Mannitol Salt Agar Classification Essay

MANNITOL SALT AGAR (MSA)

Cat. no. J330 Tryptic Soy Agar (TSA) / Cetrimide (CET) / Mannitol Salt Agar (MSA), 15x100mm Triplate, 7ml/section 10 plates/bag

INTENDED USE

Hardy Diagnostics Mannitol Salt Agar (MSA) is recommended for use as a selective and differential medium for the isolation of pathogenic staphylococci.

SUMMARY

Koch reported the use of a medium containing 7.5% sodium chloride as a selective agent for the isolation of staphylococci in 1942.(5) The results were confirmed and improved by Chapman in 1945 by the addition of this salt concentration to Phenol Red Mannitol Agar, as Staphylococcus aureus usually ferments mannitol.(3) Non-pathogenic staphylococci usually show less luxuriant growth on this medium after the incubation period.

A sodium chloride, concentration of 7.5%, is nearly ten times the usual concentration seen in most media. It serves to inhibit most organisms except staphylococci in mixed flora specimens. The beef extract and peptones supply the essential elements carbon, nitrogen, and sulfur. Mannitol is added to show the fermentation capabilities of the organisms. Acid production as the result of fermentation of this sugar results in the formation of colonies with a yellow zone. Those staphylococci that do not ferment mannitol show a purple or red zone around the colonies.

Mannitol Salt Agar (MSA) is recommended by the American Public Health Association for the enumeration of staphylococci in food and dairy products.(9,10)

FORMULA

Ingredients per liter of deionized water:*

Sodium Chloride 75.0gm
Proteose Peptone 10.0gm
Mannitol 10.0gm
Beef Extract 1.0gm
Phenol Red 0.025gm
Agar 15.0gm

Final pH 7.4 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection.(1,2,4,7) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation.

Method of Use: Allow the plates to warm to room temperature and the agar surface to dry before inoculating. Heavily inoculate and streak the specimen as soon as possible after collection. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface. Streak for isolation with a sterile loop. Incubate plates aerobically at 35-37ºC for 24-48 hours. Examine colonial morphology.

INTERPRETATION OF RESULTS

Mannitol fermentors such as S. aureus appear as yellow colonies with yellow zones in the media. Non-mannitol fermentors such as S. epidermidis, if present, will have clear pink to red colonies with no yellow color change in the medium. Consult listed references for the identification of colony morphology and further biochemical tests required for identification.(1,2,4,7)

LIMITATIONS

Most organisms other than staphylococci are inhibited by the high salt concentration found in Mannitol Salt Agar except for some halophillic marine organisms.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Staphylococcus aureus
ATCC® 6538
J 18hr 35°C Aerobic Growth; yellow colonies and media at 18-24 hours
Escherichia coli **
ATCC® 8739
B72hrs35°CAerobicPartial to complete inhibition

USER QUALITY CONTROL

Physical Appearance

Mannitol Salt Agar (MSA) should appear clear, slightly opalescent, and pinkish-red in color.

Staphylococcus aureus (ATCC® 25923) colonies growing on Mannitol Salt Agar. Incubated aerobically for 48 hours at 35ºC.

Proteus mirabilis (ATCC® 12453) growth inhibited on Mannitol Salt Agar. Incubated aerobically for 48 hours at 35ºC.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Chapman, G.H. 1945. J. Bacteriol.; 50:201.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koch, F.E. 1942. Zentr. Bakt. Labt. Orig.; 149:122.

6. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

7. Jorgensen, J.H., et al. 2015. Manual of Clinical Microbiology, 11th ed. American Society for Microbiology, Washington, D.C.

8. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

9. American Public Health Association. 1993. Standard Methods for the Examination of Dairy Products, 16th ed. APHA, Washington, D.C.

10. APHA Technical Committee on Microbiological Methods for Foods. 2001. Compendium of Methods for the Microbiological Examination of Foods, 4th ed. APHA, Washington, D.C.

ATCC is a registered trademark of the American Type Culture Collection.

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Cultivation Media for Bacteria

Isolation of bacteria is accomplished by growing ("culturing") them on the surface of solid nutrient media. Such a medium normally consists of a mixture of protein digests (peptone, tryptone) and inorganic salts, hardened by the addition of 1.5% agar. Examples of standard general purpose media that will support the growth of a wide variety of bacteria include nutrient agar, tryptic soy agar, and brain heart infusion agar. A medium may be enriched, by the addition of blood or serum. Examples of enriched media include sheep blood agar and chocolate (heated blood) agar.

Selective media contain ingredients that inhibit the growth of some organisms but allow others to grow. For example, mannitol salt agar contains a high concentration of sodium chloride that inhibits the growth of most organisms but permits staphylococci to grow.

Differential media contain compounds that allow groups of microorganisms to be visually distinguished by the appearance of the colony or the surrounding media, usually on the basis of some biochemical difference between the two groups. Blood agar is one type of differential medium, allowing bacteria to be distinguished by the type of hemolysis produced. Some differential media are also selective, for example, standard enteric agars such as MacConkey and EMB agars, which are selective for gram-negative coliforms and can differentiate lactose-fermenting and non-lactose-fermenting bacteria.

Several examples of commonly used bacteriological media, as well as examples with one or more types of bacteria cultured on them are shown below. Carefully examine the plates and observe the colony morphology, colors, and patterns of growth (or no growth) that occurs. This information can be valuable in the preliminary identification of pathogens in case studies.

Common Bacteriologic Media

Tryptic Soy Agar (TSA)

Tryptic Soy Agar - uninoculated

Type: General

Purpose: Cultivation of non-fastidious bacteria

Interpretation: Growth indicates non-fastidious bacteria present

S. aureus

Tryptic Soy Agar - Staphylococcus aureus
Note the carotenoid pigment typical of S. aureus.

E. coli

Tryptic Soy Agar - Escherichia coli

P. aeruginosa

Tryptic Soy Agar - Pseudomonas aeruginosa
Note the blue-green color due to pyocin production by the bacteria.

Chocolate Agar

Chocolate Agar - uninoculated

Type: Enriched

Purpose: Cultivation of fastidious organisms such as Neisseria or Haemophilus sp.

Interpretation: Some organisms grow on Chocolate that do not grow on standard media

S. aureus

Chocolate Agar - Staphylococcus aureus
Note: luxuriant growth with yellow pigmented colonies.

N. gonorrhoeae

Chocolate Agar - Neisseria gonorrhoeae
Note: small colonies that appear transparent on close examination.

E. coli

Chocolate Agar - Escherichia coli
Note: luxuriant growth of gray-white colonies

Thayer-Martin Agar

Thayer-Martin Agar - uninoculated

Type: Enriched and selective; contains antibiotics colistin (kills gram-negative coliforms), vancomycin (kills gram-positives), nystatin (kills fungi)

Purpose: To select for fastidious organisms, such as N. gonorrhoeae, in patient samples containing large numbers of normal flora, such as in the female genital tract

S. aureus

Thayer-Martin Agar - Staphylococcus aureus
Note: vancomycin in the medium inhibits the growth of staphylococci.

N. gonorrhoeae

Thayer-Martin Agar - Neisseria gonorrhoeae
Note: luxuriant growth of this fastidious bacterium.

E. coli

Thayer-Martin Agar - Escherichia coli
Note: colistin in the media inhibits the growth of enterics.

MacConkey (lactose) Agar

MacConkey Agar - uninoculated

Type: Selective and differential

Purpose: Contains bile salts and crystal violet which selects for gram-negative enterics, differentiates lactose-fermenters from non-fermenters. Can include sugars other than lactose for further differentiation (for example, to differentiate enterohemorrhagic E. coli (EHEC), which does not ferment sorbitol, from other E. coli types which do.)

Interpretation: Selects for non-fastidious gram-negatives; red colonies indicate fermentation of lactose, white indicates no fermentation of lactose

E. coli

MacConkey Agar - Escherichia coli
Note: Red colonies and red precipitate due to acid production as a result of lactose fermentation.

N. gonorrhoeae

MacConkey Agar - Neisseria gonorrhoeae
Note: Neisseria does not grow on MacConkey.

S. enteritidis

MacConkey Agar - Salmonella enteritidis
Note: Growth, but no fermentation of lactose. Colorless colonies, medium is slightly yellow due to the increased pH resulting from bacterial digestion of peptone in the medium.

S. aureus

MacConkey Agar - Staphylococcus aureus
Note: Gram-positives do not grow on MacConkey.

Eosin-methylene Blue Agar (EMB)

Eosin Methylene Blue Agar - uninoculated

Type: Differential (lactose) and selective (dye inhibition and precipitation at acid pH)

Purpose: Differentiates lactose fermenters (E. coli) from non-fermenters (Salmonella, Shigella)

Interpretation: Lactose fermenters blue/black; non-fermenters colorless or light purple

S. enteritidis

Eosin Methylene Blue Agar - Salmonella enteritidis
Note: pink colonies indicative of non-lactose fermentation.

E. coli

Eosin Methylene Blue Agar - Escherichia coli
Note: Green metallic sheen indicative of dye precipitation due to lactose fermentation.

K. pneumoniae

Eosin Methylene Blue Agar - Klebsiella pneumoniae
Note: Mucoid colonies with dark centers due to capsule production and lactose fermentation respectively.

Hektoen Agar

Hektoen - uninoculated

Type: Selective and differential

Purpose: Detects lactose fermentation, H2S production, inhibits non-enterics

Interpretation: Lactose fermenters yellow or salmon, non-fermenters colorless; H2S production produces black precipitate

E. coli

Hektoen - Escherichia coli
Note: Orange color indicates acid production as a result of lactose fermentation.

S. enteritidis

Hektoen - Salmonella enteritidis
Note: Clear colonies indicates a non-lactose fermentor & black precipitate in center of colonies is due to H2S production.

Mannitol Salt Agar

Mannitol Salt Agar - uninoculated

Type: Selective and differential

Purpose: Selects for Staphylococci, which grow at high salt concentrations; differentiates Staphylococcus aureus from other Staphylococci

Interpretation:Staphylococcus aureus is yellow (ferments mannitol), other staphylococci are white

S. epidermidis

Mannitol Salt Agar - Staphylococcus epidermidis
Note: growth, but no fermentation of mannitol, medium color unchanged

S. aureus

Mannitol Salt Agar - Staphylococcus aureus
Note: yellow color due to acid produced by fermentation of mannitol

E coli

Mannitol Salt Agar - Escherichia coli
Note: Streptococci, Enterics, and other organisms do not grow on Mannitol Salt Agar

Triple Sugar Iron Agar (TSI)

Triple Sugar Iron Agar - uninoculated

Type: Multi-purpose, differential

Purpose: Detects glucose, lactose, sucrose fermentation; gas and H2S production. (E. coli → A/AG; Salmonella → K/A+G; Shigella → K/A; Ps. aeruginosa → K/K)

Interpretation: Yellow butt, red slant (K/A) = ferments glucose only; yellow butt and slant (A/A) = ferments glucose + lactose and/or sucrose; red but and slant (K/K) = non-fermenter of all 3 sugars; black (+) = H2S; bubbles (G) = gas production

E. coli

Triple Sugar Iron Agar - Escherichia coli

Salmonella

Triple Sugar Iron Agar - Salmonella

Shigella

Triple Sugar Iron Agar - Shigella

P. aeruginosa

Triple Sugar Iron Agar - Pseudomonas aeruginosa

Hemolytic Reactions Observed on Blood Agar

Observation of the hemolytic reactions on sheep blood agar is a very useful tool in the preliminary identification of bacteria, particularly streptococci. The types of hemolysis are defined as follows:

alpha (α) hemolysis: An indistinct zone of partial destruction of red blood cells (RBCs) appears around the colony, often accompanied by a greenish to brownish discoloration of the medium. Streptococcus pneumoniae and many oral streptococci are α hemolytic.

beta (β) hemolysis: A clear, colorless zone appears around the colonies, in which the RBCs have undergone complete lysis. Streptococcus pyogenes, S. agalactiae, and several other species of streptococci are β hemolytic. Many other bacteria besides streptococci can be β hemolytic, including Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, etc., and hemolytic reactions can also be a useful diagnostic tool for these organisms.

no (γ) hemolysis: No apparent hemolytic activity or discoloration is produced (also called gamma hemolysis).

Sheep Blood Agar

Sheep Blood Agar - uninoculated

Type: Differential and enriched

Purpose: Determine types of hemolysis (i.e., α, β, γ)

Interpretation: α: partial clearing, green or brownish ring; β: wide zone of clearing; γ: non-hemolytic

Alpha hemolysis

Sheep Blood Agar - Streptococcus pneumoniae, alpha hemolytic

Beta hemolysis

Sheep Blood Agar - Streptococcus pyogenes, beta hemolytic

Gamma hemolysis

Sheep Blood Agar - E. coli, gamma (non) hemolytic

No growth

Sheep Blood Agar - Neisseria gonorrhoeae
Note: Neisseria are fastidious and do not grow on Sheep Blood Agar.

Credits:

Faculty: Cindy Arvidson
Culture preparation: Poorna Viswanathan
Producer: Jiatyan Chen
Scripting: Jiatyan Chen, Matt Guibord
Photography: Deon Foster, Jiatyan Chen
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