Counter Stain (Safranin
Safranin
Safranin is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic counterstain in both Gram stains, and endospore staining. It can also be used for the detection of cartilage, mucin and mast cel…
Why is counter staining necessary in Gram staining?
The safranin is also used as a counter-stain in Gram’s staining. In Gram’s staining, the safranin directly stains the bacteria that has been decolorized. With safranin staining, the gram-negative bacteria can be easily distinguished from gram-positive bacteria.
What is the counter stain in Gram staining?
What is the primary stain and counterstain stain used in the Gram stain procedure?
- Crystal Violet, the primary stain.
- Iodine, the mordant.
- A decolorizer made of acetone and alcohol (95%)
- Safranin, the counterstain.
What is used as a mordant in the Gram stain?
The proper sequence of reagents in the Gram stain procedure:
- Crystal violet.
- Iodine.
- Ethanol.
- Safranin.
What is the purpose of the primary stain in Gram staining?
The Gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. The primary stain ( crystal violet) binds to peptidoglycan, coloring cells purple. Both gram-positive and gram-negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet.
What is the function of the counterstain safranin in the Gram stain?
The safranin is also used as a counter-stain in Gram's staining. In Gram's staining, the safranin directly stains the bacteria that has been decolorized. With safranin staining, gram-negative bacteria can be easily distinguished from gram-positive bacteria.
What is the role of counter stain reagent in differential staining procedure?
The fi- nal reagent, the counterstain, has a contrasting color to that of the primary stain. Following decoloriza- tion, if the primary stain is not washed out, the coun- terstain cannot be absorbed and the cell or its com- ponents will retain the color of the primary stain.
How does counterstain work?
Strain Counterstrain is a manual therapy technique, meaning clinicians use only their hands for treatment of muscle and joint pain. It uses passive body positioning of hypertonic (spasmed) muscles and dysfunctional joints toward positions of comfort or tissue ease that compress or shorten the offending muscle.
What is the primary and counterstain for the Gram stain?
The final step in gram staining is to use basic fuchsin stain to give decolorized gram-negative bacteria pink color for easier identification. It is also known as counterstain. Some laboratories use safranin as a counterstain; however, basic fuchsin stains gram-negative organisms more intensely than safranin.
What does it mean to counterstain?
counterstain. / (ˈkaʊntəˌsteɪn) / verb microscopy. to apply two or more stains in sequence to (a specimen to be examined), each of which colours a different tissue. (tr; usually passive) to apply (one of a series of stains) to a specimen to be examinedhaematoxylin is counterstained with eosin.
Which reagent is used as a counter stain?
The most common counterstain is safranin, which colors decolorized cells pink. An alternate counterstain is basic fuchsin, which gives the decolorized cells more of a bright pink or fuchsia coloration.
Which stain reagent is the counterstain?
Answer and Explanation: The reagent that is used as a counter stain in gram staining is called safranin. This stain is pink and is added at the last step of the gram-staining process after decolorization with alcohol has taken place.
What is the purpose of a differential staining process?
Differential staining is a procedure where more than one dye is used to differentiate between different types of microorganisms on a slide. This type of staining helps to differentiate between cell types and cell structures.
What is the counter or secondary stain used in the acid-fast stain?
Methylene blueMethylene blue or potassium permanganate is used as a counterstain to provide background color.
What is the objective of gram stain?
This test differentiates the bacteria into Gram-Positive and Gram-Negative Bacteria, which helps in the classification and differentiation of microorganisms. The Gram stain separates bacteria into two groups: (1) Gram-positive microorganisms that retain the primary dye (Crystal violet) and ...
What is Gram Staining?
Gram staining is a differential bacterial staining technique used to differentiate bacteria into Gram Positive and Gram Negative types according to their cell wall composition.
How to stain a specimen with gram stain?
The procedure/steps of Gram Stain. Prepare and fix the specimen to the microscope slide before staining. Cover the smear with crystal violet, the primary stain, for 20 seconds. Gently rinse off the stain with water. Cover the smear with Gram’s iodine, the mordant, for 1 minute. Pour off the excess Gram’s iodine.
How to do Gram stain?
The procedure/steps of Gram Stain 1 Prepare and fix the specimen to the microscope slide before staining. 2 Cover the smear with crystal violet, the primary stain, for 20 seconds. 3 Gently rinse off the stain with water. 4 Cover the smear with Gram’s iodine, the mordant, for 1 minute. 5 Pour off the excess Gram’s iodine. 6 Run the acid-alcohol decolorizer over the smear until the solution appears clear. 7 Gently rinse with water. 8 Cover the smear with safranin, the secondary or counterstain, for 20 seconds. 9 Gently rinse the stain with water. 10 Blot dry with bibulous paper.
What is the primary color of Gram stain?
Gram-positive organisms contain a highly cross-linked layer of peptidoglycan that retains the primary dye, crystal violet (CV), following the application of the mordant, iodine (I). The iodine and crystal violet form ...
What stain is used to make gram negative cells pink?
The cells appear colorless. To make the colorless cells visible, a secondary stain, safranin, is applied, leaving the gram-negative cells pink. Created with BioRender.com.
How long to cover a smear with iodine?
Cover the smear with Gram’s iodine, the mordant, for 1 minute.
How does Gram stain work?
How the Gram Stain Works 1 The primary stain ( crystal violet) binds to peptidoglycan, coloring cells purple. Both gram-positive and gram-negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet. 2 Gram's iodine ( iodine and potassium iodide) is applied as a mordant or fixative. Gram-positive cells form a crystal violet-iodine complex. 3 Alcohol or acetone is used to decolorize the cells. Gram-negative bacteria have much less peptidoglycan in their cell walls, so this step essentially renders them colorless, while only some of the color is removed from gram-positive cells, which have more peptidoglycan (60-90% of the cell wall). The thick cell wall of gram-positive cells is dehydrated by the decolorizing step, causing them to shrink and trapping the stain-iodine complex inside. 4 After the decolorizing step, a counterstain is applied (usually safranin, but sometimes fuchsine) to color the bacteria pink. Both gram-positive and gram-negative bacteria pick up the pink stain, but it is not visible over the darker purple of the gram-positive bacteria. If the staining procedure is performed correctly, gram-positive bacteria will be purple, while gram-negative bacteria will be pink.
What is Gram staining?
The Gram stain is a differential method of staining used to assign bacteria to one of two groups (gram-positive and gram-negative) based on the properties of their cell walls. It is also known as Gram staining or Gram's method.
What is the primary stain for Gram positive bacteria?
The primary stain ( crystal violet) binds to peptidoglycan, coloring cells purple. Both gram-positive and gram-negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet. Gram's iodine ( iodine and potassium iodide) is applied as a mordant or fixative. Gram-positive cells form a crystal violet-iodine complex.
How to get a crystal violet stain off a slide?
If too little heat is applied, the bacteria will wash off the slide during staining. Use a dropper to apply the primary stain (crystal violet) to the slide and allow it to sit for 1 minute. Gently rinse the slide with water no longer than 5 seconds to remove excess stain.
What is the primary stain for Gram staining?
The Gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. The primary stain ( crystal violet) binds to peptidoglycan, coloring cells purple. Both gram-positive and gram-negative ...
Why is Gram stain important?
Because the bacteria are colored, not only is their Gram stain group identified, but their shape, size, and clumping pattern may be observed . This makes the Gram stain a valuable diagnostic tool for a medical clinic or lab.
What is the process of dehydrating gram positive cells?
The thick cell wall of gram-positive cells is dehydrated by the decolorizing step, causing them to shrink and trapping the stain-iodine complex inside. After the decolorizing step, a counterstain is applied (usually safranin, but sometimes fuchsine) to color the bacteria pink.
What is Gram staining?
Gram staining is initially established by the physician Hans Christian Gram, which was from Denmark. He help to distinguish Klebsiella pneumonia to pneumococci. In short, the process of gram staining comprises the use of a solution of Gram iodine or Potassium iodide to the cells which are use to stain before with Crystal violet or Gentian violet.
What are the structural changes between the cell walls of Gram positive bacteria and Gram negative answerable in a way for the?
Some the structural changes between the cell walls of gram positive bacteria and gram negative answerable in a way for the Gram stain response ? In the Gram staining, an impenetrable crystal violet with gram iodine complexes is made inside the cell of bacteria, and this complexes is extracted by alcohol from gram negative bacteria but not from gram positive. Gram positive organisms are dehydrated by alcohol, they have very dense cell wall containing of many coatings of peptido-glycan. This creates the pores/holes in the cell wall to near, avoiding the insoluble crystal violet + gram iodine complexes from evasion. In gram negative alcohol eagerly infiltrates the lipid rich external layer, and the thin peptido-glycan coating does not stop solvent channel, therefore, the crystal violet + gram iodine complexes is simply detached or removed.
How to make Gram positive and Gram negative cells purple?
Both Gram-positive and Gram-negative cells will be stained purple by the crystal violet dye. Add several drops of iodine to the smear and allow it to sit for 1 minute. Rinse the slide with water. Iodine “sets” the crystal violet, so both types of bacteria will remain purple.
What color are Gram positive cells?
Gram-positive cells resist decolorization and remain purple. The dye is released from Gram-negative cells. Counterstain (safranin) Add several drops of safranin to the smear and allow it to sit for one minute. Rinse the slide with water and blot dry. Gram-negative cells will be stained pink by the safranin.
What is Gram positive bacteria?
Bacteria retain the crystal violet and gram iodine mixtures complexes after the wash in 95% ethanol solution in purple color and are named as Gram positive Bacteria, those which miss these complexes of solution are red in color by counter stain the safranin or 10% fuchsine are named as Gram negative Bacteria. Gram Staining Results.
How many bacteria are in a glass slide?
To be observable on a glass slide, bacteria which are stained by the Gram staining procedure, it is essentially that concentrations of at least of 104 – 10 bacteria/micro liter of concentrated staining reagent. At lesser concentrations, the Gram stain solution of a clinical samples infrequently tells bacteria/organisms even, ...
How to get rid of a smear on the back of the hand?
Pour the slide with Crystal violet or Gentian solution for 1 Min or 60 Seconds. Rinse the slide in running tap water drop by drop on the back of the hand on slide so that the smear will not wash away. Pour the slide with Gram’s iodine for at least 2 Min or 180 Seconds. Again rinse the slide in running tap water.
What color are Gram positive cells?
C) Gram-positive cells would be purple, and Gram-negative cells would be pink.
Which stain is masked by the darker crystal violet stain?
C) Safranin penetrates the cell wall, but is masked by the darker crystal violet stain.
How long to cover a smear with decolorizing agent?
C) Cover the smear with decolorizing agent for 30 seconds.
What happens if you skip step of methylene blue?
If we skip step of methylene blue, the acid fast will remain red but the and the non acid fast will remain colorless. You INCORRECTLY perform an acid-fast stain on a smear from a mixed culture of Mycobacterium smegmatis (acid-fast bacilli) and Staphylococcus aureus (non-acid-fast cocci).
Which agent forms a complex with the lipopolysaccharide in the outer membrane?
C) The decolorizing agent forms a complex with the lipopolysaccharide in the outer membrane.
Which agent forms a complex with the peptidoglycan?
A) The decolorizing agent forms a complex with the peptidoglycan.
What happens to peptidoglycan as bacteria age?
As bacterial cells age, their peptidoglycan begins to break apart. What would be the effect on decolorization?