gram stain, microbio

The Gram stain is a…….

differential stain, which allows classification of bacteria into two groups based primarily on a physical difference between cell wall components of bacterial cells.

Gram staining background

technique was discovered by Hans Christian Gram in 1884 and has become the most widely utilized staining procedure used in bacteriology.

Gram staining technique is essential in..

This technique is essential in diagnostic procedures requiring bacterial identification.

Based on the differences on in the structural and chemical composition of ______ _____, bacteria are separated into two groups: gram positive or gram negative.

cell walls

Gram postive cell walls have a thick layer of?

peptidoglycan (50%), with negatively charged teichoic acids weaving through it.

Gram-negative cell walls have a much thinner layer of…

peptidoglycan (15–20%) that is accompanied by an outer membrane composed of lipids

What gives the outer membrane their negative charge?

lipopolysaccharides

The Gram stain uses four chemical reagents, including a

decolorizing agent

Is the Application and removal of the decolorizing agent is a critical step in this staining procedure?

true

Cultures prepared for gram staining must be what phase of growth?

logarithmic phase of growth (no more than 12 to 18 hours of incubation)

What is a good source of nutrition for the cells, and they will end up with holes in the cell wall, in older cultures, which allow them to be decolorized more easily.

peptidoglycan

The cell wall types of gram-positive and- negative cells can be distinguished from one another based on their ability to

retain a primary stain during decolorization with acetone-alcohol after the addition of a “mordant”-gram’s iodine, which complexes with crystal violet crystals in the lattice of peptidoglycan layers

Because of the thick complex, the decoloriser will not be able to remove the crystal violet-iodine complexes in, what kind of cell wall?

gram-positive cell wall, as the thick peptidoglycan layer is dehydrated and traps the dye molecules.

The difference in the staining is due

primarily to a structural difference between the two cell types

What cells do not decolorize readily and retain a purple dye?

gram-positive

What cells decolorize more readily as the lipid outermembrane is disintegrated when acetone-alcohol is added, and due to the thin peptidoglycan layer, crystal violet complexes will be easily decolorised and will appear pink or red after they are counterstained

gram-negative

Overview of gram staining procedure:

1. Apply primary stain (crystal violet, a basic dye that stains all bacteria purple).

2. Apply mordant (Gram’s iodine). The iodine combines with the crystal violet in the cell to form a larger salt complex (CV-I).

3. Apply decolorizing agent (acetone-alcohol mixture). The primary stain is washed out, or decolorized, from some bacteria, while others retain the stain.

4. Apply the counterstain (another basic dye, safranin) to stain the decolorized bacteria red.

Some common sources of errors in the gram staining technique

• Left primary stain on too long.

• The loop was too hot when you made the smear.

• Excessive heat was applied during heat fixing.

• The decolorizer was left on the smear too long.

• The culture was too old (more than 48 hours).

• The smear was too thick.

Performing a gram stain

1. Using aseptic technique, with a sterile metal loop, prepare a heat-fixed smear of both bacteria mixed together on a single, labeled glass slide.
Note: The gram stain takes practice, so prepare multiple slides. It is also important to let the smear air dry completely before heat fixing. Heat fixing wet smears can cause the bacteria to pop and clump together, making staining and observation difficult. This is another reason to prepare multiple slides so you do not waste time waiting for them to dry later!

2. Hold your slide with a slide holder or place it on a rack over a specimen dish and flood the smear with crystal violet stain for 60 seconds.

3. Pour off the stain and gently rinse the excess stain with a stream of water from a plastic water bottle. Note that the objective of this step is to wash off the stain, not the fixed culture.

4. Then flood the smear with iodine solution for 60 seconds.

5. Pour off the iodine solution and gently rinse the slide with running water.

6. Then hold the slide with the clothespin over the staining dish and drip decolorizer across the top of the slide, letting it run down the slide for no longer than 15 seconds. Rinse immediately again with water.

Note: DO NOT overdecolorize, as the solution will eventually remove all the stain from any cell if it is left on long enough.

7. Counterstain with safranin solution for 60 seconds.

8. Rinse off the safranin solution with water. Blot the slide gently with bibulous paper to remove the excess water.

9. Immediately wash off any spilled stain with liberal amounts of water and decolorizer to avoid leaving permanent marks in the sink, lab bench, or glassware.

10. Examine the finished slide under a microscope to determine which specimen is gram positive (purple) and which is gram negative (pink/red). Note the cell morphology and cell arrangement of your cultures.

• Note: To help you remember which color is gram negative and gram positive, call them purple (positive) and red (negative). Obviously purple and pink won’t work!
  
  

11. Draw the Gram’s stain bacteria you observed under the oil immersion lens in the space provided in the "Exercise 6: Results" page found in the Exercise 6: Gram Stain folder in the menu. Use colored pens or pencils to indicate which cells are gram positive or gram negative. Make a drawing of a representative microscopic field. Describe the cells according to their morphology and arrangement.

Which of the following choices lists the reagents for the Gram stain in the correct order they are used when staining bacteria?

Crystal violet, Gram's iodine, Acetone alcohol, Safranin

What is the Gram stain result for the red bacillus-shaped bacteria in the image

gram negative

Which step of the Gram stain procedure is considered the most critical step to ensure accurate results?

Acetone alcohol decolorizer

safranin

counterstain

crystal violet

primary stain

acetone alcohol

decolorizer

gram’s iodine

mordant