Gram Stain

The Gram stain is a very common technique used to characterize bacteria. This stain will stain bacteria one of two ways depending on the structure of their cell wall. Gram positive bacteria stain purple and have a thick cell wall. Gram negative bacteria stain pink and have a thin cell wall with an additional surrounding lipid membrane called the outer membrane. This difference in structure contributes to differences in general characteristics such as antibiotic sensitivity and hardiness. Therefore, very quickly and easily one can determine some basic characteristics about an "unknown" bacteria.

Overview:

Staining procedures generally have four major steps: smear, dry , heat fix, stain.

1. The first step, smearing, entails putting a very thin coating of bacteria on a slide.

2. The second step, drying, means the coating is allowed to air dry. The sample usually goes from being wet/shiny to being dry/dull. If the sample is not completely dry before continuing, the stain procedure will not work properly.

3. The third step, fixing, involves using either heat or chemicals to stick the bacteria to the slide. Usually the slide is gently passed thru a flame several times.

4. Lastly, the staining procedure is performed. There are a great number of staining procedures ranging from simple stains (methylene blue) that simply stain the whole bacteria, to more complicated stains that stain the various bacteria differently (Gram Stain, Acid-Fast Stain), to specific stains that stain certain parts of the organism (Flagellar Stain, Endospore Stain).

Gram Stain Procedure:

1. Smear a thin coating of sample containing bacteria on a slide. Let it air dry till it is no longer shiny but is "dull" looking.
* The thinner the coating, the faster it will dry. Putting the sample at one end of the slide will make processing easier.

2. Heat fix the bacteria by passing the slide thru a flame. The slide should be in the flame approximately to the count of one-one thousand and passed thru the flame three times. The slide should then be placed in the staining area and allowed to cool.
*If the slide is not fixed long enough the bacteria will wash off the slide during the subsequent steps. If the slide is fixed too long the bacteria will not stain (burnt) or will not stain properly.

3. The sample should be covered with Grams Crystal Violet for one minute and then the stain should be rinsed off with water.
*Bacteria and most organisms will stain purple.

4. The sample should then be covered with Grams Iodine for one minute and then rinsed off with water.
*The iodine is known as a mordant and it will form a complex with the dye.  Its purpose is to help the dye "stick" to the sample and "intensify" the color of the dye. The bacteria will still be purple.

6. The slide is held at an angle and very briefly rinsed with Grams Decolorizer till it appears that no more stain is washing off and then rinsed with water.
*The decolorizer is often a mix of acetone and alcohol. Given a short exposure (3-4 seconds) to the decolorizer, the Gram positive bacteria, due to its structure, will resist the decolorizer and retain most of the stain (mordant-dye complex) while the stain (mordant-dye complex) will be washed out of the gram negative bacteria. However, given enough time the decolorizer will also destain gram positive bacteria. Also, given enough exposure, water will also decolorize the bacteria. At this point, gram positive bacteria are still purple and gram negative bacteria are "clear".

7. The sample should be covered with Grams Safranin for one minute and then rinsed with water.
* This step is known as the counterstain. It is done to make it easier to see the gram negative bacteria. In general, unstained organisms are difficult to see and a counterstain is often used. The gram positve purple stained bacteria remain purple and the gram negative "clear" bacteria become pink in color.

8. The sample is blotted dry..
*The slide is carefully blotted as it is possible to wipe the bacteria off the slide if it is dried too vigorously. The bacteria are small enough that it is usually necessary to use 100x/oil objective (total magnification 1000x) in order to accurately see the bacteria. No coverslip is necessary but once the oil is applied it may not be easily removed without also removing the bacteria. Therefore, if no bacteria are observed at the lower magnifications do not apply the oil.

9.The last step is to  view  the specimen with a microscope.
 
 
 

Questions/problems

1. a. How stable is the gram stain characteristic of a bacteria?
    b. What factors could influence the outcome of the gram stain?

2. How would you explain a gram positive appearing gram negative?

3. How would you explain a gram negative appearing gram positive?

4. How would you explain no bacteria appearing on the slide after staining?