This stain differentiates bacteria into two groups. One is referred to as Gram positive because of its ability to retain crystal violet stain, while the other is referred to as Gram negative because it is unable to retain this stain (see Fig. 1). These organisms can be further subdivided based on their morphological characteristics.
The structure of the bacterial cell envelope determines an organism’s Gram stain characteristics. Gram-positive organisms have an inner phospholipid bilayer membrane surrounded by a cell wall composed of a relatively thick layer of the polymer peptidoglycan. Gram-negative organisms also have an inner phospholipid bilayer membrane surrounded by a peptidoglycan-containing cell wall. However, in the Gram-negative organisms, the peptidoglycan layer is much thinner. The cell wall in Gram-negative organisms is surrounded by an outer membrane composed of a phospholipid bilayer. Embedded within this bilayer are proteins and the lipid A portion of a complex molecule called lipopolysaccharide. Lipopolysaccharide is also referred to as endotoxin because it can cause a variety of toxic effects in humans.
Because of their size or cell envelope composition, certain clinically important bacteria cannot be seen on Gram stain. These include all species of the genera Mycobacterium, Mycoplasma, Rickettsia, Coxiella, Ehrlichia, Chlamydia, and Treponema. Yeasts typically stain as Gram-positive organisms, while the hyphae of molds may inconsistently take up stain but generally will be Gram positive.
Gram stains can be performed quickly, but attention to detail is important to get an accurate Gram reaction. One clue to proper staining is to examine the background of the stain. The presence of significant amounts of purple (Gram positive) in the epithelial cells, red or white blood cells, or proteinaceous material, all of which should stain Gram negative, suggests that the stain is under-decolorized and that the Gram reaction of the bacteria may not be accurate. This type of staining characteristic is frequently seen in “thick” smears. The detection of over-decolorization is much more difficult and is dependent on the observation skills of the individual examining the slide.
Staining of acid-fast organisms
Mycobacterium spp., unlike other bacteria, are surrounded by a thick mycolic acid coat. This complex lipid coat makes the cell wall of these bacteria refractory to staining by the dyes used in the Gram stain. As a result, bacteria within this genus usually cannot be visualized or, infrequently, may have a beaded appearance on Gram stain. Certain stains, such as carbol fuchsin or auramine-rhodamine, can form a complex with the mycolic acid. This stain is not washed out of the cell wall by acid-alcohol or weak acid solution, hence the term “acid-fast” bacterium.
Auramine and rhodamine are nonspecific fluorochromes. Fluorochromes are stains that “fluoresce” when excited by light of a specific wavelength. Bacteria that retain these dyes during the acid-fast staining procedure can be visualized with a fluorescent microscope (Fig. 6). In clinical laboratories with access to a fluorescent microscope, the auramine-rhodamine stain is the method of choice because the organisms can be visualized at a lower magnification. By screening at lower magnification, larger areas of the microscope slide can be examined more quickly, making this method more sensitive and easier to perform than acid-fast stains using carbol fuchsin and light microscopy.
Several other organisms are acid-fast, although they typically are not alcohol-fast. As a result, they are stained using a modified acid-fast decolorizing step whereby a weak acid solution is substituted for an alcohol-acid one. This technique is frequently used to distinguish two genera of Gram-positive, branching rods from each other. Nocardia species are acid-fast when the modified acid-fast staining procedure is used, while Actinomyces species are not. Rhodococcus equi is a coccobacillus that may also be positive by modified acid-fast stain when first isolated. The modified acid-fast stain has also been effective in the detection of two gastrointestinal protozoan parasites, Cryptosporidium and Cyclospora. It should be noted that Cyclospora stains inconsistently, with some organisms giving a beaded appearance while others do not retain the stain at all.
Trichrome stain
The trichrome stain is used to visualize protozoans in fecal specimens. This stain is particularly effective at staining internal structures, the examination of which is important in determining the identity of certain protozoans, such as Entamoeba histolytica. Modification of the trichrome stain is used in the detection and identification of microsporidia.
Direct fluorescent-antibody stains
The development of monoclonal antibodies has enhanced both the sensitivity and the specificity of staining techniques that use antibodies to detect microbes in clinical specimens. The most widely used staining technique that incorporates the use of antibodies is the direct fluorescent-antibody (DFA) stain. In this technique, a highly specific antibody is coupled to a fluorochrome, typically fluorescein, which emits an “apple-green” fluorescence. The antibody binds specifically either to antigens on the surface of the microbes or to viral antigens expressed by virally infected cells, which can be visualized under the fluorescent microscope (Fig. 7). This technique is rapid, usually requiring 1 to 2 hours. In the hands of a skilled operator, the test is highly specific, although it frequently has a sensitivity of only 60 to 70% compared with bacterial culture. Because of its rapidity, the test has been used to detect some relatively slow-growing or difficult-to-grow bacteria, such as Bordetella pertussis and Legionella pneumophila. For respiratory viruses and herpesviruses, the sensitivity of this technique approaches 90% of the sensitivity of culture. However, the development of molecular amplification techniques for the detection of viral agents has demonstrated that DFA sensitivities can be as low as 50%, but may range up to 80% for some viruses. As result, many laboratories have replaced DFA with molecular amplification for organisms such as B. pertussis, herpesviruses, and respiratory viruses.
DFA staining is frequently used for the detection of microbes that cannot be cultured. DFA is the method of choice for detection of the nonculturable fungus Pneumocystis jirovecii, a common cause of pneumonia in people with AIDS. DFA is much more sensitive than other commonly used staining techniques, such as silver, Giemsa, or toluidine blue O staining. Likewise, for the gastrointestinal protozoans Giardia lamblia and Cryptosporidium parvum, DFA staining has been found to be much more sensitive than examination of wet mounts or the use of trichrome (for Giardia) or modified acid-fast stain (for Cryptosporidium). Molecular amplification techniques are also beginning to be deployed to detect these organisms as well and may soon replace DFA testing.
Infectious disease diagnosis from peripheral blood smears and tissue sections
Not all staining used in the diagnosis of infectious disease is done in the microbiology laboratory. The hematologist and the anatomical pathologist can play important roles in the diagnosis of certain infectious diseases.
The peripheral blood smear is the method of choice for detection
