Basic microbiology: gram positive and gram-negative organisms

“The colour, the shape and the configuration”

  1. The colour

Gram staining is one of the basic staining techniques used in microbiology laboratories. It was discovered by Hans Christian Gram in 1884. This technique uses bacterial property of having peptidoglycan in their cell wall.

Hence, bacteria lacking a cell wall cannot be stained using Gram stain and are called gram indeterminate- e.g. Mycoplasma.

Gram staining process
1. Make a smear on a glass slide, heat gently to fix it.
2. Put 0.5% crystal violet (blue/purple dye) – 30 sec.
Tilt the slide to drain, and rinse with water carefully. Get rid of the excess water.
3. Put (1%) Lugol’s iodine (to fix the dye) – 30 sec.
Tilt the slide and wash off the iodine with water
4. Decolourise using 95 – 100% ethanol or acetone until colour ceases to run out of the smear – If the bacteria have a large amount of peptidoglycan in their cell wall, i.e. they have a thick cell wall, they will retain the blue dye, these are gram-positive organisms.
Rinse with water.
5. Put a red/pink stain – 0.1% counterstain safranin for 30s-1min(alternative – neutral red, carbol fuschin ) – bacteria with a thin cell wall with a small amount of peptidoglycan, would have lost the blue stain in the previous stage. These will appear red/pink; these are gram negative.
Wash with water and blot dry.
Use oil immersion objective.

Gram staining video from youtube

So, gram-positive organisms are blue/purple, gram-negatives are red/pink.
However, there are some situations where we may see an inconsistent pattern where the bacteria may show both/either colour. These are called gram variables.
It may happen during the growth phase of bacteria when the peptidoglycan content is changing. Some bacteria, however, are notoriously gram-variable – Actinomyces, Corynebacterium etc.

2. The shape

We classify bacteria based on their shape and
Common shapes are –
round = coccus (pl. cocci)
long rod-like = bacillus (pl. bacilli)
(There is a genus of bacteria called Bacillus – do not confuse these two terms. Genus Bacillus is indeed a bacillus/rod-shaped bacterium but, there are other bacteria with this shape e.g. E coli, Klebsiella, Clostridium etc).
However, some bacteria may appear somewhat in the middle of these two categories – they are called coccobacilli. Some bacilli may show branching e.g Actinomyces.

3. The configuration:

The bacteria cells spatial relation to each other can give us an important clue about the genus of bacteria. However, it is difficult to tell the species of bacteria. The proper identification process must be followed in the laboratory.

Here are some examples

Gram-positive cocci in cluster:

Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus simulans etc.

Gram positive cocci in chains:
Streptococcus pyogenes (Group A Strep), Streptococcus agalactiae (Group B Strep), Streptococcus dysgalactiae (Group C/G Strep), Streptococcus gallolyticus (Group D Strep), Streptococcus anginosus (milleri) group.

Gram positive cocci in pairs:

Pneumococcus (Streptococcus pneumoniae), Streptococcus from mouth flora (These are alpha haemolytic Strep – Strep salivarius, Strep sanguinis, Strep oralis, Strep mitis, Strep mutans etc).

Enterococcus (Enterococcus faecalis, E faecium etc) usually present as short chain or in pairs.

Gram positive rod/bacilli

Bacillus cereus, Bacillus anthracis (causes anthrax), Clostridium difficile, Clostridium tetany (causes tetanus), Clostridium botulinum, Clostridium perfringens, Clostridium novyi (causes gas gangrene), Listeria (meningitis, neonatal infection etc), Corynebacterium sp. (causes diphtheria but also some species live on our skin and normally harmless)

Gram negative cocci:

Coccobacilli: Acinetobacter, Moraxella.
Diplococci: Neisseria meningitidis, Neisseria gonorrhoea.

Gram negative rod/bacilli

Escherichia coli, Klebsiella, Enterobacter, Serratia, Salmonella, Shigella, Yersinia, Proteus, Morganella

Some gram negative bacilli may have typical appearance-

Campylobacter (curved gram negative rod)

Vibrio e.g Vibrio cholerae (comma shaped)




Fusobacterium nucleatum (an anaeorobic bacteria from the mouth) – needle shaped