Question: Did you notice zones of inhibition around the control disks? Why were control disks included in this investigation?

Ans 2). Diffrence in effectiveness of diifrent antiobacterial drugs in the diffrent species of bacteria because of diffrent mechansim of ation they act through. Tetracycline as a bacteriostatic, inhibit the protein synthesis occur in the ribosome in the cytoplam of the cell, they inhbit the synthesis of protein by binding with the 30s subunit of ribosome. As a result peptide chain falis to grow and it make the cell unprotected by the host immunity.

As for bacitracin it is bactricidal which kill the bacteria by ihibiting the cell wall synthesis and increase influx of ion inside the cell which cause movement of water inside the reulting in the cell lysis or busting of the cell.

Ans 3). If the bacteria is not susceptible to an antibacterial drug at the concentration present in the particular disk, it does not mean that the bacteria is completely resistant to the drug because may be the concentration present in the drug is higher than particular dose because in hiogher dose any drug would inhibit the bacteria. That's why a particular dose of drug is required for checking the susceptibility of bacteria for the given drug.

Ans 4)

Ans 5) It is well established that tetracyclines inhibit bacterial protein synthesis by preventing the association of aminoacyl-tRNA with the bacterial ribosome (44, 263). Therefore, to interact with their targets these molecules need to traverse one or more membrane systems depending on whether the susceptible organism is gram positive or gram negative. Hence, a discussion of the mode of action of tetracyclines requires consideration of uptake and ribosomal binding mechanisms. Also pertinent to this discussion are explanations of the joint antibacterial-antiprotozoal activity of the tetracyclines and the microbial selectivity of the class as a whole. Most of these issues have been considered at length in recent years (44, 67, 78, 263), so the focus here will be on new information.

Tetracyclines traverse the outer membrane of gram-negative enteric bacteria through the OmpF and OmpC porin channels, as positively charged cation (probably magnesium)-tetracycline coordination complexes (44, 263). The cationic metal ion-antibiotic complex is attracted by the Donnan potential across the outer membrane, leading to accumulation in the periplasm, where the metal ion-tetracycline complex probably dissociates to liberate uncharged tetracycline, a weakly lipophilic molecule able to diffuse through the lipid bilayer regions of the inner (cytoplasmic) membrane. Similarly, the electroneutral, lipophilic form is assumed to be the species transferred across the cytoplasmic membrane of gram-positive bacteria. Uptake of tetracyclines across the cytoplasmic membrane is energy dependent and driven by the ΔpH component of the proton motive force (192, 263). Within the cytoplasm, tetracycline molecules are likely to become chelated since the internal pH and divalent metal ion concentrations are higher than those outside the cell (263). Indeed, it is probable that the active drug species which binds to the ribosome is a magnesium-tetracycline complex (44, 144). Association of tetracyclines with the ribosome is reversible, providing an explanation of the bacteriostatic effects of these antibiotics (44).

Several studies have indicated a single, high-affinity binding site for tetracyclines in the ribosomal 30S subunit, with indications through photoaffinity labeling and chemical footprinting studies that protein S7 and 16S rRNA bases G693, A892, U1052, C1054, G1300, and G1338 contribute to the binding pocket (44, 180, 196, 263). However, Schnappinger and Hillen (263) have pointed out that these apparent sites for drug interaction in the ribosome may not necessarily reflect the actual binding site. Indeed, interpretation of the probing studies referred to above is complicated by the observation that binding of tetracycline (which measures approximately 8 by 12 Å) to the ribosome appears to cause wide-ranging structural change in 16 S rRNA (193). Furthermore, photoincorporation methods are subject to the limitation that upon irradiation, tetracycline photoproducts are generated which may react further with the ribosomes (196). Nevertheless, naturally occurring tetracycline-resistant propionibacteria contain a cytosine-to-guanine point mutation at position 1058 in 16S rRNA (251) (see below), which does at least suggest that the neighboring bases U1052 and C1054 identified by chemical footprinting (180) may have functional significance for the binding of tetracyclines to the 30S subunit.

The absence of major antieukaryotic activity explains the selective antimicrobial properties of the tetracyclines. At the molecular level, this results from relatively weak inhibition of protein synthesis supported by 80S ribosomes (302) and poor accumulation of the antibiotics by mammalian cells (78). However, tetracyclines inhibit protein syntheses in mitochondria (221) due to the presence of 70S ribosomes in these organelles. It has been recognized for some time that the spectrum of activity of tetracyclines encompasses various protozoan parasites such as P. falciparum, Entamoeba histolytica, Giardia lamblia, Leishmania major, Trichomonas vaginalis, and Toxoplasma gondii (28, 44, 67, 137, 214). The antiparasitic activity is explained in some cases by the finding that certain organisms, e.g., P. falciparum, contain mitochondria (67). However, a number of other protozoa which lack mitochondria nevertheless remain susceptible to tetracyclines. At present there is no satisfactory molecular explanation for these findings (67).