A. Gram stain

In many infectious diseases, pathogenic organisms (excluding viruses) can often be directly visualized by microscopic examination of patient specimens, such as sputum, urine, and CSF. The organism’s microscopic morphology and staining characteristics can provide the first screening step in arriving at a specific identification. The organisms to be examined do not need to be alive or able to multiply. Microscopy yields rapid and inexpensive results and may allow the clinician to initiate treatment without waiting for the results of a culture, as noted in the spinal fluid example in the previous paragraph. Because unstained bacteria are difficult to detect with the light microscope, most patient material is stained prior to microscopic evaluation. The most common and useful staining procedure is the Gram stain, which separates bacteria into two classifications according to their cell wall composition. If a clinical specimen on a microscope slide is treated with a solution of crystal violet and then iodine, the bacterial cells will stain purple. If the stained cells are then treated with a solvent, such as alcohol or acetone, gram positive organisms retain the stain, whereas gram-negative species lose the stain, becoming colorless (Figure 1). Addition of the counterstain safranin stains the clear, gram-negative bacteria pink or red. Most, but not all, bacteria are stainable and fall into one of these two groups. [Note: Microorganisms that lack cell walls, such as mycoplasma, cannot be identified using the Gram stain.]

Fig1.

1. Gram stain applications: The Gram stain is important therapeutically because gram-positive and gram-negative bacteria differ in their susceptibility to various antibiotics, and the Gram stain may, therefore, be used to guide initial therapy until the microorganism can be definitively identified. In addition, the morphology of the stained bacteria can sometimes be diagnostic. For example, gram-negative intracellular diplococci in urethral pus provide a presumptive diagnosis of gonorrhea. Gram stains of specimens submitted for culture are often invaluable aids in the interpretation of culture results. For example, a specimen may show organisms under the microscope but appear sterile in culture media. This discrepancy may suggest the presence of either fastidious organ isms (bacteria with complex nutrient requirements) that are unable to grow on the culture media employed or fragile organ isms, such as gonococcus or anaerobic organisms, that may not survive transport. In these cases, direct visualization with the Gram stain may provide the only clue to the nature, variety, and relative number of infecting organisms.

 2. Gram stain limitations: The number of microorganisms required is relatively high. Visualization with the Gram stain requires greater than 10⁴ organisms/mL. Liquid samples with low numbers of microorganisms (for example, in CSF), require centrifugation to concentrate the pathogens. The pellet is then examined after staining.

 B. Acid-fast stain

Stains such as Ziehl-Neelsen (the classic acid-fast stain) are used to identify organisms that have waxy material (mycolic acids) in their cell walls. Most bacteria that have been stained with carbolfuchsin can be decolorized by washing with acidic alcohol. However, certain acid-fast bacteria retain the carbolfuchsin stain after being washed with an acidic solution. The most clinically important acid-fast bacterium is Mycobacterium tuberculosis, which appears pink, often beaded, and slightly curved (Figure 2). Acid fast staining is reserved for clinical samples from patients suspected of having mycobacterial infection.

Fig2. Mycobacterium tuberculosis stained with acid-fast stain.

C. India ink preparation

This is one of the simplest microscopic methods. It is useful in detecting Cryptococcus neoformans in CSF (Figure 3). One drop of centrifuged CSF is mixed with one drop of India ink on a micro scope slide beneath a glass cover slip. Cryptococci are identified by their large, transparent capsules that displace the India ink particles.

Fig3. India ink preparation of Cryptococcus neoformans in cerebrospinal fluid. These yeast cells are identified by large transparent capsules that exclude the India ink particles.

D. Potassium hydroxide preparation

Treatment with potassium hydroxide (KOH) dissolves host cells and bacteria, sparing fungi (Figure 4). One drop of sputum or skin scraping is treated with 10 percent KOH, and the specimen is examined for fungal forms.

Fig4. Fungi in unstained nasal sinus exudate, made distinct from other materials (such as cells) with potassium hydroxide.

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