Identification of Bacteria using Staining Techniques (Simple, Gram’s & Acid-fast Staining) and Biochemical Tests (IMViC) : Pharmaceutical Guidelines -->

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Identification of Bacteria using Staining Techniques (Simple, Gram’s & Acid-fast Staining) and Biochemical Tests (IMViC)

Simple Staining, Gram Staining, Acid Fast Staining, Indole Test, Methyl Red (MR) Test, Voges-Proskauer (VP) Test, Citrate Utilization Test

Identification of Bacteria using Staining Techniques

A differential staining technique is more frequently used in microbiology to gather information about bacteria than simple stains. Several steps and multiple staining methods are required to differentiate cell types and cell structures using differential staining methods. The Gram stain is a popular example of such a technique. Besides endospore staining (to identify bacteria that form endospores), acid-fast staining (to differentiate Mycobacterium species from other bacteria), and a metachromatic stain (to identify bacteria encapsulated in phosphate granules), there are also capsule stains (to identify bacteria inside encapsulated cells). Gram staining and endospore staining procedures will be performed in the lab, and slides showing other cell structures will be made available for viewing.

Simple Staining

Bacteria can be determined quickly and easily with the simple stain by measuring cell size, shape, and arrangement. As the name suggests, the simple stain involves just one solution of stain and is very simple in its application. Crystal violet, safranin, or methylene blue are all basic dyes that can be used to dye bacteria. Stains that are hydroxide- or hydrogen-bearing readily release or accept hydroxide or hydrogen ions, leaving a positively charged stain. Most bacterial cells and cytoplasm have a negative charge, which makes these positively charged stains readily adhere to them. The morphology of the bacterial cells (shape and arrangement) can be observed after staining.


Simple staining methods are not commonly used in diagnostic microbiology laboratories. Bacterial isolates are commonly identified and distinguished using different staining procedures, including Gram stain and AFB stain. There are several instances when simple staining may be appropriate.

Inexperienced technologists and microbiologists may make an error when incubating endocervical swab cultures on blood agar; both Staphylococcus species and yeast cells can look similar on blood agar. If this is not possible, you can stain the isolate directly from the isolate with the wet mount technique.

During the identification process, the wet mount technique can be helpful. The ubiquitous presence of Bacillus spp. on culture plates may make them a contaminant. Bacillus spp., which grow in Blood Agar, but not MacConkey Agar, may be eliminated as potential contaminants or further processed for further analysis as a potential pathogen (cocci) based on the shape of the isolate (rod or cocci).

Gram Staining

The medical researcher Hans Christian Gram studied the etiology (cause) of respiratory diseases in 1884. He developed a method to identify bacteria in lung tissue taken from deceased patients, which allowed him to identify the pathogen that causes a fatal form of pneumonia. The Gram stain method was not effective at treating the disease, but it certainly helped determine the cause of death at autopsy. Using Gram's staining practices today, we identify bacteria by their color, and bacteria themselves are divided into two types: Gram positive and Gram negative.

Based on the fact that some bacteria retain a primary stain (crystal violet) to resist decolorization, Gram stains serve as differential stains. Four steps are involved in Gram staining. To begin staining cells, crystal violet is first applied, followed by a set agent (iodine). After ensuring that only Gram-negative cells are removed from the stain, alcohol is applied. The decolorized cells are then counter-stained pink with a secondary stain, safranin. It wasn't until much later that Gram realized that this was the key difference between these two kinds of bacteria. Gram-negative cells have an outer membrane covering their cell walls (the envelope). This layer dissolves with the alcohol wash. Crystal violet dye is then released. Cells that have been decolorized are the only ones that take up the pink dye safranin, explaining their different colors. Upon completion of Gram staining, Gram positive and Gram-negative cells will appear purple and pink, respectively.

You should describe the morphology and arrangement of cells when interpreting a Gram-stained smear. A Gram stain reaction and their shape and arrangement identify two distinct types of bacteria in Figure.

Acid Fast Staining

Cell walls are constructed by certain bacteria using a waxy substance called mycolic acid. By acting as a barrier, mycolic acid prevents cells from dehydrating, as well as phagocytosing by host immune cells. Gram stains not working with Mycobacterium, which cause diseases in humans and animals, is also because of this waxy barrier. These bacteria can be stained using the acid-fast method.

A slide is steamed, flooded with a primary stain, carbol fuchsin, and then stained acid-fast with the stain. In order for the dye to enter the cells, the waxy cell walls have to be melted by the heat. After the slide has cooled, it is decolored by adding an acid and alcohol solution. A cell wall containing mycolic acid is "acid-fast" and resists decolorization as a result. Nonetheless, the remainder of the cells are decolored. To counterstain the decolorization, the methylene blue dye is used. Eventually acid-fast bacteria (AFB) will appear pink, while the rest of the cells will appear blue.

Biochemical Test (IMViC)

In IMVic, each letter is associated with one of these tests. The lowercase "i" represents indole, the uppercase "M" stands for methyl red, "V" is for Voges-Proskauer, and "C" stands for citrate. Four different tests are included in IMViC. You will need to inoculate these test tubes with tryptone broth (indole test), methyl red - Voges Proskauer broth (MR-VP broth), and citrate to obtain results for these tests. It is used to identify and differentiate members of the Enterobacteriaceae family.

The following are the general procedures for conducting and interpreting IMViC tests:

The respective tests can be conducted after the cultures have grown at 37°C for 24 to 48 hours for any member of the Enterobacteriaceae.

Indole Test

In SIM or MIU, sulfide-indole, motility (SIM) medium is used, or tryptophan broth is used. Once Kovac's reagent is added, the result can be read.
  • The top of the tube turns red following the addition of Kovács reagent, indicating a positive outcome.
  • If the color of the top of the tube does not change after the Kovács reagent is added, the result is negative.
MR-VP broth is typically used for both the methyl red-Voges-Proskauer and the Voges-Proskauer test, but the reagents added depend on which test is being done.

Methyl Red (MR) Test

As the methyl red reagent is added, a red color is produced indicating a positive result.
Adding methyl red reagent does not result in any color change in a negative methyl red test

Voges-Proskauer (VP) Test

  • Adding Barritt's A and B reagents does not result in a color change in a negative test.
  • Adding Barritt's A and B reagents to Voges-Proskauer test material results in the development of a reddish-brown color.

Citrate Utilization Test

Using Simmons citrate agar, the test is performed as follows:
  • A lack of growth or a change in color does not point to a positive test for citrate utilization.
  • Citrate results indicate growth and a change in color to blue.
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Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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