The standard measurement of antimicrobial in vitro drug potency and activity is the minimum inhibitory concentration or MIC. a measurement based on testing 10^5 colony forming units per milliliter (cfu/ml) of bacteria against varying doubling dilution drug concentrations and following overnight incubation.(Clinical and Laboratory Standards Institute, 2010).

Hola Juan, muchas gracias. Tu tienes los estándares del CLSI 2010? Si los tienes podrías ayudarme con ellos?

Hi Roman Yesid,

If you are performing Broth micro dilution, the thumb rule is you have to follow the CLSI (NCCLS - old name) guidelines. The CLSI guidelines indicate that for performing Broth micro dilution method in 96 well plates, 5 X 10^4 CFU/well are required.

For this, the bacteria are suspended to achieve on Optical density of 0.080 at 625 nm - here the bacterial load is 1-2 X 10 ^ 8 CFU.mL. Then the inoculum is diluted 1: 100 in broth. Here the bacterial count becomes 1-2 X 10^6.

The compound is prepared to achieve 128 ug/100 uL in the first well. Then the compound is diluted 1:1 in remaining wells. i.e. 50ul from the first well is transfered to second well containing 50 ul of broth. This process is continued till the 9th well. 10 the well will be compound control, 11th will be inoculum control and 12th will be broth control.

Once the plates are ready, then inoculum is prepared. and 50 uL from the 1-2 x 10^6 CFU/mL is transfered into all the wells. (this becomes 5 x 10^4 CFU/ well)

Plates are incubated for 18 - 24 hrs and the well in which there is no growth of bacteria is considered as MIC.

you can contact me if you require more clarifications.

Hope it is helpful

regards

Amarnath Pisipati

Hi Amarnath , thanks for your answer. I have one little question about technique that you send me: If you add 50 uL of inoculum, I believe that concentration of extract will dilute 1:1 . What is your oppinion about it?

Hey Roman, think logically..... hmmm okay, look into this... I am sending you the details on how we can do the work..... hope it will be helpful.

Compound preparation:

(a) For 32ug/ml:

2 mg Comp + 6.25 ml solvent (as per your need usually DMSO) =

320 ug/ml - STOCK

1 ml of prepared compound Stock + 4 ml broth = 64 ug/ml solution

here 64 ug is present in 1000 uL

there fore 100 uL will have 6.4 ugms

and 50 uL will have 3.2 ugms

which implies -

from 64 ugms/mL stock compound - 100 uL dispensed into first well of 96 well plate.

In all remaining wells we add 50 uL broth.

Add 50 uL from the first wel1 into the next well containing 50 uL broth.

Further 1:1 dilutions until 9th well. remaining 50 uL discarded from the 9th well.

So, in the first well we have 3.2 ugms,/ 50 uL

When we add 50 uL of inoculum it becomes 3.2 ugms/ 100 uL

which is equal to 32 ug/ml

for you to understand, when we back calculate this to 1 mL;

3.2 ugms in 100 uL

32 ugms in 1 mL

So, you are not diluting the compound off or doing anything wrong by following this method.

Hope this is helpful.

regards

Amarnath Pisipati

hola Román, te he mandado un e-mail con la información que me pediste espero que sea útil, si el correo no llega bien me lo dices y te lo vuelvo a enviar desde mi cuenta de dropbox.

saludos.

Really helpful for me too. Can i download the CLSI Procedures anywhere?

I have a CLSI 2011 version, you can download it. Hope it is helpful.

Thanks a lot Liu ... have a nice day :)

Amarnath Pisipati,I appreciate your answer and I can guess you are a very good teacher. Well done.

Thank you Hannah, we have to appreciate the Research gate site that is helping all the researchers through out the world to come to one platform and share what ever they know...... I am happy that I was able to help....... Thank you Liu Xue.. for your copy of CLSI 2011 version.... I will download it later..

regards

Amarnath Pisipati

Thank u Marcelo !!

The commercially prepared MacFarland standard do expires and it will be good if you can prepare it in your lab as adviced by Marcelo. I must thank L Liu Xue for the latest edition of CLSI. I have downloaded it. Liu, Thanks a lot

Hello,

Currently we are at a final concentration of 1E6/mL for bacteria : the suspension test is prepared at 1E8/mL and the inoculator dispatches 1 to 3µL per well each contaning 100µL of broth. For fungi we use a final concentration of 1E5/mL. In some cases, we have studied the effect of inoculum size with concentration raging from 1E4 to 1E6/mL.

Kind regards

You must search literature and use CLSI 2011,

best regards

Reference:CLSI 2006,page:13-16

9.3.2 Dilution of Inoculum Suspension

Cultures adjusted to the 0.5 McFarland standard contain approximately 1 to 2 x 108 CFU/mL with most

species, and the final inoculum required is 104 CFU per spot of 5 to 8 mm in diameter. When using

replicators with 3-mm pins which deliver 2 μL, dilute the 0.5 McFarland suspension 1:10 in sterile broth

or saline to obtain a concentration of 107 CFU/mL. The final inoculum on the agar will then be

approximately 104 CFU per spot. When using replicators with 1-mm pins which deliver 0.1 to 0.2 μL, do

not dilute the initial suspension. Optimally, use the adjusted suspensions for final inoculation within 15

minutes of preparation.

10.2.2 Broth Microdilution Method

(1) This method is called “microdilution,” because it involves the use of small volumes of broth

dispensed in sterile, plastic microdilution trays that have round or conical bottom wells. Each well

should contain 0.1 mL of broth. To prepare microdilution trays, antimicrobial agents may be diluted

as described in Section 10.2.1 or in Tables 6 and 6A.

(2) The most convenient method of preparing microdilution trays is by use of a dispensing device and

antimicrobial dilutions made in at least 10 mL of broth. These dilutions are used to dispense 0.1

(±0.02) mL into each of the 96 wells of a standard tray. If the inoculum is to be added by pipette as

described in Section 10.3.1.2, prepare the antimicrobial solutions at twice the desired final

concentration, and fill the wells with 0.05 mL instead of 0.1 mL. Each tray should include a growth

control well and a sterility (uninoculated) well.

(3) Seal the filled trays in plastic bags and immediately place in a freezer at ≤-20 °C (preferably at

≤-60 °C) until needed. Although the antimicrobial agents in frozen trays usually remain stable for

several months, certain agents (e.g., clavulanic acid and imipenem) are more labile than others and

should be stored at ≤-60 °C. Do not store the trays in a self-defrosting freezer. Thawed antimicrobial

solutions must not be refrozen; repeated freeze-thaw cycles accelerate the degradation of some

antimicrobial agents, particularly β-lactams.