I.

BACTERIOLOGICAL EXAMINATIONS OF STOOL:

II.7 Sampling of STOOL

Fecal samples should be collected in the early stages of the disease before
antibiotic treatment is instituted, when the pathogenic germs are likely to be
present in large numbers in stools. We will collect the samples stools in a sterile
container of appropriate size, fitted with a tight-fitting cap and waterproof.
Upon receipt at the laboratory, the stools are processed as quickly as possible,
in all case no more than 2 hours after collection.

I1.2. Labeling: see above

Il.3. Stool culture on isolation medium:

Stool samples are cultured to identify enteropathogenic germs/bacteria. They

will be seeded/cultured as soon as possible upon arrival at the laboratory.
Seeding immediate is particularly used for the identification of shigella and
salmonella. If it is impossible to sow on the field, the samples must be
preserved at 4°c.
There are several media of variable selectivity suitable for a first spreading in a
box of petri which allow certain enteropathogenic bacteria to grow, while
inhibiting the growth of Gram-positive bacteria and some Gram-negative
bacteria.
The environments that we use for stool culture at the University Laboratory are:

II.3.1. Salmonella-Shigella Agar: SSA

Principle:

This medium is used for the isolation of salmonella and shigella from stools and
foodstuffs. The development of secondary flora is slowed by brilliant green,
bile salts, the high concentration of thiosulfate and citrate. Lactose, a reactive
sugar in the environment/medium, allows detection of possible growth of
coliforms (red colonies). In addition, bacteria capable of producing H2S by
reduction of thiosulfate give in the presence of iron ions colonies with a black
center.

Medium:

This medium is marketed commercially:

Presented in a bottle, ready to use, simply pour it into a petri dish.
At the end of a few months, bile salt crystals can form on the surface and deep
in the medium. They are not an indication of alteration thereof. They also
dissolve again when it is melted before pouring it into cans. Either in
dehydrated form.
The dehydrated medium corresponds to the following formula (in
gram per liter of distilled water):
- Beef meat extract…5 g/L
- Polypeptone....5 g/L
- Lactose… 10 g/L
- Bile salts...8.5 g/L
- Sodium citrate… 10 g/L
- Sodium thiosulfate…8.5 g/L
- Ferric citrate... 1 g/L
Agar... 13.5 g/L
Brilliant green… 0.00033 g/L
Neutral red… 0.025 g/L
Final pH 7.0

Put 62g of dry medium in a liter of distilled water. Leave to rest for 5 minutes,
then shake to obtain a homogeneous suspension. Heat gently, stirring
occasionally for 1 to 2 minutes. Do not autoclave. Leave to cool to around 45°C
and pour into a petri dish, of approximately 20ml per box.
Allow the medium to solidify, with the can lid slightly raised.
It should be noted that any modification in the concentration of the medium
(prolonged boiling for dissolution, significant drying of the boxes etc.) makes
the environment very inhibitory, hence an often delicate preparation,
disappointing results and the preference generally given to ready-for-use
medium.
Reading / Results:
Colonies- lactose-positive red colonies.
Lactose-negative colonies: colorless colonies
H2S-positive colonies: colonies with a black center.
The differential identification of Shigella will be considered from colorless
colonies, that of salmonella from colorless colonies with or without a black
center.
II.3.2.MacConkey Medium: 11.4.2

II.3.3.Selenite broth (soiling medium):

Principle:
The selenite present in the medium inhibits the growth of coliform bacteria and
enterococci within 12 hours after the start of incubation; then the inhibitory
action slowly decreases. Salmonella and Proteus, on the other hand, are little
inhibited from the start. It is therefore not recommended to extend the
incubation time.
Medium:
A.IPP Formula
Dissolve 4g of sodium selenite in a liter of distilled water, then add 19g of the
medium following basis:
Bacteriological peptone...5g
Disodium phosphate...10g
Lactose…4g
PH: 7

Heat to dissolve. Mix well, distribute at the rate of 10ml per tube (screw cap).
Sterilize in a boiling water bath or in flowing steam for 30 minutes. Don’t
autoclave. It is also possible to sterilize by membrane filtration. Distribute into
sterile tubes.
A. Formula (Merck, BioMérieux, Oxoid, Difco)
Selenite is incorporated into the basic medium whose formula (in grams per
liter) is
Special Peptone…5.0g
Lactose…4.0g
Sodium selenite… 4.0g
Dipotassium phosphate... 3.5g
Monopotassium phosphate...6.5g

Whatever the formula chosen:

Avoid excessive heating, do not autoclave; If the medium must be used
immediately: Completely dissolve 23g in a liter of water recently
demineralized, sterile, by stirring and possibly heating with distilled or totally
maximum at 60°c then without sterilization, in tubes, in quantities of 10 to
15ml.
Whatever the formula chosen:
-Avoid excessive heating, do not autoclave;
-The medium should have the appearance of ordinary broth: If it has a red
deposit, it is unusable. It must be stored in a cooler at 4°C in the dark.
Use/seed/sow 1 to 2g of product to be studied or 1 to 2ml of suspension (stool
for example) for 10 to 15ml of medium enrichment. Incubate 24 hours at 37°C;
-Reseed, by taking a loop from the middle, on SS
N.B: There is no enrichment medium for shigella
I1.4.Stool culture on identification medium:
The isolated germs are transplanted into Kligler's medium and incubated in the
dish at 37°C for 24 hours.
II.5. Antibiogram