Dear Mya,

You may use The disc diffusion method as follows:

J Adv Pharm Technol Res. 2015 Apr-Jun; 6(2): 53–57.

doi: 10.4103/2231-4040.154530

PMCID: PMC4397619

Investigation of cream and ointment on antimicrobial activity of Mangifera indica extract

Amgad A. Awad El-Gied, Abdelkareem M. Abdelkareem,1 and Elnazeer I. Hamedelniel1

Antimicrobial evaluation using the disc diffusion method

The original extract of MI was subject to serial dilution as follows: 5 mg/ml, 3.75 mg/ml, 2.5 mg/ml, 1.75 mg/ml, and 1 mg/ml. Filter discs (5.5/mm) were made and impregnated into each of the above dilutions. The discs were dried at 37°C for 1 h. The dried discs were therefore having the following concentration: 5 mg/ml, 3.75 mg/ml, 2.5 mg/ml, 1.75 mg/ml, and 1 mg/ml. Muller Hinton's agar plates were used the in vitroantimicrobial testing as recommended by clinical and Laboratory Standards Institute.[17] Bacterial strains were sub cultured from frozen stocks or from plates freshly obtained from Asser Central Hospital. One of three loopful of 24 h old cultures from each test strains were used to prepare 0.5 MeFard standard suspensions.

Each bacteria strain (Escherichia coli, Staphylococcus aureus, Candida albicans and Mycobacterium smegmatis) were inoculated into Mueller-Hinton agar (MHA) plates to form a bacterial lawn. Then impregnated dried discs plus positive and negative control discs were placed on the inoculated MHA agar.

The inoculated plates were incubated at 37°C and examined after 24, 48 and 72 h for inhibition zones around the wells. Zones were measured in mm by a ruler for each disc exhibiting inhibition zones under and around discs.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397619/

OR

You may use the wet disc method as described in the publication contained in the following link:

J Burn Care Rehabil. 1989 May-Jun;10(3):203-8.

The wet disc antimicrobial solution assay. An in vitro method to test efficacy of antimicrobial solutions for topical use.

Holder IA1.

Abstract

The wet disc antimicrobial solution assay, an in vitro method to test susceptibility/resistance of burn wound-isolated microorganisms against antimicrobial solutions for topical use, is described. Where antibiotic discs for Kirby-Bauer testing were available for antibiotics contained in the topical solutions, Kirby-Bauer assays were done, and the results were compared with those derived using the wet disc assay. More microorganisms were susceptible using the wet disc method compared with the Kirby-Bauer method. The wet disc method could be used to test topical antimicrobial solutions that had no Kirby-Bauer disc equivalent. The wet disc method provided more reliable information for the clinician to use in choosing appropriate topical antimicrobial solutions for treating patients' wounds than the Kirby-Bauer method.

http://www.ncbi.nlm.nih.gov/pubmed/2745497

Hoping this will be helpful,

Rafik

Perhaps you could dispense the cream using the same method used to dispense pharmaceutical ointments: squeezing it out of a collapsible ointment tube through a narrow tip, You can buy such tubes (see below):

http://shop.gohcl.com/default.aspx?page=item%20detail&itemcode=10205

Please, try to reconstitute your creamy semisolid crude in 100% DMSO, and/or absolute methanol, both of them are not lethal to bacteria. try also make different concentrations , 500, 250, 100 mg/ml. You must also, use the pure solvents as negative controls on the same tested plate.

Use disc diffusion method, it is more accurate than cup-plate (my own experience!).

Thanks so much all for your kind suggestions.

The question is how will your cream diffuse in the medium if it is not water soluble. In addition you have to test the vehicle and the active ingredients apart so you can say with certainty that any inhibitory activity results from the latter. Another pitfall may be that the inhibitory activity results from changes induced in the medium's characteristics (such as pH) and not activity on the microorganisms. Such changes are of course irrelevant for the infection.

Thank you so much, Daniel Elad

I use agar well diffusion method for antimicrobial assay. My samples are semisolid cream which can not dissolve in water.

I use two model drugs, one is hydrophobic and another one can be incorporated into both hydrophobic and hydrophilic carrier.

For positive control, I use one commercial ointment (because cream is not available in the market for my model drug).

The problem that I face is, positive control and the hydrophobic drug loaded formulations didn't show activity after putting directly into wells.  Another drug loaded preparations showed clear zones.

So, I tried to dissolve in different solvents,such as DMSO, Tween 80 and Methanol. The commercial ointment showed clear zones after dissolving into Tween 80.But hydrophobic one didn't showed activity after dissolving into these three.

Please,could someone suggest me again for that problem? Should I change solvent or change to another method?