Gram-positive bacteria are identified by their ability to retain crystal violet stain due to their thick peptidoglycan cell wall. Their cell walls lack the outer membrane found in Gram-negative bacteria. Gram-positive bacteria are traditionally classified based on cell shape and staining, though modern classification relies on molecular analysis of 16S rRNA. While Gram staining can be used to broadly classify bacteria, exceptions exist and the distinction between monoderm and diderm cell structure provides a more reliable classification system.

1. Gram-positive bacteria
Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in
contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up
the counterstain (safranin or fuchsine) and appearing red or pink. Gram-positive organisms are able
to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Gram-
positive cell walls typically lack the outer membrane found in Gram-negative bacteria.
Classification
The following characteristics are generally present in a Gram-positive bacterium:[1]
1. cytoplasmic lipid membrane
2. thick peptidoglycan layer
o teichoic acids and lipoids are present, forming lipoteichoic acids, which serve to act
as chelating agents, and also for certain types of adherence.
3. capsule polysaccharides (only in some species)
4. flagellum (only in some species)
o if present, it contains two rings for support as opposed to four in Gram-negative
bacteria because Gram-positive bacteria have only one membrane layer.
5. The individual peptidoglycan molecules are cross-linked by pentaglycine chains by a DD-
transpeptidase enzyme. In gram-negative bacteria, the transpeptidase creates a covalent
bond directly between peptidoglycan molecules, with no intervening bridge.
Both Gram-positive and Gram-negative bacteria may have a membrane called an S-layer. In
Gram-negative bacteria, the S-layer is attached directly to the outer membrane. In Gram-
positive bacteria, the S-layer is attached to the peptidoglycan layer. Unique to Gram-positive
bacteria is the presence of teichoic acids in the cell wall. Some particular teichoic acids,
lipoteichoic acids, have a lipid component and can assist in anchoring peptidoglycan, as the
lipid component is embedded in the membrane.
Classification
Along with cell shape, Gram staining is a rapid diagnostic tool of use to group species of
Bacteria. In traditional and even some areas of contemporary microbiological practice, such
staining, alongside growth requirement and antibiotic susceptibility testing, and other
macroscopic and physiologic tests, forms the full basis for classification and subdivision of
the Bacteria (e.g., see Figure, and pre-1990 versions of Bergey's Manual).

2. Species identification hierarchy in clinical settings.
As such, historically, the kingdom Monera was divided into four divisions based primarily on
Gram staining: Firmicutes (positive in staining), Gracillicutes (negative in staining),
Mollicutes (neutral in staining) and Mendocutes (variable in staining).[2]
Since 1987 and the seminal 16S ribosomal RNA phylogenetic studies of Carl Woese
(Department of Microbiology, University of Illinois) and collaborators and colleagues, the
monophyly of the Gram-positive bacteria has been challenged,[3] with striking productive
implications for the therapeutic and general study of these organisms. Based on molecular
studies of 16S sequences, Woese recognised twelve bacterial phyla, two being Gram-
positive: high-GC Gram-positives and low-GC Gram-positives (where G and C refer to the
guanine and cytosine content in their genomes),[3] which are now referred to by these names,
or as Actinobacteria and Firmicutes. The former, the Actinobacteria, are the high GC content
Gram-positive bacteria and contains genera such as Corynebacterium, Mycobacterium,
Nocardia and Streptomyces. The latter, the Firmicutes are the "low-GC" Gram-positive
bacteria, which actually have 45%–60% GC content but lower than that of the
Actinobacteria,.[1] The Firmicutes contain the well-known genera that are majority of Gram-
positives of medical interest: Staphylococcus, Streptococcus, Enterococcus (cocci), Bacillus,
Clostridium and Listeria (bacilli/rods). This group also been expanded to include the
Mycoplasma, or Mollicutes, bacteria-like organisms that lack cell walls and cannot be Gram-
stained, but appear to have derived evolutionarily from such forms.

3. Despite the wide acceptance and practical record of utility of the new molecular phylogeny, a
small group, including Cavalier-Smith, still treat the Monera as a monophyletic clade and
refer to the group as division "Posibacteria".[4]
Importance of the Outer Cell Membrane in Bacterial Classification
It is important to point out that although the bacteria are traditionally divided into two main
groups, Gram-positive and Gram-negative, based upon their Gram-stain retention property,
this classification system is ambiguous as it refers to three distinct aspects (staining result,
cell-envelope organization, taxonomic group), which do not necessarily coalesce for some
bacterial species.[5][6][7][8] The Gram-positive and Gram-negative staining response is also not
a reliable characteristic as these two kinds of bacteria do not form phylogenetic coherent
groups.[5] However, although Gram-staining response of bacteria is an empirical criterion, its
basis lies in the marked differences in the ultrastructure and chemical composition of the two
main kinds of prokaryotic cells that are found in nature. These two kinds of cells are
distinguished from each other based upon the presence or absence of an outer lipid
membrane, which is a more reliable and fundamental characteristic of the bacterial cells.[5][9]
All Gram-positive bacteria are bounded by only a single unit lipid membrane and they
generally contain a thick layer (20-80 nm) of peptidoglycan responsible for retaining the
Gram-stain. A number of other bacteria which are bounded by a single membrane, but which
stain Gram-negative due to either lack of the peptidoglycan layer (viz., mycoplasmas) or their
inability to retain the Gram-stain due to their cell wall composition, also show close
relationship to the gram-positive bacteria. For the bacterial (prokaryotic) cells that are
bounded by a single cell membrane the term Monoderm Bacteria or Monoderm
Prokaryotes has been proposed.[5][5][9] In contrast to Gram-positive bacteria, all archetypical
Gram-negative bacteria, are bounded by both a cytoplasmic membrane as well as an outer
cell membrane and they contain only a thin layer of peptidoglycan (2-3 nm) in between these
two membranes. The presence of both inner and outer cell membranes defines a new
compartment in these cells, the periplasmic space or the periplasmic compartment. These
bacteria/prokaryotes have been designated as Diderm Bacteria.[5][5][9] The distinction
between the monoderm and diderm prokaryotes is also supported by conserved signature
indels in a number of important proteins (viz. DnaK, GroEL).[5][6][9][10] Of these two
structurally distinct groups of prokaryotic organisms, monoderm prokaryotes are indicated to
be ancestral. Based upon a number of different observations including that the Gram-positive
bacteria are the major producers of antibiotics and that Gram-negative bacteria are generally
resistant to them, it has been proposed that the outer cell membrane in Gram negative bacteria
(diderms) has as a protective mechanism against antibiotic selection pressure.[5][6][9][10] Some
bacteria such as Deinococcus, which stain Gram-positive due to the presence of a thick
peptidoglycan layer, but also possess an outer cell membrane are suggested as intermediates
in the transition between monoderm (Gram positive) and diderm (Gram-negative)
bacteria.[5][10] The diderm bacteria can also be further differentiated between simple diderms
lacking lipopolysaccharide, the archetypical diderm bacteria where the outer cell membrane
contains lipopolysaccharide and the diderm bacteria where outer cell membrane is made up
of mycolic acid.[7][10][11]
Exceptions
In general, Gram-positive bacteria have a single lipid bilayer (monoderms), whereas Gram-
negative have two (diderms). Some taxa lack peptidoglycan (such as the domain Archaea, the
class Mollicutes, some members of the Rhickettsiales, and the insect-endosymbionts of the

4. Enterobacteriales) and are Gram-variable. This, however, does not always hold true. The
Deinococcus-Thermus bacteria have Gram-positive stains, although they are structurally
similar to Gram-negative bacteria with two layers (diderms). The Chloroflexi have a single
layer, yet (with some exceptions[12]) stain negative.[13] Two related phyla to the Chloroflexi,
the TM7 clade and the Ktedonobacteria, are also monoderms.[14][15]
Some Firmicute species are not Gram-positive; these belong to the class Mollicutes
(alternatively considered a class of the phylum Tenericutes), which lack peptidoglycan
(Gram-indeterminate), and the class Negativicutes, which includes Selenomonas and which
stain Gram-negative.[11] Additionally, a number of bacterial taxa (viz. Negativicutes,
Fusobacteria, Synergistetes and Elusimicrobia) that are either part of the phylum Firmicutes
or branch in its proximity are also found to possess a diderm cell structure.[8][10][11] However,
a conserved signature indel (CSI) in the HSP60 (GroEL) protein distinguishes all traditional
phyla of Gram-negative bacteria (e.g. Proteobacteria, Aquificae, Chlamydiae, Bacteroidetes,
Chlorobi, Cyanobacteria, Fibrobacteres, Verrucomicrobia, Planctomycetes, Spirochetes,
Acidobacteria, etc.) from these other atypical diderm bacteria as well as other phyla of
monoderm bacteria (e.g. Actinobacteria, Firmicutes, Thermotogae, Chloroflexi, etc.).[10] The
presence of this CSI in all sequenced species of conventional LPS-containing Gram-negative
bacterial phyla provides evidence that these phyla of bacteria form a monophyletic clade and
that no loss of the outer membrane from any species from this group has occurred.[10]
Pathogenesis
Most pathogens in humans are Gram-positive organisms. In the classical sense, six Gram-
positive genera are typically pathogenic in humans. Two of these, Streptococcus and
Staphylococcus, are cocci (sphere-shaped bacteria). The remaining organisms are bacilli (rod-
shaped bacteria) and can be subdivided based on their ability to form spores. The non-spore
formers are Corynebacterium and Listeria (a coccobacillus), whereas Bacillus and
Clostridium produce spores.[16] The spore-forming bacteria can again be divided based on
their respiration: Bacillus is a facultative anaerobe, while Clostridium is an obligate anaerobe.