Gram – staining Technique Dr Maliha Sumbul

For smears to provide valuable information, they must be stained well PLUS: Labelled clearly Spread evenly Air-dry completely (protect from dust) Fixed with care – (heat, alcohol, other chemicals)

Who could have guessed that a staining procedure devised more than a century ago would still serve as one of the most widespread methods of bacterial classification? Microbiologists of this high-tech age are still indebted to Danish physician Christian Gram, who invented the gram-staining method in 1884 In 1882, Paul Ehrlich invented the acid-fast stain.

Hans Christian Gram Name: Hans Christian Joachim Gram Birth Date: 1853 Death Date: 1938 Nationality: Danish Gender: Male Occupations: pharmacologist Hans Christian Gram

Structural Basis for the Gram Stain

Gram positive bacteria have a thick cell wall on the exterior that picks up the purple Gram stain (named after Hans Christian Gram) Gram negative bacteria, such as the E. coli cell shown in the figure, do not stain with the dye because the thinner cell wall lies between the inner and outer membranes

The difference between the two cell types appears to be in the amount of peptidoglycan in the cell wall Gram positive cell walls are about five times as rich in peptidoglycan as gram-negative cell walls In addition, gram-negatives have a second membrane (chemically different from the plasma membrane) external to the cell wall, and may also have a gelatinous sheath external to the second membrane

showing the thick peptidoglycan cell wall (cw), . Electron micrograph of a thin section of the Gram-positive M. lysodeikticus showing the thick peptidoglycan cell wall (cw), underlying cytoplasmic (plasma) membrane (cm), mesosome (m), and nucleus (n). (B) Freeze-fractured Bacteriodes cell showing typical major convex fracture faces through the inner (im) and outer (om) membranes. Bars = 1 µm; circled arrow in B indicates direction of shadowing.

Gram-positive anthrax bacteria (purple rods) in cerebrospinal fluid sample. If present, a Gram-negative bacterial species would appear pink. (The other cells are white blood cells).

Staphylococcus (Gram positive) Escherichia coli (Gram negative)

How it was evolved ? In 1884, Hans Christian Gram, a Danish doctor working in Berlin, accidentally stumbled on a method which still forms the basis for the identification of bacteria. While examining lung tissue from patients who had died of pneumonia, he discovered that certain stains were preferentially taken up and retained by bacterial cells. Over the course of the next few years, Gram developed a staining procedure which divided almost all bacteria into two large groups - the Gram stain

Staining mechanism Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which stains purple while gram-negative bacteria have a thinner layer (10% of cell wall), which stains pink. Gram-negative bacteria also have an additional outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space.

There are four basic steps of the Gram stain, which include: applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture, followed by the addition of a trapping agent (Gram's iodine), rapid decolorization with alcohol or acetone, counterstaining with safranin or basic fuchsin.

Mordant: serve to fix colors Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – ) ions. These ions penetrate through the cell wall and cell membrane of both gram-positive and gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and therefore color from the cell When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram-negative cell will lose its outer membrane and the lipopolysaccharide layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain will be removed from both gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds) After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized gram-negative bacteria a pink or red color Mordant: serve to fix colors

Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of pink and purple cells are seen The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells In cultures of Bacillus and Clostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain

The Gram stain is the most important and universally used staining technique in the bacteriology laboratory. It is used to distinguish between gram-positive and gram-negative bacteria, which have distinct and consistent differences in their cell walls. Gram-positive cells may become gram negative through mechanical damage, conversion to protoplasts, or aging, in which autolytic enzymes attack the walls.

The Gram stain procedure is as follows:  Place a slide with a bacterial smear on a staining rack. STAIN the slide with crystal violet for 30 secs – 60 secs Pour off the stain. Note: fingers stain Gram-positive - use forceps! Flood slide with Gram's iodine for 30 – 60 secs Pour off the iodine. Decolourize by washing the slide briefly with acetone (2-3 seconds). Wash slide thoroughly with water to remove the acetone - do not delay with this step. Flood slide with safranin counterstain for 2 min Wash with water. Blot excess water and dry in hand over bunsen flame.

washed briefly with 95% alcohol (destained), In other words In the Gram stain, the cells are first heat fixed and then stained with a basic dye, crystal violet, which is taken up in similar amounts by all bacteria. The slides are then treated with an I2-KI mixture (mordant) to fix the stain, washed briefly with 95% alcohol (destained), finally counterstained with a paler dye of different color (safranin). Gram-positive organisms retain the initial violet stain, while gram-negative organisms are decolorized by the organic solvent and hence show the pink counterstain. The difference between gram-positive and gram-negative bacteria lies in the ability of the cell wall of the organism to retain the crystal violet

The Gram staining method  1. A small sample of a bacterial culture is removed from a culture. In this example it is being taken from a broth culture of the pure microbe but it could be removed from a culture on solid medium or from material containing bacteria eg faeces or soil. 

2. The bacterial suspension is smeared onto a clean glass slide 2. The bacterial suspension is smeared onto a clean glass slide. If the bacteria have been removed from a culture on solid media or it is from a soil or faeces sample it will have to be mixed with a drop of bacteria-free saline solution.

The bacterial smear is then dried slowly at first and then, when dry, heated for a few seconds to the point when the glass slide is too hot to handle. This fixes ie kills the bacteria making the slide safe to handle. Care must be taken not to overheat which will char the cells.

4. Once cool, the slide is transferred to a support over a sink and flooded with a stain called Gentian Violet (a dye consisting of a methyl derivative of pararosaniline). The stain is left on the slide for about 1 minute. This stains all the bacteria on the slide a dark purple colour. Note, this stain will not penetrate the waxy cell walls of some bacteria eg mycobacteria

5. The Gentian Violet is gently washed off the slide with running water

6. The bacterial smear is then treated with Gram's solution which consists of 1 part iodine, 2 parts potassium iodide, and 300 parts water. This iodine solution reacts with the Gentian Violet turning it a very dark shade of blue. It also causes it to be retained by certain types of bacteria in a way which is not really understood.

7. After about 30 seconds the slide is gently rinsed with ethyl alcohol which causes the dye-iodine complex to be washed out of some bacteria but not others. This is called decolourisation

8. This is achieved by treating the smear with a compound which stains the Gram-negative cells a colour which contrasts markedly with the blue-black colour of the Gram-positive cells. The stain common used for this is either eosin or fuchsin, both of which are red. These are called counterstains. Bacteria in the smear which are Gram-positive are unaffected by the counterstain

9. The counter stain is left on the smear for about 30-60 seconds and then gently rinsed away with running water.

If we now looked at the smear down a microscope, the bacteria which had retained the Gentian Violet-iodine complex will appear blue-black. These are called Gram-positive. However we would not be able to see those which had lost the dye-iodine complex which are called Gram-negative. The final step in the Gram stain method is, therefore, to stain the Gram-negative cells so they can be seen.

10. After the counterstain has been rinsed off, the slide is placed between some absorbent paper and the excess water gently blotted off. Care must be taken not to rub the slide with the blotting paper because this would remove the adhering bacteria.

11. The slide is gently warmed to drive off any residual moisture and then a drop of immersion oil is placed on the stained bacterial smear. This helps transmit light through the specimen directly to the high-powered microscope lens.

12. The slide is then placed on a microscope stage and the oil-immersion lens lowered into the immersion oil. High-powered lenses are required because bacteria are very small

The results Gm + Staph epidermidis Gm – E coli

Technique: Transfer a loopful of the liquid culture to the surface of a clean glass slide, and spread over a small area. Two to four cultures may be stained on the same slide, which can be divided into 2-4 sections with vertical red wax pencil lines. To stain material from a culture growing on solid media, place a loopful of tap water on a slide; using a sterile cool loop transfer a small sample of the colony to the drop, and emulsify. Allow the film to air dry. Fix the dried film by passing it briefly through the Bunsen flame two or three times without exposing the dried film directly to the flame. The slide should not be so hot as to be uncomfortable to the touch. Flood the slide with crystal violet solution for up to one minute. Wash off briefly with tap water (not over 5 seconds). Drain. Flood slide with Gram's Iodine solution, and allow to act (as a mordant) for about one minute. Wash off with tap water. Drain. Remove excess water from slide and blot, so that alcohol used for decolorization is not diluted. Flood slide with 95% alcohol for 10 seconds and wash off with tap water. (Smears that are excessively thick may require longer decolorization. This is the most sensitive and variable step of the procedure, and requires experience to know just how much to decolorize). Drain the slide. Flood slide with safranin solution and allow to counterstain for 30 seconds. Wash off with tap water. Drain and blot dry with bibulous paper. Do not rub. All slides of bacteria must be examined under the oil immersion lens. Note: To remove immersion oil from a slide without damaging the smear, lay a piece of lens tissue on the slide, add a drop or two of xylene and draw the lens tissue across the slide. Repeat if necessary.

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