Procedure For The Preparation of Blood Agar

Procedure for the preparation of Blood Agar
1. Prepare the blood agar base as instructed by the manufacturer. Sterilize by

autoclaving at 121°C for 15 minutes.
2. Transfer thus prepared blood agar base to a 50°C water bath.
3. When the agar base is cooled to 50°C, add sterile blood agar aseptically and mix
well gently. Avoid the formation of air bubbles. You must have warmed the blood to
room temperature at the time of dispensing to molten agar base.
(Note: If you are planning to prepare a batch of blood agar plates, prepare few
blood agar plates first to ensure that blood is sterile).
4. Dispense 15 ml amounts to sterile petri plates aseptically
5. Label the medium with the date of preparation and give it a batch number (if
necessary).
6. Store the plates at 2-8°C, preferably in sealed plastic bags to prevent loss of
moisture. The shelf life of thus prepared blood agar is up to four weeks.
Preparation of MacConkey Agar
1. Suspend 49.53 grams of dehydrated medium in 1000 ml

purified/distilled water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Cool to 45-50°C.
5. Mix well before pouring into sterile Petri plates.
Triple Sugar Iron Agar
Procedure:
1. Touch a well isolated colony with a sterile straight wire.
2. Inoculate TSI by first stabbing through the centre of the medium to the bottom of
the tube and then streak the surface of the slant.
3. Leave the cap loose and incubate the tube at 35 in ambient air for 18 to 24 hours.
4. Observe the reaction
Procedure of Lysine Iron Agar Test
1. With a straight inoculating needle, inoculate LIA by stabbing

through the center of the medium to the bottom of the tube and
then streaking the slant while withdrawing needle.
2. Incubate tubes aerobically with loose caps at 35-37ºC for 18-48
hours and examine reaction of medium.
Preparation of Simmons Citrate Agar
1. Dissolve above salts in deionized water.

2. Adjust pH to 6.9.
3. Add agar and Bromothymol blue.
4. Gently heat, with mixing, to boiling until agar is dissolved.
5. The medium may be used either as slopes in test tubes or as a plate
medium in petri dishes. In both cases the surface of the medium is
lightly inoculated by streaking and, where slopes are used, the butt of
medium is inoculated by stabbing.
6. For tubes, dispense 4.0 to 5.0 ml into 16-mm tubes.
7. Autoclave at 121 degree C under 15 psi pressure for 15 minutes.
8. Cool in slanted position (long slant, shallow butt).
9. Tubes should be stored in a refrigerator to ensure a shelf life of 6 to
8 weeks.
10. The uninoculated medium will be a deep forest green due to the pH
of the sample and the bromothymol blue.
Christensen’s Urea Agar
Suspend 24.01 grams in 950 ml distilled water. Heat to boiling to dissolve

the medium completely. Sterilize by autoclaving at 10 lbs pressure
(115°C) for 20 minutes. Cool to 45-50°C and aseptically add 50 ml of
sterile 40% Urea Solution (FD048) and mix well. Dispense into sterile
tubes and allow to set in the slanting position. Do not overheat or reheat
the medium as urea decomposes very easily.
Preparation
1. Dissolve the ingredients in 100 ml of distilled water and filter sterilize

(0.45-mm pore size).
2. Suspend the agar in 900 ml of distilled water, boil to dissolve
completely.
3. Autoclave at 121 degree C and 15 psi for 15 minutes.
4. Cool the agar to 50 to 55 degree C.
5. Aseptically add 100 ml of filter-sterilized urea base to the cooled
agar solution and mix thoroughly.
6. Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the
tubes during cooling until solidified.
Procedure of Urease Test

1. Streak the surface of a urea agar slant with a portion of a
well-isolated colony or inoculate slant with 1 to 2 drops from an
overnight brain-heart infusion broth culture.
2. Leave the cap on loosely and incubate the tube at 35°-37°C in
ambient air for 48 hours to 7 days.
3. Examine for the development of a pink color for as long as 7 days.
SIM
Suspend 36.23 grams in 1000 ml distilled water. Heat to boiling to

dissolve the medium completely. Dispense in tubes. Sterilize by
autoclaving at 15 lbs pressure (121°C) for 15 minutes. Allow the tubes
to cool in an upright position.

Procedure For The Preparation of Blood Agar

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Procedure for the preparation of Blood Agar

1. Prepare the blood agar base as instructed by the manufacturer. Sterilize by

Preparation of MacConkey Agar

1. Suspend 49.53 grams of dehydrated medium in 1000 ml

Triple Sugar Iron Agar

Procedure of Lysine Iron Agar Test

1. With a straight inoculating needle, inoculate LIA by stabbing

Preparation of Simmons Citrate Agar

1. Dissolve above salts in deionized water.

Christensen’s Urea Agar

Suspend 24.01 grams in 950 ml distilled water. Heat to boiling to dissolve

1. Dissolve the ingredients in 100 ml of distilled water and filter sterilize

Procedure of Urease Test

Suspend 36.23 grams in 1000 ml distilled water. Heat to boiling to

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