Technical Data Sheet

GranuCult® plus
EMB (Eosin-Methylene blue) agar
acc. HOLT-HARRIS and TEAGUE

Ordering number: 1.03858.0500

For the isolation and differentiation of Escherichia coli and other Gram-negative bacteria from different
materials.

EMB (Eosin-Methylene blue) agar acc. HOLT-HARRIS and TEAGUE is also known as Holt-Harris &
Teague (HHT) EMB agar, as Eosin Methylene Blue Lactose Sucrose agar and as Eosin Methylene Blue
agar, modified.

Mode of Action

This culture medium is a low selective and differential medium containing lactose and sucrose
and two indicator dyes, Eosin Y and Methylene Blue. The use of these two indicators allows
differentiation between colonies of lactose and/or sucrose fermenting and non-fermenting
organisms. Sucrose is included since certain members of the coliform group of microorganisms
ferment sucrose more readily than lactose. This formulation does not discriminate between
carbohydrate utilization (lactose or sucrose).

The peptones provide nitrogen, minerals and amino acids, lactose and sucrose provide the
carbon sources. Di-potassium phosphate acts as a buffer whilst Eosin yellowish (Eosin Y) and
methylene blue serve as differential indicators and inhibitors. Agar is the solidifying agent.

Coliforms produce blue-black colonies due to the incorporation of an eosin-methylene blue dye
complex when the pH drops down. Colonies of Escherichia coli may show a characteristic green
metallic luster due to the rapid fermentation of lactose. Lactose- and sucrose-non fermenting
bacteria (like most Salmonella and Shigella) will form colorless or transparent amber coloured
colonies, but some strains of Salmonella and Shigella may not grow on this culture medium.
Some Gram-positive bacteria and yeasts may grow on this culture medium and usually form
pinpoint colonies.

Merck, Millipore, and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its
affiliates. Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Version 2021-01-26 Page 1 of 4

Typical Composition

GranuCult® plus
EMB (Eosin-Methylene blue) agar
acc. HOLT-HARRIS and TEAGUE
Peptones 10.0 g/l

K2HPO4 2.0 g/l

Lactose 5.0 g/l

Sucrose 5.0 g/l

Eosin yellowish (Eosin Y) 0.4 g/l

Methylene blue 0.07 g/l

Agar-agar* 13.5 g/l

Water n/a

pH at 25 °C 7.1 ± 0.2

* Agar-Agar is equivalent to other different terms of agar.

Preparation

Dissolve 36.0 g in 1 liter of purified water. Heat in boiling water and agitate frequently until
completely dissolved. Autoclave (15 minutes at 121 °C). Pour to plates.

The dehydrated medium is a granulate with pink color.

The prepared medium is clear to slightly opalescent and reddish-brown to violet-brown. The pH value at
25 °C is in the range of 6.9 - 7.3.

Before inoculation, allow the prepared medium to equilibrate at room temperature if it was stored at
a lower temperature.

There should be no visible moisture on the plates before use. When moisture is present, the plates
should be dried for the minimum time required to remove visible moisture, following the procedure
as described by EN ISO 11133.

Experimental Procedure and Evaluation

Depend on the purpose for which the medium is used.
Inoculate by spreading or streaking to permit discrete colonies.
Incubate 24 to 48 h, e. g. at (35 ± 1) °C, aerobic.

Merck, Millipore, and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its
affiliates. Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Version 2021-01-26 Page 2 of 4

Colonies of the most important bacteria usually have the appearance described below:

Appearance of colonies Microorganisms

Lactose- and sucrose-negative:

Translucent, amber coloured
Salmonella spp., Shigella spp. and others.

Greenish, metallic luster in reflected light,

mostly Escherichia coli
blue-black center in transmitted light

Mucoid, confluent, gray-brown centered

colonies in transmitted light, often larger Enterobacter spp., Klebsiella spp. and others
than those of E. coli

Storage
Store at +15 °C to +25 °C, dry and tightly closed. Do not use clumped or discolored medium.
Protect from UV light (including sun light). For in vitro use only.

Microbiological Performance

Specification
Test strain
Growth Typical reaction
Escherichia coli
Good to very good Metallic luster
ATCC® 25922 [WDCM 00013]

Escherichia coli
Good to very good Metallic luster
ATCC® 8739 [WDCM 00012]
Salmonella Typhimurium
Good to very good No metallic luster
ATCC® 14028 [WDCM 00031]
Bacillus cereus
None to poor No metallic luster
ATCC® 11778 [WDCM 00001]

Incubation: 22 ± 2 h at 35 ± 1 °C, aerobic.

Please refer to the actual batch related Certificate of Analysis.

Merck, Millipore, and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its
affiliates. Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Version 2021-01-26 Page 3 of 4

Literature

EN ISO International Standardisation Organisation. Microbiology of food, animal feed and water -
Preparation, production, storage and performance testing of culture media + Amendment 1 +
Amendment 2. EN ISO 11133:2014/Amd1:2018/Amd2:2020.

Holt-Harris, J.E. and Teague, O.A. (1916). A new culture medium for the isolation of Bacillus typhosus
from stools. J. Infect. Dis. 18: 596-600.

McFaddin J.F. (1985): Eosin Methylene Blue Agars. In: Media for isolation – cultivation – identification
– maintenance of medical bacteria. Volume I. pp. 292-297. Lippincott Williams and Wilkins, Baltimore,
MD, USA.

Escherichia coli Enterobacter cloacae Escherichia coli

ATCC® 25922 ATCC® 13047 ATCC® 8739
[WDCM 00013] [WDCM 00083] [WDCM 00012]

Ordering Information

Product Cat. No. Pack size

GranuCult® plus
1.03858.0500 500 g
EMB (Eosin-Methylene blue) agar acc. HOLT-HARRIS and
TEAGUE

Merck, Millipore, and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its
affiliates. Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Version 2021-01-26 Page 4 of 4