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Tube Dilution Method

The tube dilution method and agar plate method are commonly used to determine the minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of antimicrobial agents. Both methods involve growing bacteria in gradually increasing concentrations of the agent to identify the lowest concentrations that inhibit growth (MIC) and kill bacteria (MLC). The phenol-coefficient method compares the disinfectant activity of a test agent to that of phenol by measuring their ability to kill Salmonella typhi or Staphylococcus aureus over time.

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663 views4 pages

Tube Dilution Method

The tube dilution method and agar plate method are commonly used to determine the minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of antimicrobial agents. Both methods involve growing bacteria in gradually increasing concentrations of the agent to identify the lowest concentrations that inhibit growth (MIC) and kill bacteria (MLC). The phenol-coefficient method compares the disinfectant activity of a test agent to that of phenol by measuring their ability to kill Salmonella typhi or Staphylococcus aureus over time.

Uploaded by

Dipto Kumer Sarker
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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1.

Tube Dilution Method:

 Tube dilution method is the most commonly used method for determination of minimal
inhibitory concentration (MIC) and minimal lethal concentration (MLC). In this method,
a series of tubes is set up, each containing the same quantity of a standard growth liquid
medium and also a gradually increasing concentration of the antimicrobial chemical
agent (e.g., antibiotic) to be tested.

 The tubes are then inoculated with the same quantity of cell suspension of the test
microorganism and incubated till growth has appeared (usually 16 to 20 hours).
 The first tube in the series where there is complete absence of growth of the test
microorganism denotes (mark) the minimal inhibitory concentration (MIC) of the
chemical agent.

 The minimal lethal concentration (MLC) is determined by taking small quantities (usually
0.05 ml) from the tubes showing no growth and sub culturing it into fresh medium lacking
the chemical agent.

 The lowest concentration of the agent from which the microorganism does not recover
and grow when transferred to fresh medium is the minimal lethal concentration (MLC).
The tube dilution method for determination of MIC and MLC is diagrammatically
represented in Fig. 20.1.

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2. Agar Plate Method:

 Agar plate method (Fig. 20.2) is very similar to tube dilution method.
 In this method, Petri plates containing a standard growth agar medium are taken at the
place of lubes containing liquid medium.

 Antimicrobial chemical agents of various concentrations are inoculated on the agar


surface of plates already inoculated with the same quantity of the test microorganism.

 These plates are incubated and then examined for growth.

 The first plate in the series where there is complete absence of growth of the test
microorganism denotes the minimal inhibitory concentration (MIC) of the chemical agent.

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 Also, the chemical agent to be tested may be applied in the center of the Petri dish and
zone of inhibition can be observed. Zone of inhibition develops if the chemical agent is
active.

Phenol-Coefficient Method:
Suitable for disinfectant miscible with water
Strain use is Salmonella typhi or Staphylococcus aureus

 To determine the phenol coefficient, different dilutions of phenol and test disinfectants
(5 ml per tube) are added separately to tubes containing 0.5 ml of 24 hour old broth
culture (test culture) of Staphylococcus aureus or Salmonella typhi.

 All these tubes are then placed in a 20°C water both.


 At intervals of 5, 10 and 15 minutes, an aliquot (portion) from each tube is transferred to
a nutrient broth medium with a loop transfer needle for sub-culturing.

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 The inoculated subculture tubes are incubated for 2 days and subsequently examined for
visible growth.
 The highest dilution of the disinfectant and the highest dilution of phenol killing the test
organism (Staph, aureus or Sal. typhi) in 10 minutes but hot in 5 minutes are recorded.
 The number obtained by this division is the phenol coefficient of the substances tested.

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