Conventional Agar Diffusion Technique Biology Essay

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Conventional agar diffusion technique for susceptibility tests, which rely on the size of zone of inhibition surrounding a drug containing disc are not suitable for the slowly growing Mycobacterium species because the drug diffuses throughout the medium before the organism has the chance to grow. So, the following principles are recognized for the antimycobacterial screening.

The composition of medium should have a minimal effect on drug inactivation. So, Middle Brook 7H9 broth base is used.

Drug containing medium should be stored in a refrigerator shielded from light and kept in plastic tubes tightly closed in order to protect from them evaporation.

Homogenization of inoculum is essential to eliminate large clumps of cells.

SUSCEPTIBILTY TESTING:

This is done by 2 methods.

Direct method

Indirect method

DIRECT METHOD:

This was done if acid-fast bacilli are seen on the smear of the concentrated clinical specimen. Dilutions are made and inoculated.

INDIRECT METHOD

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Bacterial mass is suspended in Middle Brook 7H9 broth containing three or four small sterile glass beads. Mixture is placed on a vortex mixer and precautions are taken to prevent aerosol production. Tube is allowed to stand for 15 mts. The stock suspensions are diluted and 0.1 ml is inoculated into control and drug containing media.

The antimycobacterial screening was carried out in Maratha Mandal NGH Institute Of Dendal Sences And Research Center,Belgaum.

Materials used in antitubercular screening studies:

Middle Brook 7H9 Broth Base : Himedia, Mumbai

Alamar Blue Solution : Serotec

Media used : Middle Brook 7H9 Broth Base

Ingredients

Concentration in gm/lt

Ammonium sulphate

0.50

Disodium phosphate

2.5

Monosodium phosphate

1

Sodium citrate

0.1

Magnesium sulphate

0.05

Calcium chloride

0.0005

Zinc chloride

0.001

Copper sulphate

0.001

Ferric ammonium citrate

0.04

L-glutamic acid

0.5

Pyridoxime

0.001

Biotin

0.0005

pH at 250C

6.6±0.2

ALAMAR BLUE ASSAY METHOD

Alamar Blue is an oxidation-reduction dye used for screening of anti tubercular activity. Alamar blue (oxidized form) turns into pink colour to upon reaction. Since mycobacterium is an aerobic organism, its presence of growth turns alamar blue pink. This principle has been used to predict the presence or absence of growth of Mycobacterium tuberculosis for testing antimycobacterial agents. Pink colour indicates the presence of growth (no anti tubercular activity) and blue colour indicates the absence of growth (inhibitory activity of agents tested).

Preparation of medium:

2.35 gm of Middle Brook 7H9broth base was suspended in 450ml distilled water which contains 5ml glycerol sterillsed by autoclaving at 15 psi pressure at 1210C for 15 minutes. Cooked to 450C or below and enriched with dextrose to a final concentration of 0.5% of either bovine albumin fraction V or serum.

PROCEDURE

The anti mycobacterial activity of compounds were assessed against M. tuberculosis using microplate Alamar Blue assay (MABA).

This methodology is non-toxic, uses a thermally stable reagent and shows good correlation with propotional and BACTEC radiometric method.

To the outer perimeter of sterile 96 wells plate, 200µl of sterile deionized water were added to minimize evaporation of medium in the test wells during incubation.

The 96 wells plate received 100 µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate.

The final drug concentrations tested were 0.01 to 20.0 µl/ml.

Plates were covered and sealed with parafilm and incubated at 37°C for five days.

After this time, 25µl of freshly prepared 1:1 mixture of Alamar Blue reagent and tween 80 (10%) was added to the plate and incubated for 24 hrs.

A blue color in the well indicated abscence of bacterial growth, and pink color was scored as growth.

Lowest drug concentration (MIC) prevents the color change from blue to pink.

QUTANTITATIVE SCREENING OF ANTIMYCOBACTERIAL ACTIVITY

SL No

Samples

100

50

25

12.5

6.25

3.125

1.6

0.8

0.4

0.2

1.

2a

S

S

R

R

R

R

R

R

R

R

2

2b

S

S

S

R

R

R

R

R

R

R

3

2c

S

S

R

R

R

R

R

R

R

R

4

2d

S

S

R

R

R

R

R

R

R

R

5

2e

S

S

R

R

R

R

R

R

R

R

6

3a

S

S

S

R

R

R

R

R

R

R

7

3b

S

S

R

R

R

R

R

R

R

R

8

3c

S

S

S

R

R

R

R

R

R

R

9

3d

S

S

S

R

R

R

R

R

R

R

10

3e

S

S

S

R

R

R

R

R

R

RS-Sensitive

R-Resistant

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The names of the compounds are given in the list of newly synthesized compounds

FIGURE SHOWING ANTITUBERCULAR ACTIVITY BY ALAMAR BLUE ASSAY METHOD

RESULTS AND DISCUSSION:

In this present work, all the compounds were tested for antitubercular activity by Alamar Blue assay method. The compounds 2b, 3a, 3c, 3d, and 3e were found to be effective at 25µl/ml. This clearly establishes that antimycobacterial activity is in correlation with the docking reports.