Taylor created XL (Xylose Lysine) Agar Base to isolate and differentiate Gram-negative enteric bacteria. Sodium thiosulfate, ferric ammonium citrate, and sodium deoxycholate were added to XL Agar Base to create XLD Agar, a more selective medium. Using numerous staining chemicals, George Chapman and his colleagues at The Clinical Research Laboratory in New York produced a series of isolation media in the 1930s and 1940s.

1. CULTURE MEDIA
Asif Nawaz
M.phil 4th
Department of Microbiology
Abbottabad university of science and technology

2. Culture Media
Xylose Lysine Deoxycholate agar (XLD)
Taylor created XL (Xylose Lysine) Agar Base to isolate and differentiate Gram-
negative enteric bacteria.
Sodium thiosulfate, ferric ammonium citrate, and sodium deoxycholate were
added to XL Agar Base to create XLD Agar, a more selective medium.

3. Xylose Lysine Deoxycholate agar (XLD)
Principle
Yeast Extract gives organisms the nitrogen, carbon, and vitamins as they need it to
develop.
Fermentable carbohydrate sources include xylose, lactose, and sucrose.
Except for Shigella spp. and Providencia spp., most intestinal microbes ferment xylose.
Salmonella is distinguished by the addition of lysine.

4. Xylose Lysine Deoxycholate agar (XLD)
Why XLD is selective and differential?
XLD Agar is both a selective and differential medium. It contains yeast extract as a
source of nutrients and vitamins.
It utilizes sodium deoxycholate as the selective agent and, therefore, is inhibitory to
gram-positive micro-organisms.
 Xylose is incorporated into the medium since it is fermented by practically all enterics
except for the Shigella and this property enables the differentiation of Shigella species.
 Lysine is included to enable the Salmonella group to be differentiated from the non
pathogens since without lysine, salmonellae rapidly would ferment the xylose and be
indistinguishable from non-pathogenic species.

5. Xylose Lysine Deoxycholate agar (XLD)
Composition
Ingredients Amount (gm/L)
Lactose 7.5 gm
Sucrose 7.5 gm
Sodium Thiosulfate 6.8 gm
L-Lysine 5.0 gm
Sodium Chloride 5.0 gm
Xylose 3.75 gm
Yeast Extract 3.0 gm
Sodium Deoxycholate 2.5 gm
Ferric Ammonium Citrate 0.8 gm
Phenol Red 0.08 gm
Agar 15.0 gm
Distilled water 1000 ml

6. Xylose Lysine Deoxycholate agar (XLD)
Result interpretation
Degradation of xylose, lactose and sucrose generates acid products, causing a colour
change in the medium from red to yellow.
Hydrogen sulfide production under alkaline conditions causes colonies to develop black
centres. This reaction is inhibited by the acid conditions that accompany carbohydrate
fermentation.
Lysine decarboxylation in the absence of lactose and sucrose fermentation causes
reversion to an alkaline condition and the colour of the medium changes back to red.

7. Manitol Salt Agar (MSA)
Using numerous staining chemicals, George Chapman and his colleagues at The Clinical Research
Laboratory in New York produced a series of isolation media in the 1930s and 1940s.
A combination of bromythymol-blue lactose agar and phenol red mannitol agar was proven to be
reliable for isolation of pathogenic Staphylococci spp after work and re-examination of mannitol
fermentation.
 In 1942, it was discovered that high doses of sodium chloride in medium inhibited the growth of
most organisms except Staphylococci. MSA was created with the extra property of phenol-red
becoming yellow in acidic environments.

8. Manitol Salt Agar (MSA)
Principle
Amino acids, nitrogen, carbon, vitamins, and minerals are provided via pancreatic
digest of casein, peptic digest of animal tissue, and beef extract for the growth of
organisms.
The fermentable carbohydrate is mannitol .
Except for staphylococci, most microorganisms are inhibited by the high salt level
of 7.5 %.

9. Manitol Salt Agar (MSA)
Why MSA is selective and differential?
Mannitol salt agar (MSA) is both a selective and differential medium for culturing
staphylococci.
 The medium is selective because the presence of a high salt concentration (7·5%)
suppresses the growth of most bacteria.
However, other salt-tolerant bacteria are able to proliferate on this medium.
This agar also contains mannitol, which serves as a differential agent. Staphylococcus
aureus ferments the sugar alcohol to acidic by-products, lowering the pH of the agar.

10. Manitol Salt Agar (MSA)
Composition
Ingredients Amount (gm/L)
Pancreatic Digest of Casein 5.0 gm
Peptic Digest of Animal Tissue 5.0 gm
Beef Extract 1.0 gm
Sodium Chloride 75.0 gm
D-Manitol 10.0 gm
Phenol Red 0.025gm
Agar 15.0 gm
Distilled Water 1000 ml

11. Manitol Salt Agar (MSA)
Result interpretation
S. aureus grows in colonies that are yellow or white and surrounded by a yellow
zone.
Coagulase-negative Staphylococci produce little colourless to red colonies that are
unaffected by the medium's colour.

12. Eosin Methylene Blue Agar (EMB)
Lactose and sucrose were used as carbohydrate sources in EMB agar,
which was first described by Holt-Harris and Teague in 1916.
Levine went on to add peptone and phosphate to the medium, as well as
removing sucrose from the formula and increasing the lactose level.

13. Eosin Methylene Blue Agar (EMB)
Principle
Sucrose and lactose are used as fermentable carbohydrates substrates in EMB
agar, which promote the growth of gram-negative bacteria, particularly faecal and
non-fecal coliforms.
The inclusion of the sugars lactose and sucrose in the EMB agar, as well as the
capacity of some bacteria to ferment the lactose in the medium, allows for the
differentiation of enteric bacteria.

14. Eosin Methylene Blue Agar (EMB)
Why EMB is differential and selective?
 EMB contains the dyes eosin and methylene blue that inhibit the growth of gram-
positive bacteria therefore, EMB is selective for gram-negatives.
 In addition, the gram-negatives that grow can be differentiated based on their
ability to ferment lactose.

15. Eosin Methylene Blue Agar (EMB)
Composition
Ingredients Amount (gm/L)
Peptic Digest of Animal Tissue 10.0 gm
Dipotassium Phosphate 2.0 gm
Lactose 5.0 gm
Sucrose 5.0 gm
Eosin-Y 0.40 gm
Methylene Blue 0.065 gm
Agar 13.50 gm

16. Eosin Methylene Blue Agar (EMB)
Result interpretation
• Dual dyes, eosin Y and methylene blue, are mixed; they inhibit gram-positive bacteria
while allowing gram-negative microbes to proliferate.
• Gram-negative bacteria metabolize the lactose, resulting in dark purple colonies.
• Some lactose-fermenting bacteria generate black, flat colonies with a distinctive green
metallic sheen.
• Some lactose-fermenting bacteria form enormous mucoid colonies with a distinct purple
colony in the centre

17. Pseudomonas Cetrimide agar (PCA)
Lowburry invented Cetrimide Agar, which is a modified Tech Agar with 0.1
percent cetrimide (cetyl trimethyl ammonium bromide) added for the
selective suppression of organisms besides Pseudomonas aeruginosa.
 Cetrimide agar is mostly used to isolate and presumptively identify
Pseudomonas aeruginosa from clinical and nonclinical samples

18. Pseudomonas Cetrimide agar (PCA)
Principle
Cetrimide agar is used to test an organism's ability to grow in the presence of
cetrimide, a toxic substance that prevents the growth of many bacteria by causing
the release of nitrogen and phosphorous, slowing or killing the organisms because
organisms other than P. aeruginosa are unable to survive this germicidal activity,
whereas P. aeruginosa is resistant to cetrimide.

19. Pseudomonas Cetrimide agar (PCA)
Why PCA is differential and selective?
Cetrimide Agar, also known as Pseudomonas Cetrimide Agar or Pseudosel Agar, is a selective and
differential medium used for the isolation and identification of Pseudomonas aeruginosa from
clinical and non-clinical specimens.
Pseudomonas aeruginosa can be distinguished by their synthesis of pyocyanin, a blue, water-
soluble, non-fluorescent phenazine pigment, as well as their colony morphology and
aminoacetophenone's distinctive grape-like odour.
By slowing or killing the organisms other than P. aeruginosa which are unable to survive this
germicidal activity, whereas P. aeruginosa is resistant to cetrimide that’s why it is selective.

20. Pseudomonas Cetrimide agar (PCA)
Composition
Ingredients Amount (gm/L)
Peptone 16.0 gm
Casein Hydrolysate 10.0 gm
Potassium Sulphate 10.0 gm
Magnesium Chloride 1.4 gm
Agar 11.0 gm
Cetrimide 0.2 gm
Sodium Nalidixate 0.015 gm

21. Pseudomonas Cetrimide agar (PCA)
Result interpretation
Colonies with a blue-green tint surrounding them and UV light with a short
wavelength (254 nm) may fluoresce. Pseudomonas aeruginosa will most
likely be recognized.