Klebsiella species are Gram-negative bacteria frequently encountered in hospital settings, often linked to serious infections, including pneumonia and urinary tract infections. Initial identification in a clinical or educational laboratory often relies on a foundational growth medium. MacConkey agar (MAC) serves as an initial tool for isolating and presumptively identifying specific types of bacteria from patient samples. This guide explores how to utilize MAC’s unique properties to identify Klebsiella and details the necessary follow-up confirmation steps.
The Function and Preparation of MacConkey Agar
MacConkey agar is classified as both a selective and a differential medium. The selective function is achieved through the inclusion of compounds such as crystal violet dye and bile salts. These ingredients effectively suppress the growth of most Gram-positive bacteria, permitting the isolation of Gram-negative organisms, especially those belonging to the Enterobacteriaceae family.
The differential aspect of the medium relies on the combination of lactose and the pH indicator neutral red. When an organism ferments lactose, it generates acidic byproducts. This accumulation of acid causes the neutral red indicator to change color, resulting in a distinct pink or red hue in the growing colony and the surrounding agar. Conversely, organisms that cannot ferment lactose will not produce acid and retain a pale, translucent, or colorless appearance on the medium.
Interpreting Klebsiella Growth Colony Morphology
When Klebsiella is cultured on MacConkey agar, the first characteristic observed is its ability to ferment lactose vigorously. This metabolic activity classifies Klebsiella as a strong lactose fermenter, causing the colonies to display a bright, deep pink or red coloration. Intense acid production can sometimes cause the precipitation of bile salts in the surrounding medium, creating a slight hazy zone or halo around the colonies.
The most distinguishing visual feature of Klebsiella on this medium is its pronounced mucoid appearance. This slimy, wet look is directly attributable to the extensive polysaccharide capsule that surrounds the bacterial cell. This capsule is a defining trait of the genus, giving the colonies a characteristic spreading or “runny” quality on the agar surface.
The capsular material is composed primarily of complex sugars, and its production is often enhanced under laboratory growth conditions, maximizing the visual effect on the MacConkey plate. The high viscosity of this layer means the colonies appear significantly raised and domed compared to non-mucoid lactose fermenters. This specific morphology provides a strong presumptive identification, guiding the next steps in the laboratory process.
If a sterile loop touches a Klebsiella colony, the mucoidy nature often becomes evident as a viscous, sticky strand that stretches away from the agar. This phenomenon, sometimes referred to as a “string test,” helps rapidly distinguish Klebsiella from other pink, lactose-fermenting colonies. While other Enterobacteriaceae, such as Escherichia coli, also produce pink colonies, Klebsiella is usually separated by this highly distinctive, thick, mucoid texture.
Essential Confirmatory Testing
While the characteristic pink and mucoid growth pattern on MacConkey agar offers a strong initial suspicion, presumptive identification is never sufficient for clinical reporting. A series of biochemical tests must be performed to confirm the genus Klebsiella and differentiate it from other closely related, visually similar organisms, such as Enterobacter species.
These standard tests often follow the IMViC scheme (Indole, Methyl Red, Voges-Proskauer, and Citrate utilization). A foundational procedure is the assessment of motility, often performed in a semi-solid medium like SIM agar, which determines the organism’s ability to move independently. Klebsiella pneumoniae, the most common species, is typically non-motile, which helps distinguish it from motile relatives within the same family.
The Voges-Proskauer (VP) test is another standardized procedure, which detects the production of acetoin from glucose metabolism. The result for K. pneumoniae is typically positive, demonstrating its specific metabolic pathway. This positive VP reaction is a defining characteristic of the genus and is often paired with a negative result in the Methyl Red test, which assays for stable mixed-acid fermentation.
Conversely, the Indole test, which checks for the breakdown of tryptophan, usually yields a negative result for K. pneumoniae. This unique combination of non-motile, VP positive, and Indole negative results provides the necessary biochemical fingerprint to confirm the genus.
Species Differentiation
The Indole test also plays a significant role in differentiating between the two most clinically relevant species, K. pneumoniae and K. oxytoca. Unlike its most common counterpart, Klebsiella oxytoca is Indole positive, making this specific reaction a simple and effective way to confirm the species after the genus has been established. These detailed biochemical profiles are necessary for accurate identification, which informs treatment decisions in a clinical context.