Technical Data

Xylose-Lysine-Deoxycholate Agar MH031

Intended use
Recommended as a selective medium for the isolation and enumeration of Salmonella Typhi and other Salmonella
species from pharmaceutical products in accordance with the microbial limit testing by harmonized methodology of USP/
EP/BP/JP/IP.
Composition**
Ingredients Gms / Litre
Xylose 3.500
L-lysine 5.000
Lactose monohydrate 7.500
Sucrose 7.500
Sodium chloride 5.000
Yeast extract 3.000
Sodium deoxycholate 2.500
Sodium thiosulphate 6.800
Ferric ammonium citrate 0.800
Phenol red 0.080
Agar 13.500
pH after heating ( at 25°C) 7.4±0.2
**Formula adjusted, standardized to suit performance parameters
Directions
Suspend 54.8 grams (the equivalent weight of dehydrated medium per litre) in 1000 ml purified/ distilled water.
Heat with frequent agitation until the medium boils. DO NOT HEAT IN AN AUTOCLAVE. Transfer immediately to a
water bath at 50°C. After cooling, pour into sterile Petri plates. It is advisable not to prepare large volumes, which will
require prolonged heating and may produce precipitate.
Note: Slight precipitation in the medium may occur,which is inheritant property of the medium,and does not affect the
performance of the medium.
Principle And Interpretation
Enterobacteriaceae is a family of gram-negative, non-spore-forming bacilli that contains more than 100 species of bacteria
that normally inhabit the intestines of humans and animals. Members forming part of the normal intestinal microflora are
referred to as coliforms. The clinically significant genera of Enterobacteriaceae include Cedecea, Citrobacter,
Edwardsiella, Enterobacter, Escherichia, Ewingella, Hafnia, Klebsiella, Kluyvera, Proteus, Salmonella, Shigella and
Yersinia (1).
The Salmonellae are the most complex of all the Enterobacteriaceae. Human Salmonella infections are most commonly
caused by ingestion of food, water or milk, contaminated by human or animal excreta (2). A large number of media have
been developed for the selective isolation and identification of enteric bacilli including Salmonella. Xylose Lysine
Deoxycholate Agar is a selective as well as differential medium formulated by Taylor (3-7) for the isolation and
identification of enteric pathogens especially Shigellae from stool samples. It is also used for pharmaceutical testing and
non- sterile product testing for the detection (or absence) of Salmonella after enrichment in Rappaport Vassiliadis
Salmonella Enrichment Broth (MH1491) in accordance with the harmonized method of USP/EP/BP/JP/IP (8-12).
Deoxycholate, ferric ammonium citrate and sodium thiosulphate are selective agents that inhibit gram-positive
microorganisms. Essential nutrients, growth factors for growth of microorganism are provided by yeast extract. Xylose,
sucrose and lactose are the fermentable sugars in this medium. Xylose is fermented by almost all the enteric bacteria except
Shigellae, which enable the differentiation of Shigellae from Salmonellae. Salmonellae metabolize the xylose and
decarboxylate lysine and thus change the pH to alkaline and mimic Shigellae reaction. However to prevent this reaction by
lysine positive coliforms, lactose and sucrose are added in excess to produce acid and hence nonpathogenic H2S producers
do not decarboxylate lysine. Sodium thiosulphate helps in reactivation of sulphur containing compounds and prevents the
desiccation of these compounds during storage. It also forms the substrate for enzyme thiosulphate reductase, which breaks
it to form H2S. Thiosulphate and ferric ammonium citrate are the H2S indicators in the medium. Sodium thiosulphate is
also inactivator of halogens, mercurial and aldehyde and can minimize its toxicity in the testing sample, if any during
microbial limit tests. Sodium chloride maintains the osmotic equilibrium in this medium. Phenol red is the pH indicator.
Degradation of fermentable sugars proceed concurrently and generates acids, which cause pH indicator to give various
shades of colour,

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HiMedia Laboratories Technical Data

causing a color change in the colonies and in the medium from red to yellow on prolonged incubation. Hydrogen sulfide
production results in colonies with black centers under alkaline conditions, which can be inhibited by acid production by
carbohydrate fermentation. Alkaline condition causes the color of the medium to change back to red. This medium is an
ideal medium for screening samples containing mixed flora of enteric pathogens as recovery of Salmonella and Shigella is
not conspicuous by even profuse growth of other species (13,14).
Type of specimen
Pharmaceutical samples
Specimen Collection and Handling
For pharmaceutical samples, follow appropriate techniques for sample collection, processing as per guidelines (8-12). After
use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face
protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as
per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual
safety data sheets.
Limitations :
1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the
medium for any specific microorganism other than mentioned in the COA based on the user’s unique requirement.
3. Slight precipitation in the medium may occur,which is inheritant property of the medium,and does not affect the
performance of the medium.
4. This medium is general purpose medium and may not support the growth of fastidious organisms.
5. Some Proteus strains may give red to yellow colouration with most colonies developing black centers, giving rise to false
positive reactions. Non-enterics like Pseudomonas and Providencia may exhibit red colonies.
6. S.Paratyphi A, S.Choleraesuis, S.Pullorum and S.Gallinarum may form red colonies without H2S, thus resembling
Shigella species.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at
recommended temperature.
Quality Control
Appearance
Light yellow to pink homogeneous free flowing powder
Gelling
Firm, comparable with 1.35% Agar gel
Colour and Clarity of prepared medium
Red coloured clear to slightly opalescent gel forms in Petri plates
pH
7.20-7.60
Cultural Response
Growth Promotion is carried out in accordance with the harmonized method of USP/EP/BP/JP/IP. Cultural response
was observed after an incubation at 30-35°C for specified time. Recovery rate is considered as 100% for bacteria
growth on Soyabean Casein Digest Agar.
Growth promoting properties
Growth of microorganism comparable to that previously obtained with previously tested and approved lot of medium occurs
at the specified temperature for not more than the shortest period of time specified inoculating <=100 cfu(at 30-35°C for
<=18 hours).
Indicative properties
Colonies are comparable in appearance and indication reaction to those previously obtained with previously tested and
approved lot of medium occurs for the specified temperature for a period of time within the range specified inoculating
<=100cfu (at 30-35°C for 18-72 hours).
Inhibitory properties
No growth of the test microorganism occurs for the specified temp for not less than longest period of time specified
inoculating >=100cfu (at 30-35°C for >= 72 hours).

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HiMedia Laboratories Technical Data

Cultural Response
Cultural characteristics observed after incubation at 30-35 °C for 18-48 hours. Recovery rate is considered as 100% for
bacteria growth on Soyabean Casein Digest Agar.
Organism Inoculum Growth Observed Lot Recovery Colour of Incubation
(CFU) value (CFU) Colony temperature
Growth Promoting +
Indicative
Salmonella Typhimurium 50 -100 luxuriant 25 -100 >=50 % red with black 18 -72 hrs
ATCC 14028 (00031*) centres
Salmonella Abony 50 -100 good-luxuriant 25 -100 >=50 % red with black 18 -72 hrs
NCTC 6017 (00029*) centres
Additional Microbiological
testing
Escherichia coli 50 -100 fair 10 -30 20 -30 % yellow 18 -72 hrs
ATCC 8739 (00012*)
Escherichia coli 50 -100 fair 10 -30 20 -30 % yellow 18 -72 hrs
ATCC 25922 (00013*)

Escherichia coli NCTC 9002 50 -100 fair 10 -30 20 -30 % yellow 18 -72 hrs
Proteus vulgaris ATCC 50 -100 good-luxuriant 25 -100 >=50 % grey with black 18 -72 hrs
13315 centres
Salmonella Paratyphi A 50 -100 good-luxuriant 25 -100 >=50 % red 18 -72 hrs
ATCC 9150
Salmonella Paratyphi B 50 -100 good-luxuriant 25 -100 >=50 % red with black 18 -72 hrs
ATCC 8759 centres
Salmonella Enteritidis 50 -100 good-luxuriant 25 -100 >=50 % red with black 18 -72 hrs
ATCC 13076 (00030*) centres
Salmonella Typhi 50 -100 good-luxuriant 25 -100 >=50 % red with black 18 -72 hrs
ATCC 6539 centres
Shigella dysenteriae 50 -100 good-luxuriant 25 -100 >=50 % red 18 -72 hrs
ATCC 13313
Shigella flexneri 50 -100 fair-good 15 -40 30 -40 % red 18 -72 hrs
ATCC 12022 (00126*)
Shigella sonnei ATCC 25931 50 -100 fair-good 15 -40 30 -40 % red 18 -72 hrs
# Klebsiella aerogenes 50 -100 fair 10 -30 20 -30 % yellow 18 -72 hrs
ATCC 13048 (00097*)
Enterobacter cloacae 50 -100 fair 10 -30 20 -30 % yellow 18 -72 hrs
ATCC 13047
Staphylococcus aureus >=10³ inhibited 0 0% >=72 hrs
subsp. aureus ATCC
25923 (00034*)
Staphylococcus aureus >=10³ inhibited 0 0% >=72 hrs
subsp. aureus ATCC
6538 (00032*)
Enterococcus faecalis >=10³ inhibited 0 0% >=72 hrs
ATCC 29212 (00087*)
Key: (#) Formerly known as Enterobacter aerogenes (*) Corresponding WDCM numbers
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. For better performance. it
is advised to store the plates at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored
dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product.
Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of
temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated
expiry period.

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HiMedia Laboratories Technical Data

Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product.
Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with
sample must be decontaminated and disposed of in accordance with current laboratory techniques (2,13).
Reference
1. Pelczar M. J. Jr., Reid R. D., Chan E. C. S., 1977, Microbiology, 4th Ed., Tata McGraw-Hill Publishing Company
Ltd, New Delhi.
2.Koneman E. W., Allen S. D., Janda W. M., Schreckenberger P. C., Winn W. C. Jr., 1992, Colour Atlas and
Textbook of Diagnostic Microbiology, 4th Ed., J. B. Lippinccott Company.
3.Taylor W. L., 1965, Am. J. Clin. Pathol., 44:471-475.
4.Taylor W. L. and Harris B., 1965, Am. J. Clin. Pathol., 44:476.
5.Taylor W. L. and Harris B., 1967, Am. J. Clin. Pathol., 48:350.
6.Taylor W. L. and Schelhart B., 1967, Am. J. Clin. Pathol., 48:356.
7.Taylor W. L. and Schelhart B., 1968, Am. J. Clin. Pathol., 16:1387.2.
8..The British Pharmacopoeia, 2022, Medicines and Healthcare products Regulatory Agency.
9.European Pharmacopoeia, 2022, 10 th volume, European Directorate for the quality of medicines & Healthcare.
10.Indian Pharmacopoeia, 2022, Indian Pharmacopoeia Commission, Ministry of Health and Family Welfare Government of
India.
11.The Japanese Pharmacopoeia, 17th edition, 2016, The Ministry of Health, Labour and welfare.
12.The United States Pharmacopoeia-National Formulatory (USP-NF), 2022.
13.Isenberg H.D., Kominos S. and Siegal M., 1969, Appl. Microbiol., 18:656.
14.McCarthy M.D., 1966, N.Z. J. Med. Lab. Technol., 20:127.

Revision : 05/2022

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User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and
other related HiMedia™ publications. The information contained in this publication is based on our research and development work and is to the best
of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to specifications and information related
to the products at any time. Products are not intended for human or animal or therapeutic use but for laboratory,diagnostic, research or further
manufacturing use only, unless otherwise specified. Statements contained herein should not be considered as a warranty of any kind, expressed or
implied, and no liability is accepted for infringement of any patents.

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