Pili comprise several types of morphologically similar thin appendages growing out from the surface of gram-negative bacteria.'-3 Type I pili are composed of protein subunits of molecular weight 17,000 polymerized into rigid right-handed helices of diameter 70 A and pitch 24 A, having an axial hole 20-25 A in diameter.4 Other types of pili with different external diameters exist2 but their composition and fine structure is unknown.It had been considered previously that pili might be involved in the fertility of male bacteria.3 This speculation was based on the frequent occurrence of pili on male E. coli K 12 strains and on the plausibility of chromosomal transfer being mediated by a rod-like structure which could actively traverse the cell membrane and wall. However, no correlation of piliation with maleness was found. Many female strains were richly piliated and some cultures of male strains contained only a few sparsely piliated cells. The hypothesis could not be disproved, however, since none of the many male strains subsequently examined were ever completely nonpiliated. Although the majority of cells in a culture may have had no pili, at least a few cells could always be found with a few attached pili.A new method of approach to chis problem has been provided by the recent electron microscopic observations of Crawford and Gesteland5 who noted that a male-specific bacteriophage, R-17, adsorbed to pili of an Hfr and an F+ strain of E. coli but not to pili of an F-strain.We have investigated the adsorption of another male-specific bacteriophage, M 12, isolated by P. H. Hofschneider.8 Our studies revealed that M 12 adsorbs to some of the pili present on male bacteria and that phage adsorption can be used to distinguish them in electron micrographs from other types of pili occurring on the same cell. It is possible to demonstrate that these "F pili" are genetically controlled by the fertility factor of E. coli K 12.Materials and Methods.-Phage: M 12 phage, a small (about 270 A diameter), spherical, RNA-containing phage infecting Hfr and F+ strains but not F-strains, was obtained from Dr. P. H. Hofschneider.Electron microscopy: Bacteria were grown in tryptone yeast extract calcium broth (10 gm tryptone, 5 gin yeast extract, 5 gm NaCl, 0.75 gm CaCl2 2H20 per liter of water, the CaC12 sterilized separately). Overnight, unshaken, unaerated cultures were diluted 1:10 into fresh medium and grown for 3-4 hr under the same conditions until the bacterial density was about 5.108 cells per ml. M 12 phage was added at multiplicities from 5 to 100 and the mixture incubated at 370C. After 10 min, the mixture was rapidly chilled in an ice bath and prepared for the electron microscope by the collodion agar filtration method of Kellenberger.7 Tests for phage susceptibility: (A) Plaque formation: Standard phage plaque methods using tryptone yeast extract calcium soft agar (0.7%) and bottom agar 776

The effect of mechanical agitation (blending) on the removal of F pili, type I pili, and flagella from Hfr (high-frequency recombinant) and resistance transfer factor (RTF) fi+ Escherichia coli cells was studied by electron microscopy. The reduction in number and length of appendages was measured as a function of blendor speed under standard conditions of temperature, medium, cell density, and blendor configuration. F pili and flagella were removed within the same narrow range of blendor speeds. Type I pili were removed within a higher and broader range of speeds. The speed which reduced the average length of type I pill to 50% was 3.5 times the speed which reduced the average length of F pili to 50 %. None of the speeds employed inhibited cell growth, viability, or the ability to produce cell appendages. The kinetics of reappearance of F pili and type I pili after removal by blending were also different. F pili grew out very rapidly, reaching 50% of their full length in 30 sec and their full length in 4 to 5 min. The number of attached F pili per cell also increased rapidly, reaching a constant value in 4 to 5 min. After 5 min, F pilus lengths were distributed around a modal value of about 1.2,um, and the numbers of F pili per cell were distributed according to a Poisson distribution, with an average of 1.0 per cell. These reappearance kinetics, length distributions, and number distributions are consistent with a model of F-pilus outgrowth in which new F pili appear at random locations on the cell surface at an average rate of about once every 4 min, grow to their characteristic length in about 4 min, and then separate from the cell. F pili which had separated could absorb to the cells, leading to the presence of two classes of F pili on cells: those in the process of natural outgrowth and those attached by absorption. Type I pili increased in length much more slowly than did F pili, although the fraction of cells having visible type I pili increased very rapidly after blending because of the large number of type I pili per cell. The fraction of flagellated cells increased even more slowly, reaching only 30% of the unblended fraction in 30 min. The application of blending spectra and reappearance kinetics to the identification of cell functions with surface structures is discussed.