Lab C

Name: Lylene Belle M.
Carcedo
Section: BSN-1B____________________ Date: ________
1. What is the difference between a simple and differential stain?
Simple Stain:
 In a simple stain, only one type of stain is used.

 It involves staining the entire microorganism with a single dye, usually a basic dye like crystal
violet, methylene blue, or safranin.
 Simple stains are used to provide contrast between the microorganism and the background,
making it easier to observe their morphology, size, and arrangement.
 Simple stains do not distinguish between different types of microorganisms; they color all cells
in the sample uniformly.
Differential Stain:
 Differential staining involves using multiple stains to differentiate between different types of
microorganisms or different parts of a single microorganism.
 The most common type of differential stain is the Gram stain, which differentiates between
Gram-positive and Gram-negative bacteria based on differences in cell wall structure.
 Another example of a differential stain is the acid-fast stain, which distinguishes acid-fast
bacteria (such as Mycobacterium species) from non-acid-fast bacteria.
 Differential stains utilize a primary stain, a counterstain, and sometimes decolorizing agents to
achieve differential staining patterns.
 These stains are particularly useful for identifying and classifying bacteria based on their
specific characteristics or structures.
2. Name the reagent used and the purpose of each of the following in the Gram stain.
a. Mordant:
Reagent: Gram's iodine or iodine-potassium iodide solution.
Purpose: The mordant is used to enhance the interaction between the primary stain and the bacterial
cells by forming a complex with the crystal violet or other primary stain. It helps to fix the primary stain
within the thick peptidoglycan layer of Gram-positive bacterial cell walls, aiding in the retention of the
stain during the subsequent decolorization step.
b. Primary stain:
Reagent: Crystal violet or gentian violet.

Purpose: The primary stain is applied first and stains all cells in the sample. In the Gram stain, it
imparts a purple color to both Gram-positive and Gram-negative bacteria. However, it is primarily
retained by Gram-positive bacteria due to their thicker peptidoglycan layer.
c. Decolorizer:
Reagent: Typically, 95% ethanol or ethanol-acetone solution.
Purpose: The decolorizer is used to remove the primary stain from the Gram-negative bacteria while
allowing the primary stain to remain trapped in the thick peptidoglycan layer of Gram-positive
bacteria. This step differentiates between Gram-positive and Gram-negative bacteria based on
differences in cell wall structure. Gram-positive bacteria retain the purple color, while Gram-negative
bacteria lose the purple color and become colorless.
d. Counterstain:
Reagent: Safranin or basic fuchsin.
Purpose: The counterstain is applied after decolorization to stain the Gram-negative bacteria, which
lost the primary stain during decolorization. It imparts a pink or red color to the Gram-negative
bacteria, allowing them to be visualized under the microscope. Gram-positive bacteria retain the
primary stain and do not take up the counterstain, so they remain purple.
3. Which step is the most crucial or most likely to cause poor results in Gram stain? Why?
The decolorization step is the most crucial and most likely to cause poor results in the Gram stain
technique. This step is essential for differentiating between Gram-positive and Gram-negative
bacteria based on their cell wall properties. However, if the decolorization is not performed correctly, it
can lead to inaccurate staining outcomes. Over-decolorization may result in the removal of the
primary stain from Gram-positive bacteria, leading to false-negative results, while insufficient
decolorization can cause Gram-negative bacteria to retain the primary stain, yielding false-positive
results. Thus, precise timing and technique during the decolorization step are paramount for accurate
interpretation of Gram stain results.
4. What is meant by gram variable?
Gram-variable" refers to a classification used to describe bacteria that exhibit characteristics of both
Gram-positive and Gram-negative staining reactions. In Gram staining, bacteria are typically
classified as either Gram-positive or Gram-negative based on the color they retain after staining and
decolorization steps. Gram-positive bacteria retain the crystal violet stain and appear purple, while
Gram-negative bacteria lose the crystal violet stain but retain the counterstain (safranin or basic
fuchsin) and appear pink or red.
However, some bacterial species may not consistently exhibit one staining characteristic over
another. Gram-variable bacteria are those that may appear Gram-positive under certain conditions or
in certain growth phases and Gram-negative under different conditions or growth phases. This
variability in staining reaction can occur due to variations in the thickness of the peptidoglycan layer in
the cell wall, differences in the composition of the outer membrane (in Gram-negative bacteria), or
other factors influencing the uptake and retention of stains during the staining process.
Gram-variable bacteria pose challenges in classification and identification, as their staining

characteristics may not provide clear-cut differentiation. It underscores the importance of considering
other methods, such as biochemical tests or molecular techniques, for accurate identification of these
bacterial species.
5. Which part of the bacterial cell is most involved with Gram staining, and why?
The cell wall of the bacterial cell is the part most involved with Gram staining. This is because the
primary differentiation between Gram-positive and Gram-negative bacteria lies in the structure of their
cell walls. Gram-positive bacteria have a thick layer of peptidoglycan in their cell walls, which retains
the crystal violet-iodine complex during the staining process, imparting a purple color to the cells. In
contrast, Gram-negative bacteria have a thinner layer of peptidoglycan surrounded by an outer
membrane, which is disrupted by the decolorizing agent, causing them to lose the primary stain and
take up the counterstain, appearing pink or red. Therefore, the composition and structure of the cell
wall play a central role in determining the outcome of the Gram stain and are the primary focus of this
staining technique.

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Name: Lylene Belle M.

1. What is the difference between a simple and differential stain?

 In a simple stain, only one type of stain is used.

Reagent: Gram's iodine or iodine-potassium iodide solution.

Reagent: Crystal violet or gentian violet.

Reagent: Typically, 95% ethanol or ethanol-acetone solution.

Reagent: Safranin or basic fuchsin.

Gram-variable bacteria pose challenges in classification and identification, as their staining

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