Differential Bacterial Stains and Controls

There are many staining techniques that can be used to view bacteria. Simple stains employ only one dye, and provide information limited to cell shape and grouping. The use of differential stains is more complex. Differential protocols employ a series of dyes to distinguish different types of bacteria based on some chemical or structural attribute of the cell.

Differential stains are often used for general identification of bacteria, rather than identifying the exact species. Examples include the Gram stain, Acid-fast stain and Endospore staining protocols.

When doing a differential stain, it is best to use controls to help identify the stain reaction of the unknown bacteria. Controls are types of bacteria that are known to show a certain specific stain reaction.

* Gram Stain Controls *

The Gram stain classifies nearly all bacteria into one of two groups (Gram+ or Gram-), based on characteristics of the bacterial cell wall, specifically the location and amount of the cell wall’s primary structural molecule, peptidoglycan.

Bacteria that have cell walls entirely made of peptidoglycan will stain Gram-positive. Gram-negative cells have one layer of peptidoglycan sandwiched between two membranes. The location of this key molecule plays a key role in bacterial vulnerability to different classes of antibiotics.

When preparing a Gram stain slide, a positive control would be a type of bacterium that is known to stain Gram+, such as Staphylococcus. A negative control would be any known Gram- organism, such as Esherichia coli.

* Acid Fast Stain Controls *

The Acid-fast stain is specifically used to identify bacteria that possess a waxy lipid within the structure of their cell walls. Due to the presence of this lipid, water-based stains, such as the Gram stain, do not work well on Acid-fast organisms. This protocol utilizes heat to drive the primary stain into waxy bacterial cell walls.

When doing an Acid-fast stain, the positive control would be an acid-fast bacterium with the characteristic waxy cell wall, such as members of the genera Mycobaterium or Nocardia. Most bacteria do not possess these special waxy lipids, so a nonacid fast organism would be any bacterium other than Mycobacterium and Nocardia.

* Endospore Stain Controls *

The Endospore stain is used for identifying bacteria that can produce tough, dormant spores. Endospores enable bacteria to withstand many hardships, such as excessive heat, lack of nutrients, and some bacteriocidal chemicals.

Very few types of bacteria can form endospores; most notably Bacillus and Clostridium. So a positive control for the Endospore stain would be a bacterium that produces endospores; a negative control would be any type of bacteria that are not endospore producers.

* Sources *

Schauer Cynthia (2007) Lab Manual to Microbiology for the Health Sciences, Kalamazoo Valley Community College.

Bauman, R. (2005) Microbiology. Pearson Benjamin Cummings.