3.

2Culture
3.2.1 Nutrient agar medium:

Nutrient agar medium / HIMEDIA Labs Ltd. / Mumbai, India.

This medium used in the cultivation of the bacterial isolates of the study
and composed of the following components:

Contents Gram/L
Peptic digest 5.00
Sodium chloride 5.00
Beef extract 1.50
Yeast extract 1.50
Agar 15.00

3.2.2 Nutrient agar medium preparation method:

This medium prepared by dissolving (28 g) of its components powder in
(1000ml) of distilled water. ‘Then, pH was adjusted to (7.2) and medium
sterilized by autoclave and poured on petri dishes as it was recommended
by the medium container instructions.

3.2.3 Blood agar medium:

Rashmi Diagnostics , Bangalore, India.
Blood agar medium composed of composed of the following components
Contents Quantity
Tryptone 15.00 g
Sodium chloride NaCl 5.00 g
Phytone or soytone 5.00 g
Agar 15.00g
Distilled water 1 litter

3.2.4 Blood agar medium preparation method:

Blood agar base was prepared according to the manufacturing company
.it was autoclaved at 121 C for 15 minutes , and cooled to 50 C ,and 5 %
of human blood was added .This medium was used to cultivate bacterial
strains and to detect their ability for blood hemolysis.
3.2.5 Mac Conkey agar medium:
HiMedia Laboratories Limited , Mumbai — 400 086 , India

Composition of media
Contents Quantity
Peptone l7 grams
Protease peptone 3 grams
Lactose 10 grams
Bile salts 1.5 grams
Sodium chloride NaCl 5 grams
Neutral red 0.03 grams
Crystal violet 0.001 grams
Agar 13.5 grams

3.2.6 Mac Conkey agar medium preparation method:

This medium prepared by dissolving (50 grams) of its component powder
in(1000ml) of distilled water . medium sterilized by autoclave and poured
on petri dishes .final PH 7.1.

3.3 Methods
3.3.1 Procedure
While observing growth on culture media smear must be prepared from
colonies of bacterial on clean slide.

1. Add one drop of normal saline on a slide by dropper.

2. Take single isolated colony by a wire loop and mix it with the normal
saline.
3. Let the smear dry and fix it through a flame.
4. Stain the slide by Gram stain
3.3.2 Gram stain procedure
1. Add few drops of erystal violet on the slide for 1-2 minutes.
2. Wash by distilled water.
3. Add few drops of iodine on the slide for 1-2 minutes.
4. Wash the slide by distilled water
5. Add alcohol on the slide for 30 seconds.
6. Rinse the slide with distilled water.
7. Gently flood the smear with safranin and let stand for 1-2 minutes.
8. Wash the slide by distilled water, and let it dry
9. Examine the slide microscopically by 100X oil immersion

3.3.2.1 Interpretation of the result:

Detect of gram -positive bacteria ,which stain purple , or gram-
negative bacteria which stained pink .

3.3.3 Procedure of API

3.3.3.1 Preparation of strip:
1.Prepare an incubation box (tray and lid) and distribute about Sml of
distilledwater or demineralized water into honeycombed wells of the tray
to create a humid atmosphere.
2.Record strain reference on the elongated flap of the tray.
3. Remove the strip from its packaging.
4. Place the strip in the incubation box.

3.3.3.2 Preparation of inoculums:

1. Open an ampule of API suspension medium (Sml) .or uses any tube
containing 5ml of sterile distilled water without additives.
2. Using pipette, remove a single well isolated colony from an isolation
plate. It isrecommended to use young cultures (18-24 hours) old.
3. Carefully emulsify to achieve a homogeneous bacterial suspension.
This suspension must be used immediately after preparation.
3.3.3.3 Inoculation of the strip:
1. Using the same pipette, fill both tube and cupule of the tests |CIT| , |
VP} , and |GEL| with the bacterial suspension.
2. Fill only the tube (and not the cupule) of other tests.
3. Create anaerobiosis in the tests ADH. LDC , H2S . URE by overlaying
with mineral oil,
4. Close the incubation box.
5. Incubate at 36° C + or — 2° C for 18-24 hours.

3.3.3.3.1 Interpretation:
1. Add the proper reagents to the components:
 drop of kovac’s reagent to the IND (read within a couple of minutes)
 1drop of barritt’s A and B reagents to VP (a positive reaction may
take up to10 minutes).
 drop of FeCl; to TDA.
2. Read all other tests without reagents by using table.
3. Record results on the diagram. The oxidase test reaction should be
negative, and is added as the last test result.
4. Take your result sheet to the computer to INPUT OUR REACTION:

The API 20E website database will provide with potential organisms.