BAC-1 - Screening of 2-aminothiazoles against Actinomycetes

[KlementineJBS]

to prep

• [ ] prepare MH II broth

• [ ] prepare amikacin in solvent (distilled water or DMSO) and dilute in MH broth (1 in 100) to double desired final conc.

• [ ] prepare compounds in solvent and dilute in MH broth (1 in 100) to double desired final conc.

• [ ] sweep bacterial growth into small volume of sterile water/saline (PBS?) using an applicator stick - put into small glass culture tube

• [ ] break up clumps by using 7 - 10 small 3mm glass beads and vortexing vigorously

• [ ] allow to sit 10-15 minutes so clumps can settle to the bottom

• [ ] use pasteur pipette to add supernatant to a plastic tube with 2 mL water/saline

• [ ] check turbidity and adjust to achieve 0.5 McFarland standard (equivalent to 1.5 x 10^8 colony forming units (CFU/ml)

[KlementineJBS]

29.10.24

Rihab uses MH broth instead of water/saline when preparing inoculum.

• Remove bacterial growth and add to tube with 1 mL MH broth
• Mix well and break up using glass beads (vortex with beads).
• Allow to settle 10-15 minutes so large clumps sink to bottom
• Take ~0.5 mL and add to a clean tube
• Compare against McFarland 0.5 standard and adjust as needed with MH broth
• Use a sterile swab to spread over MH agar
• (prepare 1 in 200 dilution of this inoculum: 60 uL of the McFarland suspension in 1140 uL MH broth) <- unsure about this??
• Use suspension in plate

Bacteria should be no more than 2 weeks old. S aureus should be subcultured the day before using.

[KlementineJBS]

Questions:

• should anything be autoclaved??
• where do I find: tubes, beads, amikacin, swabs (sterile), McFarland standard/chemicals required, sterile pipette
• do I need to do that dilution step? It's not mentioned in protocol so perhaps was a one-off?
• how to prepare amikacin controls
• how to prepare my own compounds

[KlementineJBS]

Wendy has previously published a method using the resazurin assay here.

[KlementineJBS]

Amikacin stock should be 10 mg/mL (approx. 17 mM)

[KlementineJBS]

31.10.24

Weighed out 12.3 mg of amikacin hydrate (CAS = 1257517-67-1, batch number = 125M4764V/0000103289) so need to dissolve in 1.23 mL of aquadest according to Mickey's procedure.

[KlementineJBS]

31.10.24

Made up plate with compounds and amikacin.

• Started off by making 200 uM solutions of each compound to be screened (600 uL each - 12 uL of 10 mM stock dissolved in DMSO added t0 588 uL MH II broth). This gives 2% DMSO i.e. 1% after addition of bacterial suspension
• made amikacin solution (32 ug/mL in MH II broth, using 10 mg/mL stock in DMSO)
• added 125 uL of compound solutions to columns 1, 3, 5, 7, 9
• added 75 uL of MH broth supplemented with DMSO** to columns 2, 4, 6, 8, 10 (used multi channel)
• transferred 25 uL from column 1 -> column 2 using multi channel, repeated for remaining columns
• NCs: 100 uL of MH broth with DMSO, 100 uL of plain MH broth each
• PCs: 100 uL of MH broth with DMSO each
• amikacin controls: made according to Mickey's protocol exactly i.e. started with 200 uL of 32 ug/mL in row 1, 100 uL of MH broth in all others, serial diluted down the column and removed 100 uL from row H

**DMSO supplementation of broth: for controls, made up MH broth with 6 uL DMSO in 1794 uL MH broth (0.33% DMSO). For others, mistakenly made up broth with 10 uL of DMSO in 1490 uL MH broth (0.66%, should be 2%).

NB MYOS733 was made using a 2.5 mM solution so all concentrations will be 1/4 of the rest. MYOS422, 424, 427, 428 and 429 were already made into solutions - it was assumed that these were 10 mM stocks as is typical, but couldn't find Ma's notes on this.

[KlementineJBS]

01.11.24

Made some changes to protocol as written on recommendation from Marleen, who works with NTMs and showed me how to use the McFarland reader etc.

For N brasiliensis, we scraped a large amount of bacterial colonies into 2 mL of PBS in a sterile tube, vortexed with ~7 glass beads (3 mm) and allowed to settle. Approximately 70 uL of the supernatant in 2 mL PBS gave a McFarland reading of 0.5.

For S aureus, we used only a tiny smear of the bacteria and didn't vortex with beads. Made up McFarland using ~ 2 mL PBS as before, adjusted to 0.5.

For both, we diluted the McFarland solution 1 in 200 times (50 uL in 10 mL MH II broth) and used this as the inoculum. 5 uL of this inoculum was diluted in 10 mL broth and 100 uL was plated on MH agar to check viability.

N brasiliensis was subbed directly and from the McFarland solution onto blood agar. S aureus was subbed only from the McFarland solution onto blood agar.

Nocardia should be read at the same time as S aureus

[KlementineJBS]

04.11.24

Reading after 72 hours growth indicates some promising results, but controls are uncertain.

S aureus control looks good, with an MIC of 2 ug/mL (amikacin). N brasiliensis shows no sign of growth at any of the given concentrations of amikacin - next time, plate should probably be made from 4 ug/mL instead of 16 ug/mL

NC has signs of bacterial growth - likely this is due to a mistake on my part. Need to be careful when preparing plates on Thursday.