What is the Difference Between Gram Stain and Acid Fast

The main difference between Gram stain and acid fast stain is that Gram stain helps to distinguish bacteria with different types of cell walls whereas acid-fast stain helps to distinguish Gram-positive bacteria with waxy mycolic acids in their cell walls. Furthermore, in Gram stain, the Gram-positive bacteria retain the crystal violet stain, appearing purple while in acid-fast stain, the acid-fast bacteria retain carbofuchsin stain, appearing in red color.

Gram stain and acid-fast stain are two types of differential, bacteriological stain methods used to characterize bacteria.

Key Areas Covered

1. What is Gram Stain
     – Definition, Steps, Bacterial Characterization
2. What is Acid Fast Stain
     – Definition, Steps, Bacterial Characterization
3. What are the Similarities Between Gram Stain and Acid Fast
     – Outline of Common Features
4. What is the Difference Between Gram Stain and Acid Fast
     – Comparison of Key Differences

Key Terms

Acid Fast Stain, Counter Stain, Gram-Positive Bacteria, Gram Stain, primary Stain

What is Gram Stain

Gram stain is a common type of differential staining method used to distinguish between two large groups of bacteria: Gram-positive and Gram-negative bacteria. Danish microbiologist Hans Christian Gram developed this staining method in 1884. The characteristic feature of bacteria used in the Gram stain is the presence of peptidoglycan in the cell wall. Therefore, the bacteria with peptidoglycan in their cell wall are characterized as Gram-positive bacteria and bacteria without peptidoglycan in their cell walls are characterized as Gram-negative bacteria.

Figure 1: Gram Staining

The steps of the Gram stain are as follows.

1. Applying the primary stain, crystal violet on to a heat-fixed bacterial smear.
2. The addition of Gram’s iodine as a mordant. Here, the mordant is responsible for stabilizing the stain by acting as a trapping agent. Generally, this involves the formation of the crystal violet-iodine complex clump in the thick layers of peptidoglycan in the cell wall. This gives Gram-positive bacteria a purple color.
3. The addition of a decolorizing agent such as ethanol or acetone\ethanol solution, which washes out the dyes remaining in the thin peptidoglycan layers. This makes Gram-negative bacteria colorless.
4. Counterstaining with safranin, which stains the decolorized cells in pink color.

Finally, Gram-positive bacterial cells or crystal-violet stained cells appear in the smear in purple color while Gram-negative cells or safranin-stained cells appear in the smear in red color.

What is Acid Fast Stain

Acid-fast stain is another type of differential staining technique which help to distinguish between Gram-positive bacteria with waxy mycolic acids in their cell walls. Also, Ziehl-Neelsen technique and Kinyoun technique are the two methods of acid-fast staining. The main difference between the two acid-fast staining methods is the use of heat during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the primary stain into the acid-fast cells. However, the Kinyoun method does not use heat.

Figure 2: Acid-Fast Staining

Moreover, the common steps of the acid-fast staining are as follows.

1. Applying the primary stain, carbolfuchsin on to the bacterial smear. Here, the waxy, acid-fast cells retain the red color of the carbolfuchsin.
2. Applying the decolorization agent, which is mainly an acid-alcohol solution. Importantly, the primary stain retains in the acid-fast cells even after the decolorization.
3. Counterstaining with methylene blue, which renders non-acid-fast cells blue.

Therefore finally, the acid-fast cells or carbolfuchsin-stained cells appear in red color while the non-acid-fast cells or methylene blue-stained cells appear in blue color.

Similarities Between Gram Stain and Acid Fast

• Gram stain and acid-fast stain are two types of differential staining methods used to stain bacteria.
• Both methods allow the characterization of Gram-positive bacteria.
• Also, both get the use of multiple stains during the staining process. Therefore, organisms appear in two colors in the stained sample.
• Besides, both staining methods are used in clinical settings to diagnose diseases.

Difference Between Gram Stain and Acid Fast

Definition

Gram stain refers to a staining technique for the preliminary identification of bacteria, in which a violet dye is applied, followed by a decolorizing agent and then a red dye while acid-fast stain refers to a differential stain used to identify acid-fast organisms such as members of the genus Mycobacterium. Thus, this is the main difference between Gram stain and acid fast stain.

Significance

Gram stain is a common technique used to characterize bacteria into two large groups while acid-fast stain is the technique used to differentiate Gram-positive bacteria.

Type of characterization

While gram stain characterizes bacteria with different types of cell walls, acid-fast stain characterizes bacteria with mycolic acid in the cell wall. Hence, this is another difference between Gram stain and acid fast stain.

Primary Stain

The primary stain used in Gram stain is crystal violet while the primary stain used in acid-fast stain is carbofuchsin.

Mordant

Mordant is also a difference between Gram stain and acid fast stain. Iodine is used by the Gram stain as a mordant while no mordant is used in the acid-fast stain.

CounterStain

One other difference between Gram stain and acid fast stain is the counterstain. Safranin is the counterstain used in Gram stain while methylene blue is the counterstain used in the acid-fast stain.

Differentiation

Furthermore, Gram-staining differentiates bacteria as Gram-positive and Gram-negative while acid-fast stain differentiates Gram-positive bacteria as acid-fast and non-acid-fast bacteria. So, this is another difference between Gram stain and acid fast stain.

Appearing

Moreover, in Gram stain, Gram-positive bacteria appear in blue color while Gram-negative bacteria appear in red color. In contrast, in acid-fast stain, acid-fast bacteria appear in red color while non-acid-fast bacteria appear in blue color.

Conclusion

Gram stain is one of the main bacteriological staining method used to distinguish between Gram-positive and Gram-negative bacteria. It is a differential staining technique which uses crystal violet as the primary stain and safranin as the counter stain. In comparison, the acid-fast stain is another differential staining method used to characterize Gram-positive bacteria, especially the members of the genus Mycobacterium. It uses carbofuchsin as the primary stain and methylene blue as the counter stain. However, the main difference between Gram stain and acid fast stain is the type of characterization.

References:

1. “Staining Microscopic Specimens|Microbiology.” Lumen Learning, Lumen, Available Here.

Image Courtesy:

1. “Gram stain 01” By Y tambe – Y tambe’s file (CC BY-SA 3.0) via Commons Wikimedia  
2. “Mycobacterium tuberculosis Ziehl-Neelsen stain 02” By CDC/Dr. George P. Kubica – phil.cdc.gov CDC-PHIL ID #5789 (Public Domain) via Commons Wikimedia