Anti-bacterial growth inhibition assay

Marbio

Jun 11, 2026

Anti-bacterial growth inhibition assay

DOI

https://dx.doi.org/10.17504/protocols.io.81wgbjjk3vpk/v1

Marbio ¹

¹UiT The Arctic University of Norway

Marbio

Marbio

UiT The Arctic University of Norway

DOI: https://dx.doi.org/10.17504/protocols.io.81wgbjjk3vpk/v1

Protocol Citation: Marbio 2026. Anti-bacterial growth inhibition assay . protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbjjk3vpk/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: May 15, 2026

Last Modified: June 11, 2026

Protocol Integer ID: 317181

Keywords: Anti-bacterial assay, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, growth inhibition, human pathogen, growth, assay, tested concentration

Abstract

The protocol for the measurement of growth inhibition at tested concentration on a selection of human pathogens.

Materials

**Table 1: Equipment and consumables**

AB
EquipmentVendor, product number
Incubator 37 °CPanasonic Healthcare
Shaking incubator, 37 °CHeidolph Instruments GmbH
Victor Multilabel CounterPerkinElmer

**Table 2: growth media and antibiotic**

AB
Growth media/chemicalsVendor, product number
MilliQ H2OMerck Millipore
Mueller-Hinton (MH) brothDifco, 275730 (21g/L)
Brain Heart Infusion (BHI)Sigma Aldrich, 53286 (37g/L)
Blood agar platesSUMP Media, UNN
GentamicinMerck Biochrom GmbH, A2712



**Table 3: pathogens, recommended growth media and bacterial density**

ABC
Bacterial strainVendor, product numberRecommended growth media
Enterococcus faecalisLGC Standards, ATCC 29212BHI
Escherichia coliLGC Standards, ATCC 25922MH
Pseudomonas aeruginosaLGC Standards, ATCC 27853MH
Staphylococcus aureusLGC Standards, ATCC 25923MH
Streptococcus agalactiaeLGC Standards, ATCC 12386BHI

**Additional Materials**:
- Autoclave 5 Erlenmeyer flasks, 100 mL

Safety warnings

The final DMSO concentration in the microtiter plate should not exceed 0.25%.

Method

ASSAY PREPARATIONS
Blood agar plates.
Autoclave MQ-H2O
Autoclave 5 Erlenmeyer flasks, 100 mL.
If needed, prepare and autoclave MH-broth and BHI according to pack instructions.

Transferring pathogens from biofreezer stock to blood agar plates
Streak pathogens from biofreezer on blood agar plates. Keep pathogens on ice during the streaking procedure. Transfer the stock vials back to the biofreezer.
Incubate blood agar plates over night at 37 °C
Transfer blood agar plates to the fridge. These plates can be kept there and used for 2 weeks, before a scoop of bacterial colonies are transferred to a new plate and incubated as in step 1 and 2.
After a total of 4 weeks storage of pathogens on blood agar plates, new stock is streaked out on fresh plates from biofreezer stock.

RUNNING THE ASSAY
Day 1
Transfer a scoop of pathogens from blood agar plate to 8 mL growth media, according to table 3.
Incubate cultures at 37 °C for approximately 20 hours. (**No shaking**)

Day 2
Preparation of stock solution of samples for testing
Prepare working stocks that are twice the final test concentration_. The final DMSO concentration in the samples should not exceed 0.25%.
Transfer 50 µL of sample in duplicate to a microtiter plate according to Figure 1. Transfer 50 µL ddH2O to column 1 and 12. Transfer 50 µL growth medium to column 1.

Preparation of media, equipment and gentamycin control
For each pathogen, prepare the following equipment:
25 mL growth media in 100 mL autoclaved Erlenmeyer flask
9.9 mL growth media in 14 mL Falcon tube
36 mL growth media in 50 mL Falcon tube

Dilution of overnight cultures
Transfer 2 mL overnight culture of each pathogen to separate Erlenmeyer flasks containing 25 mL growth media, according to table 3.
Incubate the diluted pathogens for 1.5 h (E. faecalis, E. coli, S. agalactiae) or 2.5 h (P. aeruginosa, S. aureus) at 37 °C, 100 rpm. 
Measure the OD600 of 5 bacterial cultures by 96-well microtiter plate, adjust the bacterial OD600 to E. faecalis is 0.125; to E. coli is 0.08; to P. aeruginosa is 0.075; to S. aureus is 0.08; to S. agalactiae is 0.08.
Transfer 100 µL of the bacterial suspension to 9.9 mL growth medium (dilution factor 1:100).
Transfer 4 mL bacterial suspension from step 3 to 36 mL growth medium (dilution factor 1:10).
Transfer 50 µL bacterial suspension from step 4 to column 2-11 in the designated microtiter plate according to Figure 1. In the gentamycin plate, the gentamycin has 2-fold dilution from 32ug/ml to 0.0156ug/ml Figure 2. The transfer of pathogens to microtiter plates should be completed within 30 min to maintain viable bacterial density.
Incubate the microtiter plates at 37 °C over night (19-20 h).
1.      
ABCDEFGHIJKLM
   
    1
    2
    3
    4
    5
    6
    7
    8
    9
    10
    11
    12
  
  A
  medium blank  1-1  1-1
    2-1
    2-1
    3-1
    3-1
    4-1
    4-1
    5-1
    5-1
  growth control
  B
  medium blank  1-2
    2-2
    2-2
    3-2
    3-2
    4-2
    4-2
    5-2
    5-2
  5-2growth control
  C
  medium blank  1-3
    2-3
    2-3
    3-3
    3-3
    4-3
    4-3
    5-3
    5-3
  5-3growth control
  D
  medium blank  1-4
    2-4
    2-4
    3-4
    3-4
    4-4
    4-4
    5-4
    5-4
  5-4growth control
  E
  medium blank  1-5
    2-5
    2-5
    3-5
    3-5
    4-5
    4-5
    5-5
    5-5
  5-5growth control
  F
  medium blank  1-6
    2-6
    2-6
    3-6
    3-6
    4-6
    4-6
    5-6
    5-6
  5-6growth control
  G
  medium blank  1-7
    2-7
    2-7
    3-7
    3-7
    4-7
    4-7
    5-7
    5-7
  5-7growth control
  H
  medium blank  1-8
    2-8
    2-8
    3-8
    3-8
    4-8
    4-8
    5-8
    5-8
  5-8growth control
Figure 1: The layout of 96-well microtiter plate for samples.   
 
ABCDEFGHIJKLM
    1
32ug/ml  2
16ug/ml  3
8ug/ml  4
4ug/ml  5
2ug/ml  6
  1ug/ml  7
  0.5ug/ml  8
0.25ug/ml  9
0.125ug/ml  10
0.63ug/ml  11
0.0315ug/ml  12
0.0156ug/ml
  A
  E. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalis
  B
  E. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalisE. faecalis
  C
  E. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coli
  D
  E. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coli
  E
  P. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosa
  F
  P. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosa
  G
  S. aureusS. aureusS. aureusS. aureusS. aureusS. aureusS. aureusS. aureusS. aureusS. aureusS. aureusS. aureus
  H
  S. agalactiaeS. agalactiaeS. agalactiaeS. agalactiaeS. agalactiaeS. agalactiaeS. agalactiaeS. agalactiaeS. agalactiaeS. agalactiaeS. agalactiaeS. agalactiae
Figure 2: The layout of microtiter plate for antibiotic reference (gentamycin) control.   
 

Day 3
Perform a visual inspection of the microtiter plates. Readings from plates where there are growth in column 1 or no growth in column 12 should not be reported as valid results. These assays will have to be repeated due to contamination, or insufficient growth of the pathogen.
Measure the absorbance in the wells using the Victor plate reader. Plates are read at OD600.

Results

Evaluation of results
The results are scored as Active (A), Inactive (I) or Questionable (Q) according to the measured absorbance.
A < 0.05
Q 0.05 - 0.09
I > 0.09

Protocol references

EUCAST Discussion document E.Dis 5.1. March 2003
European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID)

	A	B
	Equipment	Vendor, product number
	Incubator 37 °C	Panasonic Healthcare
	Shaking incubator, 37 °C	Heidolph Instruments GmbH
	Victor Multilabel Counter	PerkinElmer

	A	B
	Growth media/chemicals	Vendor, product number
	MilliQ H2O	Merck Millipore
	Mueller-Hinton (MH) broth	Difco, 275730 (21g/L)
	Brain Heart Infusion (BHI)	Sigma Aldrich, 53286 (37g/L)
	Blood agar plates	SUMP Media, UNN
	Gentamicin	Merck Biochrom GmbH, A2712

A	B	C
Bacterial strain	Vendor, product number	Recommended growth media
Enterococcus faecalis	LGC Standards, ATCC 29212	BHI
Escherichia coli	LGC Standards, ATCC 25922	MH
Pseudomonas aeruginosa	LGC Standards, ATCC 27853	MH
Staphylococcus aureus	LGC Standards, ATCC 25923	MH
Streptococcus agalactiae	LGC Standards, ATCC 12386	BHI

A	B	C	D	E	F	G	H	I	J	K	L	M
	1	2	3	4	5	6	7	8	9	10	11	12
A	medium blank	1-1	1-1	2-1	2-1	3-1	3-1	4-1	4-1	5-1	5-1	growth control
B	medium blank	1-2	2-2	2-2	3-2	3-2	4-2	4-2	5-2	5-2	5-2	growth control
C	medium blank	1-3	2-3	2-3	3-3	3-3	4-3	4-3	5-3	5-3	5-3	growth control
D	medium blank	1-4	2-4	2-4	3-4	3-4	4-4	4-4	5-4	5-4	5-4	growth control
E	medium blank	1-5	2-5	2-5	3-5	3-5	4-5	4-5	5-5	5-5	5-5	growth control
F	medium blank	1-6	2-6	2-6	3-6	3-6	4-6	4-6	5-6	5-6	5-6	growth control
G	medium blank	1-7	2-7	2-7	3-7	3-7	4-7	4-7	5-7	5-7	5-7	growth control
H	medium blank	1-8	2-8	2-8	3-8	3-8	4-8	4-8	5-8	5-8	5-8	growth control

A	B	C	D	E	F	G	H	I	J	K	L	M
	1 32ug/ml	2 16ug/ml	3 8ug/ml	4 4ug/ml	5 2ug/ml	6 1ug/ml	7 0.5ug/ml	8 0.25ug/ml	9 0.125ug/ml	10 0.63ug/ml	11 0.0315ug/ml	12 0.0156ug/ml
A	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis
B	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis	E. faecalis
C	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli
D	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli	E. coli
E	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa
F	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa	P. aeruginosa
G	S. aureus	S. aureus	S. aureus	S. aureus	S. aureus	S. aureus	S. aureus	S. aureus	S. aureus	S. aureus	S. aureus	S. aureus
H	S. agalactiae	S. agalactiae	S. agalactiae	S. agalactiae	S. agalactiae	S. agalactiae	S. agalactiae	S. agalactiae	S. agalactiae	S. agalactiae	S. agalactiae	S. agalactiae