MUELLER HINTON MEDIUM (General Information)
 

INTENDED USE
Mueller Hinton media are used for testing the susceptibility of microorganisms to antimicrobial agents.
 

HISTORY
In an attempt to develop a simple transparent medium containing no heat-labile materials and capable of withstanding autoclaving, Mueller and Hinton selected the complex Gordon and Hine pea meal extract agar as the most suitable complete medium available and attempted to break it into its essential components.  The authors found that starch could replace the growth-promoting properties of pea extract, acting as a “protective colloid” against toxic substances present in the medium.  Further, they found that the tryptic digest of meat could be replaced by casamino acids technical.

Bauer and Kirby recommended Mueller Hinton medium for performing antibiotic susceptibility tests using a single disc of high concentration.

The World Health Organization Committee on Standardization of Susceptibility Testing has accepted Mueller Hinton medium for determining the susceptibility of microorganisms because of its reproducibility and acceptability to workers and the field.
 

MUELLER HINTON BROTH FORMULA

     INGREDIENTS                                                                                               PER LITER

 Beef, infusion from.................................................................................................300.0 g

 Casamino Acids, Technical....................................................................................17.5 g

 Starch............................................................................................................................1.5 g
 

Typical Culture Response in/on Mueller Hinton Medium
 

   Organism                                                                      Growth
  Escherichia coli ATCC  25922                          good to excellent
  Neisseria meningitidis ATCC  13090              good to excellent
  Staphylococcus aureus ATCC  25923          good to excellent
  Enterococcus fecalis ATCC  33186               good to excellent
  Streptococcus pneumoniae ATCC  6303       good to excellent
  Pseudomonas aeruginosa ATCC  27853      good to excellent
 

GENERAL DIRECTIONS FOR PREPARATION

1.  Suspend 21 grams of the  dehydrated medium  in 1 liter distilled or deionized water.

 NOTE:  Sometime a gently heat ing is necessary to completely dissolve the powdered material. 

 NOTE:  If this were an agar medium the heating process to dissolve would be to   boiling

2.  Dispense as desired.

 NOTE:  If this were an agar medium meant  to be dispensed directly into agar   plates, then the medium must be sterilized first.

3.  Sterilize in the autoclave for 15 minutes at 15 lbs pressure (121^oC).

 NOTE: Remember the above is a broth (not an agar) medium.  Preparation of an agar medium is some what different as you will see in the below  exercise.
 
 

PREPARATION OF MUELLER-HINTON BROTH AND AGAR MEDIA
 

Each student will be required to prepare 500 ml of the Mueller-Hinton medium.  Subsequently this volume will be dispensed as broth and agar.  Each student will use these media in the Antibiotic Sensitivity Exercise the following week.
 

1.   Prepare 500 ml broth in 1 liter flask as directed in the above directions.

 NOTE:  Remember that you are only preparing 500 ml, whereas the above instruction is for a liter of the medium.

2.   Dispense approximately 75 ml into each of two prescription bottles.

 NOTE:  Use the volume measure on the prescription bottle as an indication of 75 ml.  Label fully and place them with the materials to be sterilized.

3.   Accurately measure the remaining volume using a 500 graduated cylinder.

4.   Return the volume to the liter flask.

5.  Add agar to a final concentration of 1.5%.
 

 NOTE:                                 1.5                                        X
                                          ______             =               ______

                                            100                         volume remaining broth

6. This medium containing agar is to be melted in a microwave oven (about 5 minutes at full power)

 NOTE:  The medium must come to a full boil so the the agar is completely    dissolved before it can be dispensed.  Additionally, be careful not to let the    medium to boil over in the microwave.

7.  Dispensed into large unsterile tubes with 20 mls in each.

 NOTE: You will need 15 tubes of this agar medium.  The easiest way to accurately dispense this medium into the tube is to use a dummy tube with 20 ml   of water as an indicator of the volume level in each tube.  Additionally, use a beaker  to dispense the medium  into each of the tubes.

 NOTE:  Be sure to immediately rinse any glassware that has contained agar or had been in contact with the agar medium.  Also if any left over agar is poured into  the sink drain  be sure to follow it with ample water so as to not let it solidify in the   drain trap.

8. Cap and combine your tubes with another individual in one test tube rack.

9.  Label the rack with masking tape so that your tubes are on one end and are identifiable.

10. Place with materials to be sterilized.