The Gram Stain
   
Introduction

Gram's Stain is a widely used method of staining bacteria as an aid to their identification. It was originally devised by Hans Christian Joachim Gram, a Danish doctor. Historical note.

Gram's stain differentiates between two major cell wall types. Bacterial species with walls containing small amounts of peptidoglycan and, characteristically, lipopolysaccharide, are Gram-negative whereas bacteria with walls containing relatively large amounts of peptidoglycan and no lipopolysaccharide are Gram-positive.

 
It's a mystery

Although it may seem strange, the reason why bacteria with these two major types of bacteria cell walls react differently with Gram's stain appears to be unconnected with the wall structure itself. The exact mechanism of the staining reaction is not fully understood, however, this does not detract from its usefullness.
   
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. 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.

3. 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.

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.

    

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

  

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    
Gram positive
Staphylococcus epidermidis
 
Typical Gram-positive bacteria

1.

staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which is a common cause of boils

2.

streptococci such as the many species of oral streptococci, Streptococcus pyogenes which causes many a sore throat and scarlet fever and Streptococcus pneumoniae which causes lobar pneumonia

3.

clostridia such as Clostridium tetani which cause tetanus (lockjaw)

4.

actinomyces such as Actinomyces odontolyticus which is found in mouths

5.

species of the genus Bacillus such as Bacillus subtilis which are common microbes living in soil
 
 
Gram negative
Escherichia coli
 
Typical Gram-negative bacteria

the bacilli that cause

1.

whooping cough, Bordetella pertussis

2.

typhoid, Salmonella typhi

3.

cholera, Vibrio cholerae

4.

the normally benign, ubiquitous, gut-dwelling Escherichia coli

Generally cocci are Gram-positive but there are exceptions. The most significant from a clinical point of view is the gonococcus, Neisseria gonorrhoea which typically appears as a Gram-negative diplococcus looking very much like a pair of kidney bean.

 
 
 
 

And finally

It is important to recognise that not all species of bacteria can be usefully stained by Gram's method. Some species are Gram variable and some what is called Gram indeterminant. It is also worth mentioning that the growth conditions may also affect a bacterial species Gram reaction. For example, Gram positive cells growing in batch culture which are into the decline phase of the growth cycle often show numerous Gram negative cells present.

Another major exception is the genus Mycobacterium which includes such important human pathogens as Mycobacterium tuberculosis (TB) and Mycobacterium leprae (leprosy). Bacteria such as these have very different cell walls which contain a great deal of waxy material, although they still contain peptidoglycan. This waxy material prevents stain penetration rendering the cells invisible.

Special methods have been devised to stain these bacteria involving the use heat to allow the stain to permeate the cell walls. Once cooled even dilute acid fails to remove it hence the name "acid-fast" to describe these species of bacteria.

Since these acid-fast bacteria are not stained by Gram's method they have been described as "Gram-negative". Strictly speaking this is true but it can be misleading to refer to them this way.

 

 

 

Footnote

The division of all life into prokaryotes and eukaryotes was challenged by Carl Woese in 1977. He pioneered the use of 16S ribosomal RNA in the study of taxonomy which was a major breakthrough in our understanding of the relationships between all forms of life and used this technique to reveal major differences in prokaryotic micro-organisms.

In 1990 Woese proposed that the existing Kingdom of Prokaryotes be divided into Archaea and Bacteria. Based on his research, which showed that these two groups evolved quite independently from a common ancestor, the consensus view now is that the Kingdoms called Prokaryota and Eukaryota be replaced by three domains (sometimes called Superkingdoms or Empires) called Archaea, Bacteria and Eukarya.

Back to "And finally"

 

  

 

 

 

SUMMARY

1.

Although apparently unconnected with the bacterial cell wall structure, Gram's stain differentiates between two major cell wall types. Bacterial species with walls containing small amounts of peptidoglycan and, characteristically, lipopolysaccharide, are Gram-negative whereas bacteria with walls containing relatively large amounts of peptidoglycan and no lipopolysaccharide are Gram-positive.

2.

Examples of Gram-negative bacteria are Escherichia coli, Salmonella typhi, Vibrio cholerae and Bordetella pertussis

3.

Examples of Gram-positive bacteria are Staphylococcus epidermidis, Streptococcus pyogenes, Actinomyces odontolyticus and Clostridium tetani

4.

Not all bacteria can be stained by Gram's method, the best-known exception belong to the genus Mycobacterium which have waxy cell walls.
   

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