Question

Choose the item that correctly matches the microorganism with an appropriate stain or preparation. A. Mycobacterium tuberculosis with India ink B. Fungi with \(\mathrm{KOH}\) C. Cryptococcus neoformans in cerebrospinal fluid with Ziehl-Neelsen (classic acid-fast stain) D. Chlamydia with Gram stain E. Escherichia coli (gram-negative bacterium) with crystal violet followed by treatment with acetone

Short answer

The correct microorganism-stain pair is 'E. Escherichia coli (gram-negative bacterium) with crystal violet followed by treatment with acetone'

Step-by-step solution

Understanding Microorganisms and Stains

Different microorganisms have a distinct appearance under a microscope when they are stained with a particular stain. For instance, Mycobacterium tuberculosis stains well with Ziehl–Neelsen or an acid-fast stain. Fungi are often prepared with KOH (potassium hydroxide) to dissolve surrounding cells and expose the chitin in the fungal cell walls. Cryptococcus neoformans in cerebrospinal fluid can be stained with India ink to allow a clear view of the organism's capsule.

Analyzing the Options

Each option provided should be scrutinized according to the given stains and preparations commonly utilized for each microorganism.

Choosing the Correct Pair

Escherichia coli is a gram-negative bacterium, meaning it will not retain the crystal violet stain in the Gram stain procedure but will rather take up the red counterstain (safranin). The acetone (also known as decolorizer in the Gram stain procedure) helps to wash out the crystal violet stain from gram-negative bacteria making them appear red under a microscope. Hence, the option 'E. Escherichia coli (gram-negative bacterium) with crystal violet followed by treatment with acetone' is the correct pair.

Key concepts

Mycobacterium tuberculosis Staining Techniques

Mycobacterium tuberculosis, the causative agent of tuberculosis, has a unique cell wall structure rich in lipids which renders it impervious to most common stains. However, the Ziehl-Neelsen (ZN) stain, also known as the acid-fast stain, is specifically designed to highlight these bacteria. The dye used in the ZN stain is applied with heat, allowing it to penetrate mycobacterial walls. After the application of a decolorizer, which removes the dye from non-acid-fast cells, a counterstain (commonly methylene blue) is used. Thus, acid-fast bacteria like Mycobacterium tuberculosis appear bright red against a blue background, making them easily detectable under a microscope.

When identifying Mycobacterium tuberculosis, it's crucial not to confuse this test with an India ink preparation, which is really not suitable for this bacteria but rather used for staining certain fungi, such as Cryptococcus neoformans. This technique showcases the importance of the correct staining protocol for accurate diagnosis and treatment of diseases.

KOH Preparation for Fungi

Fungi can be tricky to identify due to their complex structure and often require a special preparation to be adequately visualized. The Potassium Hydroxide (KOH) preparation is widely used because it clears the material around the fungi, providing a stark contrast that makes the fungal elements stand out. When a sample is mixed with KOH and gently heated, the KOH dissolves the keratin in tissue cells but not the chitin in the fungal cell walls. As a result, under the microscope, the fungal cells, which retain their structure due to their chitinous walls, become more visible against the cleared background. This technique is fundamental in diagnosing fungal infections, especially those affecting the skin or nails.

Correctly identifying the need for a KOH preparation can significantly improve the detection of various fungi, leading to more accurate diagnoses.

Cryptococcus neoformans Staining

Cryptococcus neoformans is a fungus known for causing severe infections, particularly in immunocompromised individuals. It is encased in a polysaccharide capsule which is a key virulence factor. The India ink stain is routinely used to visualize Cryptococcus neoformans in cerebrospinal fluid (CSF). This stain colors the background but not the fungal cells or their capsules. The capsules repel the ink, creating a clear halo around the yeast cells, which allows for easy visualization of the characteristic encapsulated yeast cells against the dark background.

This staining technique is especially significant in diagnosing cryptococcal meningitis, with the India ink stain serving as a rapid, although not definitive, diagnostic method. It's a prime example of the adaptation of staining techniques to highlight specific microbial features that are instrumental for clinical diagnoses.

Gram Stain Procedure

The Gram stain is a fundamental tool in microbiology used to classify bacteria into two major groups: Gram-positive and Gram-negative, based on their cell wall structure. This staining technique involves four key steps: application of the primary stain (crystal violet), the addition of a mordant (iodine), decolorization with alcohol or acetone, and counterstaining with safranin or another dye like methylene blue. Gram-positive bacteria retain the crystal violet color and appear purple due to their thick peptidoglycan layer, while Gram-negative bacteria, with their thinner cell wall and outer membrane, do not retain the purple stain and instead take up the counterstain, appearing red or pink.

This method is exceedingly important for diagnosing bacterial infections and guiding antibiotic treatment, as it provides critical information on the bacterial cell wall's structural differences. It clearly exemplifies the principle that the steps followed in staining are as crucial as the stains themselves for the accurate visualization and identification of microbial organisms.