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Introduction of Various Bacteria Gram Stain

Gram stain is the most common and very useful tool in Microbiology to know the bacterial structures, yeasts of fungi whether the organisms are cocci or bacilli or other forms, Gram-positive or negative for further identification, to check the purity of growth, etc. The main objective of the topic ‘Various Bacteria Gram Stain’ is to make familiar with common bacteria generally encountered in clinical laboratories.

List of Various Bacteria Gram Stain

1. Micrococcus
2. Staphylococcus aureus
3. CoNS
4. Enterococcus
5. Beta-hemolytic streptococci ( Streptococcus pyogenes and Streptococcus agalactiae)
6. Streptococcus pneumoniae
7. Viridans streptococci
8. Diphtheroids
9. Bacillus species
10. Escherichia coli
11. Klebsiella pneumoniae
12. Proteus mirabilis
13. Proteus vulgaris
14. Salmonella Typhi
15. Shigella
16. Vibrio cholerae
17. Pseudomons aeruginosa
18. Acinetobacter
19. Neisseria gonorrhoeae

Various Bacteria Gram Stain short Description with Visual Demonstration

• Micrococcus in  Gram-stained smear showing Gram-positive cocci in tetrads. The spherical form of Gram-positive cells ranging from about 0.5 to 3 micrometers in diameter and typically appear in tetrads. Micrococcus species are both catalase and oxidase test positive.

• Staphylococcus aureus Gram Stained smear of culture showing Gram-positive cocci in clusters ( groups) but also singly and pairs. They are Gram-positive cocci of uniform size. The diameter of the cells ranges from 0.5 to 1.0 μm. S . aureus is catalase, cogulase and DNAse positive.

• Coagulase-negative staphylococci (CoNS) are a type of Staphylococci that are commonly found in our skin as normal flora. The Gram stain properties are the same as Staphylococcus aureus.

• Enterococcus species- They are Gram-positive cocci, occurring in pairs or short chains. They are non-capsulated and the majority are non-motile except E. gallinarum and E. casseliflavus/flavescens.
• Beta-hemolytic streptococci – They are pin pint colonies with beta-hemolysis. They are Streptococcus pyogenes and Streptococcus agalactiae. S. pyogenes is 0.04 U bacitracin sensitive while S. agalactiae is resistant. S. agalactiae is penicillin-sensitive and gentamycin-resistant and it is normally encapsulated also called Group B Streptococcus. It is Gram-positive cocci, occurring characteristically in chains but also in pairs and singly. S. pyogenes also possesses the same Gram stain features except long chains are found in fluid culture.

• Streptococcus pneumoniae: It is a Gram-positive elongated ( lanceolate) diplococcus. Gram-stained smear of culture also shows short chains. The encapsulated form encountered in Gram stain from clinical specimens while from the culture are non-capsulated. It is sensitive to optochin( 5µg) and bile soluble while catalase-negative. S. pneumoniae is in a Gram-stained smear of sputum sample as shown below video clip.

S. pneumoniae is in a Gram-stained smear of culture as shown below video clip.

• Viridans streptococci- They often show alpha-hemolytic on blood agar but occasionally may be beta or non-hemolytic optochin resistant, non- bile soluble, non-pathogenic to mice, negative Quellung test, and no fermentation of inulin. They are normal flora of the oropharynx and gastrointestinal tract but still some species may cause endocarditis, bacteremia, and dental caries. Few species of viridans streptococci are Streptococcus mutans, S. anginosus, S. mitis, S. sanguinis and S. salivaruis. They are  Gram-positive cocci in short chains and pairs. The size of cocci is around 1 to 1.5 µm.
  The chains are not as long as beta-hemolytic streptococci.
• Diphtheroids-Diphtheroids are aerobic, non-sporing, pleomorphic Gram-positive rods that are more uniformly stained than C. diphtheriae, lack the metachromatic granules, and are arranged in a palisade manner. They are normal commensals of the skin and mucous membranes and they differ from Corynebacterium diphtheriae in biochemical tests as well as in toxin production. Because, they are usually found as commensals on the skin, they are often considered as only contaminants when isolated from clinical specimens. However, there are becoming etiological agents being associated with various infections too.

• Bacillus species: They are ubiquitous in nature and remain both free-living (non-parasitic) species, and two parasitic pathogenic species, B. anthracis causes anthrax; and B. cereus causes food poisoning and thus these two Bacillus species are medically significant. The word, Bacillus is Latin which means stick. Bacillus is a genus of Gram-positive,rod-shaped bacteria, a member of the phylum Firmicutes, with 266 named species. Bacillus species are obligate aerobes as well as facultative anaerobes and catalase test positive. Bacillus can reduce themselves to oval endospores and they can remain in this dormant state for years. They are generally motile with peritrichous flagella. The genus includes psychrophilic, mesophilic, and thermophilic species. The maximum temperatures for vegetative growth ranging from about 25°C to above 75 °C and the minimum from about 5 °C to 45°C. Their salt tolerance varies from less than 2 % to 25 % NaCl. Their spores are ubiquitous, being found in the soil, dust, water, and air, and constitute the commonest contaminants in bacteriological culture media.
• E. coli in a Gram-stained smear of culture is shown below. They are bacteria that come under the family Enterobacteriaceae. They are normally found in the environment, foods, and intestines of people and animals. They are a large and diverse group of bacteria. They are normal flora when they are in our intestines. But if they spread to another part of your body, they can cause severe infections like urinary tract infections (UTIs), diarrhea, pyogenic infections, septicemia, respiratory illness and pneumonia, and other illnesses. The family Enterobacteriaceae has the following general phenotypic features-
  1. They are small gram-negative rods.
  2. Having usual cell structure (non sporulated)
  3.  Motile by peritrichous flagella.
  4. Grow on ordinary medium.
  5. Grow both aerobically and anaerobically.
  6. Grow without the addition of sodium chloride or other supplements.
  7. Grow well on MacConkey agar.
  8.  Active biochemically.
  9. Ferments D-glucose and other sugar, often with gas production.
  10. Catalase test positive.
  11. Oxidase test negative.
  12. Reduce nitrate to nitrite
  13. Contain the enterobacterial  common antigen (ECA) and
  14. They have a 39-59% guanine plus cytosine (G+C) content of DNA.

Klebsiella pneumoniae: They are short, plump, Gram-negative, encapsulated, non-motile rods. They are about 1-2 µm x 0.5-0.8 µm. The classification of Klebsiella has been undergone various modifications. They have been classified into two species and they are Klebsiella pneumoniae and Klebsiella oxytoca. Klebsiella pneumoniae is further subdivided into four subspecies ( subsp.) and they are-

• Klebsiella pneumoniae subsp. aerogenes
• Klebsiella pneumoniae subsp. pneumoniae
•  Klebsiella pneumoniae subsp. ozaenae
• Klebsiella pneumoniae subsp. rhinoscleromatis

• Proteus mirabilis: They are Gram-negative rods having a size of 1-3 µm × 0.5 µm. They are non-encapsulated, non-sporing, and actively motile. They possess peritrichate flagella. They may show as short cocobacillary forms, long and filamentous type, especially in young culture. Many strains possess fimbriae. They are commensals of our intestine. Proteus species are also saprophytic and widely distributed in nature. They are opportunistic pathogens and may cause the following infections-
  • Urinary tract infection (UTI)
  • Pyogenic lesions
  • Infection of the ear ( otitis media)
  • Respiratory tract infections (RTIs)
  • Pyelonephritis

  Note: Some strains are also multi-drug resistant due to having R plasmids.

Keynotes on Various Bacteria Gram Stain

1. Anaerobic cocci are generally susceptible to penicillin, and all are susceptible to metronidazole (5 µm disk).
2. The anthrax bacillus is non-motile, unlike most other members of this genus.
3.  Unlike other subsp. , Klebsiella oxytoca is indole positive.
4. Indole helps to differentiate P. vulgaris ( positive) from P. mirabilis ( negative).
5. Several methods have been used to inhibit warming of Proteus are increasing agar concentration up to 6%, incorporating alcohol 5-6%, chloral hydrate, sodium azide, sulphonamide, surface-active agents, and boric acid.

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