Macromolecular Biosynthesis Assay for Evaluation of Influence of an Antimicrobial on the Synthesis of Macromolecules

Materials and Reagents

1. [³H] N-acetylglucosamine (Hartmann Analytic, catalog number: ART 0101 )
   
2. [³H] Uridine (Hartmann Analytic, catalog number: MT-602 )
   
3. [³H] Thymidine (PerkinElmer, catalog number: NET35500 )
   
4. [³H] Leucine (Hartmann Analytic, catalog number: MT-672 )
   
5. Triptic soy broth (TSB) (Becton Dickinson, catalog number: 211825 )
   
6. Control antimicrobials for the influence on synthesis of:
   1. Peptidoglycan: vancomycin (Vancocin 500 mg) (Teva Pharma)
   2. RNA: actinomycin D (Sigma-Aldrich, catalog number: A1410 ) 
   3. DNA: ciprofloxacin (Ciproxin Infusion 0.2 g) (Bayer) 
   4. Proteins: chloramphenicol (Applichem, catalog number: A1806 )
   5. All macromolecules (antiseptic activity): chlorhexidine dihydrochloride (Sigma-Aldrich, catalog number: C8527 ) 
      Note: All substances prepared according to the manufacturer’s instructions; minimal inhibitory concentration (MIC) of each antimicrobial can be determined according to Clinical and Laboratory Standards Institute guidelines (see Reference 1) (The following MICs were determined for S. aureus WSPPA: MICvancomycin = 2 μg/ml, MICactinomycin D = 6.25 μg/ml, MICciprofloxacin = 2 μg/ml, MICchloramphenicol = 8 μg/ml, MICchlorhexidine = 0.78 μg/ml)
7. Scintillation cocktail (Ultima Gold, catalog number: 6013321 ).
   
8. Phosphate-buffered saline (PBS) (Reagens, catalog number: 9007695 ).
   
9. Na₂HPO₄ (Sigma-Aldrich, catalog number: S3264 )
   
10. KH₂PO₄ (Sigma-Aldrich, catalog number: P8416 )
    
11. MgSO₄^.7H₂O (Fluka, catalog number: 63138 )
    
12. NH₄Cl (Sigma-Aldrich, catalog number: A9434 )
    
13. NaCl (Sigma-Aldrich, catalog number: S3014 )
    
14. Sodium citrate tribasic dihydrate (Sigma-Aldrich, catalog number: S4641 )
    
15. Glucose (B. Braun Medical, catalog number: 395176 )
    
16. Various amino acids (i.e., L-alanine (Sigma-Aldrich, catalog number: A7469 )
    
17. L-Valine (Sigma-Aldrich, catalog number: V0513 )
    
18. L-Isoleucine (Sigma-Aldrich, catalog number: I7403 )
    
19. L-Aspartic acid (Sigma-Aldrich, catalog number: A7219 )
    
20. L-Glutamic acid (Sigma-Aldrich, catalog number: G8415 )
    
21. L-Serine (Sigma-Aldrich, catalog number: S4311 )
    
22. L-Threonine (Sigma-Aldrich, catalog number: T8441 )
    
23. L-Cysteine hydrochloride (Sigma-Aldrich, catalog number: C6852 )
    
24. L-Arginine (Sigma-Aldrich, catalog number: A8094 )
    
25. L-Leucine (Sigma-Aldrich, catalog number: L8912 )
    
26. L-Lysine (Sigma-Aldrich, catalog number: L9037 )
    
27. L-Proline (Sigma-Aldrich, catalog number: P5607 )
    
28. L-Phenylalanine (Sigma-Aldrich, catalog number: P5482 )
    
29. L-Tryptophan (Sigma-Aldrich, catalog number: T8941 )
    
30. L-Histidine monohydrochloride (Sigma-Aldrich, catalog number: H5659
    
31. Glycine (Sigma-Aldrich, catalog number: G7126 )
    
32. Cyanocobalamine (Sigma-Aldrich, catalog number: V2876 )
    
33. p-Aminobenzoate (Fluka, catalog number: 06940 )
    
34. Biotin (Sigma-Aldrich, catalog number: B3399 )
    
35. Nicotinic acid (Sigma-Aldrich, catalog number: N0761 )
    
36. D-pantothenic acid hemicalcium salt (Sigma-Aldrich, catalog number: P5155 )
    
37. Pyridoxine hydrochloride (Sigma-Aldrich, catalog number: P6280 )
    
38. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T1270 )
    
39. Riboflavin (Sigma-Aldrich, catalog number: R9504 )
    
40. ZnCl₂ (Sigma-Aldrich, catalog number: 208086
    
41. MnCl₂^.4H₂O (Sigma-Aldrich, catalog number: M5005 )
    
42. BH₃O₃ (Fluka, catalog number: 15665 )
    
43. CoCl₂^.6H₂O (Sigma-Aldrich, catalog number: C8661 )
    
44. CuCl₂^.2H₂O (Sigma-Aldrich, catalog number: C3279 )
    
45. NiCl₂^.6H₂O (Sigma-Aldrich, catalog number: 223387 )
    
46. Na₂MoO₄^.2H₂O (Sigma-Aldrich, catalog number: M1003 )
    
47. FeCl₂^.4H₂O (Fluka, catalog number: 44939 )
    
48. NaOH (Sigma-Aldrich, catalog number: S5881 )
    
49. Uracil (Sigma-Aldrich, catalog number: U0750 )
    
50. Cytosine (Sigma-Aldrich, catalog number: C3506 )
    
51. Adenine (Sigma-Aldrich, catalog number: A2786 )
    
52. Guanine (Sigma-Aldrich, catalog number: G11950 )
    
53. Sodium dodecyl sulphate (SDS) (Sigma-Aldrich, catalog number: L4390 )
    
54. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T9159 )
    
55. 15 ml TPP centrifuge tubes (TPP 91015 )
    
56. 10% TCA, 5% TCA, 5% TCA/1.5 M NaCl (see Recipes)
    
57. Completely defined medium (CDM) (see Recipes)
    Note: This medium has been optimised for Staphylococcus (S.) aureus and may require further optimisation if other bacteria are investigated. 

Equipment

1. 37 °C bacterial culture incubator
   
2. Sterile capped glass tubes (to reduce bacterial adherence) (GlasKeller, catalog number: 2613111 )
   
3. Ultracentrifuge tubes (2 ml) (Eppendorf, catalog number: 0030120.094 )
   
4. Scintillation tubes (PerkinElmer, catalog number: 6000288 )
   
5. Benchtop centrifuge
   
6. Vacuum pump
   
7. Liquid scintillation analyser (Tri-CARB, catalog number: 1900TR )
   

Procedure