Listeria monocytogenes is a pathogenic bacterium that causes listeriosis, a serious foodborne disease. This Gram-positive, rod-shaped organism is found widely in nature, including in soil, water, and the feces of some animals. A notable characteristic of L. monocytogenes is its ability to survive and multiply at refrigerator temperatures, making it a challenge in food safety. To detect its presence, scientists use culturing, a process that creates specific laboratory conditions to encourage the bacterium to grow from a sample for identification from food, environmental, or clinical sources.
Purpose of Culturing Listeria Monocytogenes
The reasons for culturing Listeria monocytogenes include protecting public health and diagnosing illness. In the food industry, culturing is used to ensure products are safe for consumption. Production facilities test food items like soft cheeses, deli meats, and fresh produce, as well as the manufacturing environment. Swabs are taken from surfaces like floor drains and processing equipment, where the bacteria can form biofilms and create a source of contamination.
In a clinical setting, culturing is used to diagnose an active infection, known as listeriosis. When a person has symptoms like fever, diarrhea, or meningitis, doctors collect samples for testing. These specimens can include blood, cerebrospinal fluid (CSF), amniotic fluid, or placental tissue. Isolating L. monocytogenes from these samples provides a definitive diagnosis and helps guide medical treatment.
The Laboratory Culturing Process
Culturing L. monocytogenes is a multi-step process to isolate it from other microorganisms. It begins with collecting a sample, such as a portion of a food product, an environmental swab, or a clinical specimen. The first step is enrichment, where the sample is placed into a liquid broth that encourages Listeria to grow while suppressing competing bacteria.
Enrichment is performed in two stages to maximize recovery. First, the sample is placed in a primary enrichment broth, like Half-Fraser Broth, and incubated at 30°C for 24 hours. This broth contains agents that inhibit non-Listeria bacteria. A small amount of this culture is then transferred to a secondary enrichment broth, like Fraser Broth, which has more selective agents and is incubated at 35-37°C for another 24 to 48 hours. This process increases the Listeria population to detectable levels.
After enrichment, the concentrated liquid culture is streaked onto a solid agar plate. Laboratories use selective and differential agars, like Oxford Agar or PALCAM Agar, which contain substances that prevent the growth of most other bacteria. These agars also have compounds that react to Listeria’s metabolic activity, causing its colonies to develop a distinct appearance. The plates are incubated at 35-37°C and examined after 24 and 48 hours for potential colonies.
Identification and Confirmation of Cultures
After potential colonies grow on the agar plates, a microbiologist performs tests to confirm they are L. monocytogenes. The first step is a visual inspection of the colony morphology. On selective media like Oxford or PALCAM agar, L. monocytogenes forms small, grayish colonies surrounded by a black halo from esculin hydrolysis. On chromogenic media like ALOA, pathogenic Listeria colonies appear blue-green and are surrounded by an opaque white halo, indicating specific enzyme activity.
Further confirmation involves microscopic examination. A Gram stain is used to confirm the bacterium is Gram-positive and rod-shaped. Another identification method is observing its movement in a liquid medium at room temperature (around 25°C). Under a microscope, the bacterium exhibits a unique end-over-end “tumbling motility” that helps distinguish it from other bacteria.
A series of biochemical tests provides the final confirmation. One is the Catalase test, where adding hydrogen peroxide to a colony causes bubbling, indicating a positive result. Another is the CAMP test, where the colony is streaked on a blood agar plate near Staphylococcus aureus. L. monocytogenes enhances the hemolytic activity of the Staphylococcus, creating an arrowhead-shaped zone of clearing on the agar.
Biosafety Considerations for Handling Cultures
Handling Listeria monocytogenes in a laboratory requires safety protocols because it is a human pathogen. The bacterium is classified as a Biosafety Level 2 (BSL-2) organism, which applies to microbes that pose a moderate hazard to personnel and the environment. Specific precautions are necessary to prevent accidental infection or release.
All work with live cultures, especially procedures that could generate aerosols, must be performed within a biological safety cabinet (BSC). Personnel must wear personal protective equipment (PPE), including a lab coat, gloves, and eye protection. Individuals who are pregnant or immunocompromised should be informed of the risks associated with this pathogen.
Decontamination and waste disposal are enforced in a BSL-2 laboratory. All cultures, contaminated supplies, and used PPE must be decontaminated before being discarded. The most common sterilization method is an autoclave, which uses high-pressure steam at 121°C to kill the bacteria. Surfaces and equipment must be disinfected with an agent like 1% sodium hypochlorite or 70% ethanol to contain the organism.