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TTC CHAPMAN AGAR (Lactose Agar TTC with Tergitol 7) ISO 9308-1

CAT N: 1076 For the detection and enumeration of E.coli and coliforms in
drinking water by membrane filtration technique


FORMULA IN g/ l
Lactose 20.00 Sodium Heptadecyl Sulfate (Tergitol 7) 0.10
Peptone 10.00 Bromothymol Blue 0.050
Yeast Extract 6.00 Bacteriological Agar 15.00
Beef Extract 5.00
Final pH 7.2 0.1 at 25C




PREPARATION
Suspend 56.15 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent
agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121C for 15 minutes. Cool to 45-50C
and aseptically add 2.5 ml of TTC 1% Supplement (Cat. 6030) previously reconstituted in 5 ml of sterile distilled water.
Homogenize gently and dispense into Petri dishes. DO NOT OVERHEAT THE MEDIUM. The prepared medium should be
stored at 2-8C. The color is green.

The dehydrated medium should be homogeneous, free-flowing and greenish-beige in color. If there are any physical
changes, discard the medium

TTC 1% SUPPLEMENT (CAT. 6030)
1 Vial reconstructed in 5 ml of distilled water

Triphenyltetrazolium Chloride 50 mg


USES
TTC CHAPMAN AGAR (Lactose Agar TTC with Tergitol 7) is a selective and differential medium prepared according to
ISO 9308-1, is used for the presumptive control of E.coli and coliforms in waters for human consumption by the
membrane - filtration technique.

Peptone and Beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is
source of vitamins, particularly the B-group. Lactose is a fermentable carbohydrate providing carbon and energy. Sodium
heptadecylsulfate (Tergitol 7) and TTC inhibit most gram positive bacteria. Bromothymol blue is a pH indicator.
Bacteriological agar is the solidifying agent.

The norm ISO 9308-1 recommends:
Two samples of water must be taken on two membranes and incubated in TTC Chapman Agar at 362C and
44 4 C respectively. After 21 3 hours of incubation:
- E. coli and Citrobacter spp present yellow colonies with orange-colored center.
- Enterobacter spp form red colored colonies and dark yellow with orange-colored center. The medium is yellow.
- Klebsiella spp form red colored or yellow, but without center. The medium is yellow.
- Lactose non fermentative bacteria grow with purple colonies and change the medium to blue.
Non fermenting species
Escherichia coli
ATCC 25922





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- Klebsiella and Enterobacter species can also produce yellow-green colonies.

The results will always refer to counts per 100 ml of sample (considering if it has been necessary to make dilutions). The
colonies that grow at 362C will be considered as fecal coliforms and the colonies that grow at 44 4C are considered
as E. coli.

Confirmation of the colonies in EMB Agar (Cat. 1039), Kligler Iron Agar (Cat. 1042), etc. is necessary for the verification
of the biochemical characteristics. The Indole and Oxidase tests are carried out in the following media respectively:
Tryptophan Culture Broth (Cat. 1237) and Tryptone Soy Agar (Cat. 1138).


MICROBI OLOGI CAL TEST
The following results were obtained in the performance of the medium from type cultures, with the Supplement added,
after incubation at a temperature of 36 2C and 444C and observed after 21 3 hours.


Microorganisms
Growth
44C
Growth
36C
Colony Color
Inoculum
cfu/ ml
Recovery
Rate
36C
Recovery
Rate
44C
Escherichia coli ATCC 25922 Good Good
Yellow with orange
center
10
3
-10
5
50 %
50 %
Citrobacter spp Null Good
Yellow with orange
center
10
3
-10
5
50 %
0.01 %
Klebsiella spp Null Good Red to yellow 10
3
-10
5
50 % 0.01 %
Enterobacter aerogenes ATCC
13048
Null Good
Red to dark yellow
with orange center
10
3
-10
5
50 %
0.01 %
Non fermenting species Null Good Light violet 10
3
-10
5
50 % 0.01 %
Enterococcus faecalis ATCC
11700
Total to
partially
inhibited
Total to
partially
inhibited
--- 10
5
0.01 % 0.01 %


BIBLIOGRAPHY
Chapman G.H. 1946. A single culture medium for selective isolation of plasma coagulating staphylococci and for improved testing of
chromogenesis (J . Bacteriol. 51: 409-410).
Tittsler R.P. and L.A. Sandholzer. 1936. The Use of Semi-Solid Agar for the detection of bacteria motility. (J . Bacteriol 31: 575-580)
ISO 9308-1.Water quality. Detection and enumeration of Escherichia coli and coliform bacteria.PART.1. Membrane filtration method




STORAGE
Once opened keep powdered medium closed to avoid hydration.

2C
25C

ISO