Experiment 3
Enteric bacteria
Contens
• Growth on identification culture
medium of enteric rods
• Growth on Double sugar-ion agar
• Five sugar fermentation
• Widal Test
Growth on identification culture
medium of enteric rods
 Salmonella-Shigella agar
E.coli-- lactose fermenters--acid-their colonies large, red and opaque.
Shigella, Salmonella -- not lactose
fermenters, their colonies small,
uncolored, transparent.
E.coli
Salmonella
Growth on Double sugar-ion agar
Double sugar-iron agar
solid medium-slant
(Lactose )
Ferrous sulphate
semi-solid mediumlocomotion (glucose)
Media that differentiate characteristics. (a) double-sugar iron agar in a
slant. This medium contains two fermentable carbohydrates, phenol red to
indicate pH changes, and a chemical (iron) that indicates H2S gas production.
Reactions (from left to right) are: a tube in which no acid was produced (red); a
tube in which acid production (yellow) has occurred in the bottom (butt) of the
slant; a tube in which acid has been produced throughout the slant; and a tube in
which H2S has been produced, forming a black precipitate.
Double sugar-iron(DSI) agar
DSI results:
agar change
bacteria
E.coli
S. dysentery
S. typhi
S. paratyphi B
slant butt
H2S
gas
flagellum
Five sugar fermentation
Five sugar fermentation
Glucose （red)
Lactose (yellow)
Maltose (blue)
mannitol (white)
Sucrose (black)
Indicator: bromcresol purple
alkalescence: purple
If sugar is fermented, produce acid, pH
purple
yellow
E.coli
S. paratyphi B
S. typhi
Indicator: bromcresol purple-acid:yellow
-alkalescence: purple
bacteria
G L M M S
S. typhi
S. paratyphi B
+ - + +  -   -
S dysenteriae
+ - + + -
E.coli
    -
+: sugar is fermented with acid production
-: sugar is not fermented
: sugar is fermented with acid and air production
Widal Test
To detect the unknown sera with given Ag
(O,H,HA,HB) – millipore plate or tube
agglutination, so to determine whether there is
relative antibody and its titer, help to the
diagnosis of typhoid and paratyphoid.
– H, O antigen from S. typhi
– HA,HB from S. paratyphi A and B
MATERIALS
Reagents:
(1)S typhi H-antigen suspension and O-antigen
suspension , HA,HB suspension from S
paratyphi A and B
(2) The patient’s serum in 1:10 dilution,
(3) NS.
Equipment：
Millipore plate , pipette，wax pencil，vial,
thermostatic incubator.
MATERIALS
Each student：1 millipore plate ,
1 pipette ， 1 vial
Each group：
Antigens（H、O、PA、PB）and
1:10 serum, wax pencil，NS.
PROCEDURES
1. Dilution of antiserum
Pipette 12 drops of antiserum into vial, then
pipette 12 drops of NS, mix thoroughly by
pulling the fluid up and down. The antiserum
has been diluted doubly (1:20).
Continue this procedure through to 1:640.
2. Add 3 drops of corresponding Ag in each
row.
Mix contents by shaking the millipore-plate.
3. Incubate at 50ºC for 1.5h.
– Gently move the millipore-plate onto the bench
tops, avoid moving for 10 minutes.
4. Observe results
1:10 serum
12 d
1:20
Serum
1:20
12 d
Serum
+NS 12d
3d hole
NS
12d
Antigen
1:40
Serum
12 d
+NS 12d
1:80
Serum
12d
+NS 12d
1:160
Serum
12 d
+NS 12d
NS
H
O
PA
PB
1:40
1:80
1:160
1:320
1:640
Control
Notice
1.Each pore of same column adding 3 drops
same diluted serum.
2. Each pore of same row adding 3 drops
same antigen.
3. Pipette 3 drops of saline into each pore in last row．
4. Pipette is kept upright.
5. When taking NS、serum or antigen, you must use
corresponding pipette.
Result
－
No clumping is observed
+
+++
A few clumping are observed to scatter
at the bottom
A moderate amount of clumping are
observed to scatter at the bottom
Almost all clumping
++++
All clumping
++
Normal value
O < 1:80;
H < 1:160;
H (A/B) < 1:80
O. and H. antibody’s diagnosing
O---IgM H---IgG
O Ab↑, H Ab↑
enteric fever
O Ab↓, H Ab↓
not enteric fever
O Ab↑, H Ab↓
early infection or
cross reaction with other salmonellae
O Ab↓, H Ab↑
prophylactic vaccination
or non-specific anamnestic reaction
Report of experiment
Record and analyze the result of Widal test.