When performing gram staining reaction you noticed you were out of one of the reagents. You still we

Here are the steps to Gram-staining:

- Applying a primary stain (crystal violet) to a heat-fixed (death by heat) smear of a bacterial culture
- Add a trapping agent (Gram's iodine)
- Add alcohol or acetone to rapidly decolor, and ...
- Counterstain with safranin

It can't be crystal violet because that's the first step!

I think the student may have run out of safrainin because after you apply the alcohol, a Gram-negative cell will lose its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. So you can tell that the smear is made up of Gram-negative bacteria if all the colour is gone after alcohol treatment. Thus, after decolorization, the Gram-positive cell remains purple and the Gram-negative cell loses its purple color. The counterstain is used only for optical reasons really, so that the cells become more visible under a microscope.

I hope I answered your Q.

So "I'm still confused, if alcohol was omitted, gram positive organisms will appear purple? and gram negative would be red?

Which is likely to cause some or all of the gram positive bacteria to appear Gram negative?
prolonging the alcohol wash step to 30 min during gram staining
treating the culture w/ .1% lysozyme for 10 min before gram staining
eliminating CV step during gram staining of a fresh culture

When you add alcohol, you remove the outer surface of the bacteria being examined. You need alcohol, and so without it, you wouldn't be able to make an educated guess.

Which textbook (& Edition) is this? I could probably look up the answer. I think Sarah is right, btw.

so if you see red cocci and purple rods, what does that mean?

Why should this matter?

Does that mean one forgot to add alcohol if the result is red cocci and purple rods? can anyone confirm this for me?

In Gram-(+), the alcohol/acetone mixture displaces water in the peptidoglycan layer, resulting in dehydration. This loss of water causes the thick peptidoglycan layer to shrink, tightening the matrix created by the crosslinking of polysaccharides and proteins.

Because of its larger size, the crystal violet-iodine complex is blocked from moving easily through the thick layers of dehydrated peptidoglycan and exiting the bacterial cell. The dye complex remains trapped inside the cell.

It is important to know that this decolorizing step is a critical step in the Gram stain protocol. Exposure to the alcohol for too long can cause cells that are gram-positive to lose too much of the dye complex due to damage to the peptidoglycan layer. These cells will not appear gram-positive when the staining procedure is complete.

The counterstain, normally safranin, is added.  Like crystal violet, safranin is a weakly water-soluble, basic dye that produces cationic stain particles in solution that bind negatively charged moieties such as the techoic acids, peptides and phospholipid heads within the envelope and in the cytoplasm.

Safranin, because of its small size, is able to penetrate the dehydrated peptidoglycan layer and bind to negatively charged moieties. Because the safranin is much lighter in color than the crystal violet-iodine complex, it is not visualized well.

Sarah, I believe, is correct in that safrainin is least important.

A friend interrupted you while you were performing gram staining.  When you looked at the specimen under the light microscope you obtained both red cocci and purple rods.  Based only on this information which of the following statements is correct?  Circle all correct answers.  You must circle at least one answer for this question to be graded.

   a.  There are at least 2 species in this specimen.
   b.  Forgot to add alcohol.
   c.  One of the organisms is gram-negative.
   d.  Forgot to add iodine.
   e.  Added safranin before adding crystal violet.
   f.  Added both crystal violet and safranin before adding iodine.

Is the right answer A and C?

A friend interrupted you while you were performing gram staining.  When you looked at the specimen under the light microscope you obtained both red cocci and purple rods.  Based only on this information which of the following statements is correct?  Circle all correct answers.  You must circle at least one answer for this question to be graded.

   a.  There are at least 2 species in this specimen.
   b.  Forgot to add alcohol.

- I'm assuming they added alcohol because in Gram-(-), adding the alcohol removes the purple. Adding the safranin causes the bacteria to turn red.

   c.  One of the organisms is gram-negative.
   d.  Forgot to add iodine.

- No, not enough information provided for this.

   e.  Added safranin before adding crystal violet.

- No

   f.  Added both crystal violet and safranin before adding iodine.

- No

Is the right answer A and C?