0.5 McFarland Standard

Use bacterial suspension with 0.5 McFarland turbidity (CLSI recommends) for antimicrobial testing of new antimicrobial agents (CLSI . The Number of bacterial cells present in the McFarland standard vary based on the cell size.
0.5 McFarland standard is equal to bacterial density of  1.5 x 10⁸  CFU/ml.

Uses :

For  Antimicrobial susceptibility testing, the inoculum does not contain a concentration of bacteria approximates the 0.5 McFarland turbidity standard, antimicrobial susceptibility test results will be affected. 

Resistant organism may appear susceptible if too less bacteria are used in the inoculum.

Procedure: 

McFarland Standard is mixing of 1% solution of barium chloride and 1% of sulfuric acid.

0.05 ml of 1 % barium chloride mixing with 9.95 ml of sulfuric acid to make 0.5 McFarland standard.

This standard can compare with bacterial suspension (saline) in another tube. If the bacterial suspension turbidity is less than McFarland standard add more bacteria can be added. If the bacterial suspension is more turbid than Macfarland standard add diluent. 

McFarlands standards and bacterial suspensions can be measured the OD at 600nm.

Mc Farland Standards

McFarland Std. No.	0.5	1	2	3	4
1% barium chloride (1ml)	0.05	0.1	0.2	0.3	0.4
1% Sulfuric acid  (1ml)	9.95	9.9	9.8	9.7	9.6
Cell density Approx. (1.5×108 CFU/ml)	1.5	3	6	9	12
Absorbance at 600nm	0.08 to 0.1	0.257	0.451	0.582	0.669

McFarland Std.

Antimicrobial Activity - Disk Diffusion method ( Kirby- Bauer method)

Disc diffusion method is to determine the antimicrobial activity  or Antibiotic susceptibility of testing organism . This test can be used to check the therapeutic potentials of various sources like., plant, microbes, nanoparticles etc.,

Requirements:

Mueller Hinton agar plate
Sterile cotton swab
Stock solution of plant extract/ other extracts (antimicrobial agent)
Bacterial broth culture (8 Hrs old)
Sterile disc (Whatman No.1 filter paper)


Procedure:

1. Preparation of Muller Hinton agar (MHA) plate :
       38 gm of MHA medium suspend in 1 liter of distilled water. pH is neutral.

       Heat (boil) and agitate frequently to dissolve.

       Autoclave the medium 121° c for 15 min.

       Pour into sterile petri dishes. 

2. Swabbing bacterial suspension (0.5 McFarland std. or equal to 1-2x10⁸   CFU/mL) on agar pales  

    by sterile cotton swabs.

3.Stock solution of plant extracts/ other sources to prepare at required concentration. (Eg.    concentration of solution 1mg/mL).

4. Soak the sterile discs in 10-50 µL (desired volume) of plant extracts.

5. Place the discs on agar medium.

6. Keep positive control (standard antibiotic disc) and negative control (eg.DMSO) on the plate.

7. Incubate the plate at 37° c for 24 Hrs.

8. After the incubation the diameter of zone of inhibition (mm) to be measured. 

Ref:
NCCLS

Antimicrobial Activity - Agar Well Diffusion Method

Agar well diffusion method is to determine the antimicrobial activity (NCCLS). This test can be used to check the therapeutic potentials of various sources like., plant, microbes, nanoparticles etc.,

Requirements:

Mueller Hinton agar plate
Sterile cotton swab
Stock solution of plant extract/ other extracts (antimicrobial agent)
Bacterial broth culture (8 Hrs old)
Sterile cork borer

Procedure:

1. Preparation of Muller Hinton agar (MHA) plate :
       38 gm of MHA medium suspend in 1 liter of distilled water. pH is neutral.

       Heat (boil) and agitate frequently to dissolve.

       Autoclave the medium 121° c for 15 min.

       Pour into sterile petri dishes. 

2. Swabbing bacterial suspension (0.5 McFarland std. or equal to 1-2x10⁸   CFU/mL) on agar pales  

    by sterile cotton swabs.

3.Wells to be made by using sterile cork borer (size of the well range between 6mm to 10mm    

     diameter).

4. Stock solution of plant extracts/ other sources to prepare at required concentration. (eg.  

    concentration of solution 1mg/mL) and  add into the well (volume 50 -100µL range).

5. Incubate the plate at 37° c for 24 Hrs.

6. After the incubation the diameter of zone of inhibition (mm) to be measured. 

7. Three replicates to be performed against each organism.

Ref:
NCCLS

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