Central University of Paraguay
Question
Asked 22nd Feb, 2023
What are the common reasons that makes gram-negative bacteria appear as gram-positive bacteria?
How often does this happen in the laboratory?
Most recent answer
There are several reasons why Gram-negative bacteria may appear as Gram-positive bacteria in laboratory tests. Some of the common reasons include:
- Over-decolorization: If the decolorizing step is prolonged, the crystal violet stain can be removed from both the Gram-positive and Gram-negative bacteria, making them both appear as Gram-positive.
- Cell wall damage: The cell wall of Gram-negative bacteria is thinner and more complex than that of Gram-positive bacteria. If the cell wall of a Gram-negative bacterium is damaged, it may become more permeable and take up the crystal violet stain, resulting in it appearing as Gram-positive.
- Culture conditions: The culture conditions can affect the appearance of bacteria. For example, if a Gram-negative bacterium is grown in an environment with a high concentration of peptidoglycan, it may appear as Gram-positive.
- Atypical cell wall structure: Some Gram-negative bacteria have atypical cell wall structures that can cause them to appear as Gram-positive. For example, some bacteria may have a thicker peptidoglycan layer or an outer layer of lipopolysaccharides that can mask the Gram-negative characteristics.
The frequency of Gram-negative bacteria appearing as Gram-positive in the laboratory depends on several factors, including the quality of the staining technique used, the experience of the technician performing the test, and the characteristics of the bacteria being tested. However, with proper staining techniques and trained personnel, the occurrence of Gram-negative bacteria appearing as Gram-positive should be rare.
All Answers (4)
FEU-NRMF
Hi trina!
I read about a study entitled 'Assessing Gram-stain error rates within the pharmaceutical microbiology laboratory' (Sandle, T., 2020) and their results imply that gram-negative bacteria may appear as gram-positive due to the following reasons:
1. The smear made may be thick. This can cause the inability of the gram-negative bacteria to become fully decolorized during the decolorization step, which then makes it appear gram-positive.
2. Iodine solution is inadequate.
3. The smear is not heat-fixed properly.
I hope this helps!
FEU-NRMF
Hi Trina!
Errors in the results of gram staining particularly "gram-negative bacteria appearing as gram-positive bacteria" or vice versa, could include the following:
- Use of antibiotics before collecting a specimen
- Inappropriate age of culture: too young or too old
- Fixing the smear before its dry
- The smear is too thick
- Low concentration of crystal violet
- Excessive heat fixation
- Excessive washing between steps
- Insufficient exposure to iodine
- Prolonged decolorization
- Excessive counterstaining
- Lack of experience in preparing the slide, and reviewing the slide
Reference: Tripathi N, Sapra A. Gram Staining. [Updated 2022 Aug 8]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022
Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK562156/
Central University of Paraguay
The main reason why Gram-negative bacteria may appear Gram-positive is due to either technical errors during the staining process or the presence of atypical cell wall structures.
Some of the technical errors that can result in Gram-negative bacteria appearing Gram-positive include over-decolorization, improper fixation, or using old or contaminated stains. Over-decolorization can cause the crystal violet to be removed from both Gram-positive and Gram-negative bacteria, making them both appear pink under the microscope.
Alternatively, some Gram-negative bacteria may possess atypical cell wall structures that can cause them to appear Gram-positive. For example, some bacteria may produce a thick layer of peptidoglycan or an outer capsule that can trap the crystal violet stain, making them appear purple or blue instead of pink under the microscope.
The occurrence of Gram-negative bacteria appearing Gram-positive in the laboratory is relatively rare if the staining procedure is properly executed. However, technical errors during the staining process or variations in bacterial cell wall structures can lead to false results. Therefore, it is important to carefully follow the staining protocol and properly identify bacterial morphology to avoid misinterpretation of results.
Central University of Paraguay
There are several reasons why Gram-negative bacteria may appear as Gram-positive bacteria in laboratory tests. Some of the common reasons include:
- Over-decolorization: If the decolorizing step is prolonged, the crystal violet stain can be removed from both the Gram-positive and Gram-negative bacteria, making them both appear as Gram-positive.
- Cell wall damage: The cell wall of Gram-negative bacteria is thinner and more complex than that of Gram-positive bacteria. If the cell wall of a Gram-negative bacterium is damaged, it may become more permeable and take up the crystal violet stain, resulting in it appearing as Gram-positive.
- Culture conditions: The culture conditions can affect the appearance of bacteria. For example, if a Gram-negative bacterium is grown in an environment with a high concentration of peptidoglycan, it may appear as Gram-positive.
- Atypical cell wall structure: Some Gram-negative bacteria have atypical cell wall structures that can cause them to appear as Gram-positive. For example, some bacteria may have a thicker peptidoglycan layer or an outer layer of lipopolysaccharides that can mask the Gram-negative characteristics.
The frequency of Gram-negative bacteria appearing as Gram-positive in the laboratory depends on several factors, including the quality of the staining technique used, the experience of the technician performing the test, and the characteristics of the bacteria being tested. However, with proper staining techniques and trained personnel, the occurrence of Gram-negative bacteria appearing as Gram-positive should be rare.
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