Gram staining is one of the basic staining technique 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 this method – e.g. Mycoplasma.
Gram staining process
1. Make a smear on a glass slide using bacterial colony, heat fix.
2. Put a crystal violet (blue dye)
3. Put iodine (to fix the dye)
4. Decolourise using ethanol/acetone – If the bacteria have 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.
5. Put a red/pink stain (safranin, neutral red etc) – bacteria with a thin cell wall with small amount of peptidoglycan, would have lost the blue stain in the previous stage. These will appear red, these are gram negative.
So gram positive organisms are blue, gram negatives are red.
Occasionally it becomes difficult in some bacteria as they may stain inconsistently – we call them gram variable (we would exclude them from this discussion).
We classify bacteria based on their shape and how they appear in relation to each other.
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 shaped bacteria but, there are other bacteria with this shape – E coli, Klebsiella, Clostridium etc).
However, some bacteria may appear somewhat in the middle of these two categories – they are called coccobacilli.
These bacteria may form a particular type of configuration, which help us to make an educated guess of their genus. They may form cluster, chains, pairs, Chinese letter appearance. However, it is difficult, if not impossible, to predict the species of gram stain only.
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: Acinetobacter, Moraxella - they can often be seen as coccobacilli Neisseria meningitidis (diplococci - two coccus together), Neisseria gonorrhoea (diplococci).
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
Yeast- like Candida, appears gram positive.