article : Differential staining for Clostridium bacteria

The final differential stain that we will be learning in lab is called an Endospore stain 7/09. It is a special staining procedure that allows us to stain both vegetative (active) cell and endospores.

What are Endospores?

Two genera of bacteria, Clostridium and Bacillus, produce endospores. Endospores are tough, resistant structures that allow the bacteria to essentially exist in ‘suspended animation.’ Endospores do not metabolize and do not reproduce but merely exist, much like plant seeds, until exposed to environmental conditions suitable for bacterial growth.

Example of Clostridium bacteria with characteristic drumstick-shaped endospore-producing cells. The dark rod-shaped cells are vegetative, actice cells. The clear ovals are endospores, and the objects consisting of both dark rod and clear oval are vegetative cells producing endospores.

Endospore Sporulation

Endospores are made through a process known as sporulation in response to extreme environmental conditions. Extreme environments include high temperatures, drying out (dessication), extremes in pH, bacteriocidal chemicals and lack of food. When environmental conditions are favorable, the endospore germinates. Upon germination, the cell returns to its normal metabolic state, capable of reproduction.

Staining Endospores

Normal staining techniques will not stain the resistant endospores. Here are the steps that are required:

• Malachite Green: The stain, malachite green, is forced into the spore with heat much like the carbol fuschsin was forced through the waxy mycolic acid layer of Mycobacterium. After flooding the slide with malachite green, it is suspended over a boiling water bath for 5 minutes and then rinsed with water.
• Safrinin: Once the endospore is stained, the counter stain, Safranin, provides color for the vegetative (i.e. metabolically active) cells.

What Vegetative Cells and Endospores Look Like

Below is a photo of Bacillus bacteria that have been stained using the endospore stain. At the end of this differential staining process the vegetative cells (active, metabolizing cells) are pink and the endospores, if present, are green.

ADDITIONAL ARTICLES ABOUT ENDOSPORE STAINING

Follow the links below to articles explaining the Endospore stain procedure and the differences between vegetative cells and endospores.

Bacterial Endospore Stain Protocol: Differential Test to Identify Bacteria Genera Bacillus & Clostridium 7/09

Endospore staining involves application of a series of dyes. Malachite green stains endospores and safrinin dyes vegetative cells pink. Here's endospore stain procedure.

Differential Staining & Bacterial Controls: Positive & Negative Controls: Gram, Acid-fast & Endospore Stains 7/09

Bacterial controls are often used with differential stains as examples of typical positive and negative stain reactions; helpful references when identifying unknowns.

Sources:

Microbiology class laboratory material appearing on this website is adapted from the Applied Microbiology laboratory manual by Cynthia Schauer.

Images:

Heat fixed slide prepared for Gram staining. Positive control is on the left and negative control is on the right, T. Port.