BIOS242 Lab 5
Name:
Lab 5: Differential Staining
Learning Objectives:
Explain the principle of differential staining
Apply differential staining to identify bacteria
Differential stain uses two or more stains to specifically stain certain structures or cellular components which cannot be easily observed using simple stains.
Differential staining principles are based upon the specific chemical nature and composition of cellular components and therefore, different structures are
observed using different stains and staining procedures. Differential staining often becomes the basis of identification of the bacteria in clinical labs.
In this lab, we will learn about four separate staining procedures.
Gram Staining
Endospore Staining
Capsule Staining (Special Staining)
Exercise 1: Gram Staining
Gram staining is used to identify cells based on the differences of the cell wall. While some bacteria may contain a thick layer of peptidoglycan that forms a thick
rigid cell wall (Gram positive), other bacteria have a very thin layer of peptidoglycan forming a thin cell wall sandwiched between two cell membranes (Gram
negative). The outer membrane is rich in
Bacteria with Bacteria with lipopolysaccharides (LPS). Gram staining
thin cell wall thick cell wall takes advantage of these differences in the
cell wall of bacteria. Using two different
Primary Stain: Crystal
Violet stains that offer a lot of contrast, bacteria
Mordant: Iodine
containing a thick cell wall are stained
1
, Decolorizing agent:
Acetone/Alcohol
BIOS242 Lab 5
Name:
purple. They are called Gram-positive cells. Bacteria containing a thin cell wall are stained pink and are called Gram-negative cells. In Gram staining, bacteria are
first treated with a primary stain, crystal violet. Upon treatment with crystal violet, all cells are stained purple irrespective of presence of thick or thin cell wall.
Stained bacteria are then treated with a mordant, iodine. Iodine
Counterstain: helps crystalize crystal violet on the cell surface and therefore, cells retain crystal violet with
Safranin
higher affinity.
GramAfter that, cells are rinsed with a decolorizing agent, a solution of acetone and alcohol. The decolorizing agent dissolves lipids and peptidoglycan
negative
Gram positive
from both gram cell
positive and gram negative cell walls. However, the thicker peptidoglycan layer of the Gram positive bacteria helps to retain some of the crystal
cell
violet. This step is the most CRITICAL step in the staining process. Over decolorization can completely decolorize Gram positive bacteria as well. Under
decolorization will make Gram negative bacteria appear as Gram positive.
Lastly, bacteria are stained with a counterstain, safranin. Both Gram positive and Gram negative cells will stain with safranin but the purple crystal violet will
override the pink color in Gram positive cells. The Gram negative cells, which are colorless cells after decolorization, will appear pink after using safranin.
Materials:
Broth cultures of S. epidermidis, E. coli, a mixed culture containing one Gram positive and one Gram negative bacteria; sterile loop, Gram staining kit, glass slides,
Incinerator, DI water, marker, immersion oil, lens paper, bibulous paper, microscope
Note to students: Wear gloves and use PPE before starting the lab work. Use aseptic technique to prevent contamination. Use only 1-2 drops of stain per slide.
Avoid using excess.
Method:
1. Obtain glass slides and pure cultures. Write the name of the bacteria on the side of the glass slide.
2. Using aseptic technique make a thin smear. Allow it to air dry and then heat fix it.
3. Add a drop or two of crystal violet and allow staining for 1 minute.
4. Gently wash off crystal violet using a few drops of DI water.
5. Add a few drops of iodine solution to the slide and wait for 1 minute.
6. Wash iodine solution using a few drops of water.
2
Name:
Lab 5: Differential Staining
Learning Objectives:
Explain the principle of differential staining
Apply differential staining to identify bacteria
Differential stain uses two or more stains to specifically stain certain structures or cellular components which cannot be easily observed using simple stains.
Differential staining principles are based upon the specific chemical nature and composition of cellular components and therefore, different structures are
observed using different stains and staining procedures. Differential staining often becomes the basis of identification of the bacteria in clinical labs.
In this lab, we will learn about four separate staining procedures.
Gram Staining
Endospore Staining
Capsule Staining (Special Staining)
Exercise 1: Gram Staining
Gram staining is used to identify cells based on the differences of the cell wall. While some bacteria may contain a thick layer of peptidoglycan that forms a thick
rigid cell wall (Gram positive), other bacteria have a very thin layer of peptidoglycan forming a thin cell wall sandwiched between two cell membranes (Gram
negative). The outer membrane is rich in
Bacteria with Bacteria with lipopolysaccharides (LPS). Gram staining
thin cell wall thick cell wall takes advantage of these differences in the
cell wall of bacteria. Using two different
Primary Stain: Crystal
Violet stains that offer a lot of contrast, bacteria
Mordant: Iodine
containing a thick cell wall are stained
1
, Decolorizing agent:
Acetone/Alcohol
BIOS242 Lab 5
Name:
purple. They are called Gram-positive cells. Bacteria containing a thin cell wall are stained pink and are called Gram-negative cells. In Gram staining, bacteria are
first treated with a primary stain, crystal violet. Upon treatment with crystal violet, all cells are stained purple irrespective of presence of thick or thin cell wall.
Stained bacteria are then treated with a mordant, iodine. Iodine
Counterstain: helps crystalize crystal violet on the cell surface and therefore, cells retain crystal violet with
Safranin
higher affinity.
GramAfter that, cells are rinsed with a decolorizing agent, a solution of acetone and alcohol. The decolorizing agent dissolves lipids and peptidoglycan
negative
Gram positive
from both gram cell
positive and gram negative cell walls. However, the thicker peptidoglycan layer of the Gram positive bacteria helps to retain some of the crystal
cell
violet. This step is the most CRITICAL step in the staining process. Over decolorization can completely decolorize Gram positive bacteria as well. Under
decolorization will make Gram negative bacteria appear as Gram positive.
Lastly, bacteria are stained with a counterstain, safranin. Both Gram positive and Gram negative cells will stain with safranin but the purple crystal violet will
override the pink color in Gram positive cells. The Gram negative cells, which are colorless cells after decolorization, will appear pink after using safranin.
Materials:
Broth cultures of S. epidermidis, E. coli, a mixed culture containing one Gram positive and one Gram negative bacteria; sterile loop, Gram staining kit, glass slides,
Incinerator, DI water, marker, immersion oil, lens paper, bibulous paper, microscope
Note to students: Wear gloves and use PPE before starting the lab work. Use aseptic technique to prevent contamination. Use only 1-2 drops of stain per slide.
Avoid using excess.
Method:
1. Obtain glass slides and pure cultures. Write the name of the bacteria on the side of the glass slide.
2. Using aseptic technique make a thin smear. Allow it to air dry and then heat fix it.
3. Add a drop or two of crystal violet and allow staining for 1 minute.
4. Gently wash off crystal violet using a few drops of DI water.
5. Add a few drops of iodine solution to the slide and wait for 1 minute.
6. Wash iodine solution using a few drops of water.
2
