Griffith’s experiment (Discovery of transforming principle)

Griffith’s experiment (Discovery of transforming principle)

  • Griffith experiment was a stepping stone for the discovery of genetic material. Frederick Griffith experiments were conducted with Streptococcus pneumoniae (Pneumococcus), a bacterium that causes pneumonia.
  • Griffith was working with two strains* of Pneumococcus, that differ from one another in their microscopic appearance and in their ability to cause disease.

(*A bacterial strain is a population of bacterial cells descended from a single parent cell; strains may differ in one or more inherited characteristics)

  • Each strain of Pneumococcus may be one of dozens of different types called serotypes that differ in the precise chemical structure of the polysaccharide constituent of the thick slimy capsule.
  • Serotypes are identified by immunological techniques and are usually designated by Roman numerals.(Griffith used types IIR and IIIS Strain of Pneumococcus for his experiments).

Streptococcus pneumoniae strains used by Griffith for his experiment

1) S strain (capsulated)

  • S strain is virulent and pathogenic (Cause disease)
  • When grown on an agar plate with required nutrients in the laboratory, they form colonies that look dome-shaped and smooth. Hence it is designated as the S form.
  • The S strain has a capsule outside its cell wall. As a result it has a smooth appearance under the microscope.

Capsule character: Gelatinous polysaccharide coating that contains the binding sites (known as O-antigens) through which it recognizes the cells it infects.

2) R strain (non – capsulated)

  • The R strain is avirulent and non-pathogenic (does not cause disease)
  • When grown on an agar plate with required nutrients in the laboratory, they form colonies that appear flat and rough. Hence it is designated as the R form.
  • The outer surface of R strain is cell wall and lacks a capsule. As a result, the cell surface appears uneven under the microscope.

(Virulent S strain and avirulent R strains are easily distinguished by standard microbiological culture techniques. The presence or absence of the capsule causes a visible difference between colonies of virulent and avirulent strains).

Capsule is the major virulence factor for S strain: The non encapsulated bacteria are readily engulfed and destroyed by phagocytic cells in the host animal’s circulatory system. Capsulated bacteria are not easily engulfed, so they multiply and cause pneumonia.

Table 1 summarizes the characteristics of Griffith’s two strains.
Figure 1: Petriplate with smooth and rough colonies of Streptococcus pneumoniae (R strain colonies appear on the left and S strain colonies appear on the right of the same agar)

Griffith experiment

In a series of experiments, Griffith analyzed the effects of live R, live S, and heat-killed S strains of S. pneumoniae on live mice.

Control 1: Mice injected with live S strain

  • When mice were injected with the live S strain (type IIIS), the mice developed pneumonia and died.
  • On autopsy, he found large amounts of type IIIS bacteria in the blood of the dead mice

Control 2: Mice injected with live R strain

  • When Griffith injected type IIR bacteria into mice, the mice lived healthy and no bacteria were recovered from their blood

Control 3: Mice injected with heat killed S strain

  • Griffith knew from the work of others that only living virulent cells would produce pneumonia in mice.
  • Griffith knew that boiling will kill all the bacteria and will destroy their virulence. When he injected large amounts of heat-killed type IIIS bacteria into mice, the mice lived and no type IIIS bacteria were recovered from their blood

Griffith’s critical experiment

  • Griffith’s critical experiment involved an injection of living IIR (avirulent) cells combined with heat-killed IIIS (virulent) cells into healthy mice.
  • Since neither cell type caused death in mice when injected alone, Griffith expected that the double injection would not kill the mice.
  • Surprisingly, 5 days after the injections, the mice became infected with pneumonia and died
  • When Griffith examined blood from the hearts of these mice, he observed live type IIIS bacteria.
  • Furthermore, when he injected this isolated S strain into fresh mice, the mice developed pneumonia and died.

 Conclusions from his experiments

1) Something had passed from the heat-killed S strain into the live R strain and “transformed” it into the pathogenic S strain; he called this the “transforming principle.”

2) Since these bacteria retained their type IIIS characteristics through several generations, the infectivity was heritable.

(Transformation is now defined as a change in genotype and phenotype due to the assimilation of external DNA by a cell).

These experiments are now famously known as Griffith’s transformation experiments.

Figure 2: Griffith’s transformation experiment demonstrating genetic transformation in bacteria. The photographs shown are bacterial colonies containing cells with capsules (type IIIS) and without capsules (type IIR).

Summary of the experiment

Griffith’s results had several possible interpretations, all of which he considered.

  • First, it could have been the case that he had not sufficiently sterilized the type IIIS bacteria and thus a few live bacteria remained in the culture. Any live bacteria injected into the mice would have multiplied and caused pneumonia.

Griffith knew that this possibility was unlikely, because he had used only heat-killed type IIIS bacteria in the control experiment, and they never produced pneumonia in the mice.

  • A second interpretation was that the live, type IIR bacteria had mutated to the virulent S form. Such a mutation would cause pneumonia in the mice, but it would produce type IIS bacteria, not the type IIIS that Griffith found in the dead mice.

Many mutations would be required for type II bacteria to mutate to type III bacteria, and the chance of all the mutations occurring simultaneously was impossibly low.

Griffith finally concluded that the type IIR bacteria had somehow been transformed, acquiring the genetic virulence of the dead type IIIS bacteria. This transformation had produced a permanent, genetic change in the bacteria. Though Griffith didn’t understand the nature of transformation, he theorized that some substance in the polysaccharide coat of the dead bacteria might be responsible.

 

 

 

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