Gram Stain
Last updated 15/01/19
Definition
- Micorbiological staining procedure used to seperate bacteria into two distinct groups
Features
- Developed by Hans Christian Gram to differentiate between Strep pneumoniae and Klebsiella pneumonias
- Staining separates based on chemical and physical properties of cell wall
- Gram positive (G+)
- Gram negative (G-)
- Gram variable from damaged cells
- Mycobacterium
- Cornebacterium
- Actinomyces
- Quicker results than culture (MCS)
Procedure
- Prepare slide smear with culture
- Add Crystal Violet stain
- Let stand for 10-60 seconds
- Wash off Crystal Violet stain with water
- Add Iodine solution
- Fixes stain to the cell wall
- Wash off iodine with water
- Add decoloriser
- Ethanol or Acetone
- Gram negatives will lose the stain
- Gram positives will retain the stain
- Wash off decoloriser with water
- Counterstain with Basic Fuchsin
- Gram positives will not absorb more stain
- Gram negatives will take up Basic Fuchsin stain
- Let stand for 40-60 seconds
- Wash off decoloriser with water
- Dry slide
- View slide under microscope
Gram Negative
- Usually more pathogenic
- Cell Wall
- Often have a lipopolysaccaride (LPS) in cell wall which lead to greater inflammatory response
- Thinner peptidoglycan layer than gram positives (10% of cell wall)
- Absorbs Crystal Violet stain but is removed by decoloriser
- Absorbs and retains Basic Fuchsin stain to appear red pink under microscope
- Cocci
- Neisseria
- Moraxella
- Veilonella
- Bacilli (Rods)
- Medium to long
- Enterobacteriacae
- Bacteroides
- Prevotella
- Fusobacterium
- Pseudomonas
- Capnocytophaga
- Tiny
- Haemophilus
- Acinetobacter
- Moraxella
- Prevotella
- Prophyromonas
- Curved
- Campylbacter
- Helicobacter
Gram Positive
- Usually more sensitive to beta-lactams: Penicillins
- Cell Wall
- Thick mesh like cell wall of peptidoglycan (50-90% of cell wall)
- Absorbs Crystal Violet stain and resists decoloriser
- Dose not absorb Basic Fuchsin
- Remains violet color under microscope
- Cocci
- Clusters
- Staphylcocci
- Micrococcus
- Stomatococcus
- Peptostreptococcus
- Pediococcus
- Aerococcus
- Chains
- Bacilli (Rods)
- Large
- Clostridium
- Bacillus
- Lactobacillus
- Medium
- Corynebacertium
- Cutibacterium
- Listeria
- Bifidobacterium
- Eubacetrium
- Small
- Cornybacterium
- Cutibacterium
- Listeria
- Gardnerella
- Rhodococcus
- Branching
- Nocardia
- Actinomyces
- Streptomyces
- Cutibacterium
References
- Freeman J, Roberts S. Approach to Gram stain and culture results in the microbiology laboratory. UpToDate. 02/10/2018