall. • Safranin is used as a secondary counterstain to make Gram-negative organisms red.

Figure 9 will show the Gram-stain procedure in the order it is done:

Figure 9.

Gram staining is used only on bacteria and can help define what bacterial species is the pathogen in a clinical situation. There will be Gram-positive and Gram-negative bacterial organisms.

Acid-fast staining is also used to define different types of bacteria and can be used to define two separate types of gram-positive cells. There are two techniques used: the Ziehl-Neelsen technique and the Kinyoun technique. The main stain used in both is called carbolfuchsin. It is preferentially retained by cells that have mycolic acids in the cell wall, which will retain the stain after decolorization. Methylene blue is the counterstain. The main difference between the two techniques is that the Ziehl-Neelsen technique uses heat, while the other does not. Acid-fast bacteria will be red or pink against a blue background. Figure 10 shows what an acid-fast organism looks like:

Figure 10.

Some yeasts and bacteria will be surrounded by a capsule, which can affect their virulence. Because capsules do not absorb basic dyes, a negative staining technique is used to show up the background with capsule staining. The capsule looks like a halo on a colored background. Heat fixation Is not necessary. Dyes used for this purpose are nigrosin and India Ink. Positive staining can be used to highlight inner cell structures but these will not reveal the actual capsule.

Endospore staining will show bacterial endospores. Endospores are structures that can survive harsh environments. There are two stains used in this type of staining. There is the Schaeffer-Fulton method that uses heat to infuse malachite green into the endospore. A safranin counterstain is used to stain the rest of the cell. The endospore will resist decolorization and will remain green.

Flagella staining will reveal flagella structures. These are not otherwise easily seen with light microscopy. The mordant, usually tannic acid, is applied, which will show the flagellum. Then, a counterstain with pararosaniline or basic fuchsin will reveal the rest of the bacterium. Because flagella are delicate, the specimen must be carefully handled during the staining process.

A very thin specimen slice is necessary for transmission electron microscopy. Because of this, the cells need to be imbedded in a plastic resin and later dehydrated. Ethanol is used instead of water inside the cells, and the resin solidifies the cells. The sections are sustained using an ultramicrotome. The dyes are not colored but include heavy metalbased stains, such as uranyl acetate and osmium tetroxide.

With scanning electron microscopy, the specimen also needs to be dehydrated using ethanol. They need to be even drier than the TEM specimens and must be dried under liquid carbon dioxide. Gold or palladium are sputtered onto the surface of the specimen so it can more easily be seen.