emb agar e coli results

Classification: Oldest method based on their action on lactose. Groups. Lactose fermentation. Colonies on MacConkey agar. Examples. Lactose fermenters (LF)-. Pink, mucoid colonies. sucrose in the EMB agar and the ability of certain bacteria to ferment A smaller amount of acid production, which is a result of slow.

Emb agar e coli results -

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

Tryptic Soy Agar - uninoculated

Type: General

Purpose: Cultivation of non-fastidious bacteria

Interpretation: Growth indicates non-fastidious bacteria present

S. aureus

S. aureus

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

E. coli

E. coli

Tryptic Soy Agar - Escherichia coli

P. aeruginosa

trypticsoy_ps.aeruginosa

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

Chocolate Agar

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

S. aureus

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

N. gonorrhoeae

N. gonorrhoeae

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

E. coli

E. coli

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

Thayer-Martin Agar

Chocolate 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

no growth

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

N. gonorrhoeae

N. gonorrhoeae

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

E. coli

no growth

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

MacConkey (lactose) Agar

MacConkey 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

E. coli

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

N. gonorrhoeae

N. gonorrhroeae

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

S. enteritidis

Salmonella

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

N. gonorrhroeae

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

Eosin-methylene Blue Agar (EMB)

Eosin Methylene Blue

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

Salmonella

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

E. coli

E. coli

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

K. pneumoniae

E. coli

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

Hektoen Agar

Hektoen

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

E. Coli

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

S. enteritidis

Salmonella

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

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

S. epidermidis

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

S. aureus

S. aureus

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

E coli

Mannitol Salt Agar

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

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

E. coli

Triple Sugar Iron Agar - Escherichia coli

Salmonella

Salmonella

Triple Sugar Iron Agar - Salmonella

Shigella

Shigella

Triple Sugar Iron Agar - Shigella

P. aeruginosa

Ps. 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

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

Alpha haemolysis

Sheep Blood Agar - Streptococcus pneumoniae, alpha hemolytic

Beta hemolysis

Beta haemolysis

Sheep Blood Agar - Streptococcus pyogenes, beta hemolytic

Gamma hemolysis

Gamma haemolysis

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

No growth

N. gonorrhoeae

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

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Faculty: Cindy Arvidson
Culture preparation: Poorna Viswanathan
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Scripting: Jiatyan Chen, Matt Guibord
Photography: Deon Foster, Jiatyan Chen
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Источник: https://learn.chm.msu.edu/vibl/content/differential/

EMB Plate Results:


Staphylococcus epidermidis (no growth), E. coli (dark purple), Pseudomonas aeruginosa (bottom), Enterobacter aerogenes (left)


Enterobacter aerogenes


E. coli


P. aeruginosa


E. coli - note metallic sheen = lactose fermentor


E. coli - note metallic sheen = lactose fermentor


P. aeruginosa (lactose nonfermentor)

Источник: https://www.uwyo.edu/virtual_edge/results/emb_results.htm

American Society for Microbiology

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PowerPoint Contents

Figure 1: Escherichia coli on EMB (Enlarged view). FIG. 1. Eosin-methylene blue (EMB) agar plate inoculated with Escherichia coli (a gram-negative coliform bacterium) showing good growth of dark blue-black colonies with metallic green sheen indicating vigorous fermentation of lactose and acid production which precipitates the green metallic pigment. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 2: Enterobacter aerogenes on EMB (Enlarged view). FIG. 2. EMB agar plate inoculated with Enterobacter aerogenes (a gram-negative coliform bacterium) showing good growth of brown, dark-centered, mucoid colonies indicating lactose fermentation and acid production. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 3: Klebsiella pneumoniae on EMB (Enlarged view). FIG. 3. EMB agar plate inoculated with Klebsiella pneumoniae (a gram-negative coliform bacterium) showing good growth of brown, dark-centered, mucoid colonies (smaller than Enterobacter) indicating lactose fermentation and acid production. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 4: Pseudomonas aeruginosa on EMB (Enlarged view). FIG. 4. EMB agar plate inoculated with Pseudomonas aeruginosa (a gram-negative noncoliform bacterium) showing good growth but no fermentation of sugars or acid production. (Naowarat Cheeptham and Carolynne Fardy, Thompson Rivers University, Kamloops, BC, Canada)

Figure 5: Proteus vulgaris on EMB (Enlarged view). FIG. 5. EMB agar plate inoculated with Proteus vulgaris (a gram-negative coliform bacterium) showing growth of pink colonies indicating non-lactose fermentation and some acid production. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 6: Escherichia coli and Pseudomonas aeruginosa on EMB (Enlarged view) FIG. 6. EMB agar plate inoculated with a mixed culture of Escherichia coli and Pseudomonas aeruginosa. Note the metallic green sheen of the strong lactose-fermenting Escherichia coli and the pinkish colonies of nonfermenter Pseudomonas aeruginosa. (Archana Lal, Independence Community College, Independence, KS)

Figure 7: Acinetobacter baumannii on EMB (Enlarged view). FIG. 7. EMB agar plate inoculated with Acinetobacter baumannii (a gram-negative non-glucose-fermenting bacillus) showing a colony with a classic blue-grey center. This should not be mistaken for evidence of lactose fermentation on EMB agar. (Bobbi Pritt, Mayo Clinic, Rochester, MN)

Figure 8: Stenotrophomonas maltophilia on EMB (Enlarged view). FIG. 8. EMB agar plate inoculated with Stenotrophomonas maltophilia (a gram-negative non-glucose-fermenting bacillus) showing good growth and non-lactose-fermenting morphology. (Bobbi Pritt, Mayo Clinic, Rochester, MN)

Figure 9: Escherichia coli and Salmonella enteritidis on EMB (Enlarged view). FIG. 9. EMB agar plate inoculated with mixed enteric flora. The lactose fermenter Escherichia coli grew with purple-centered colonies while the lactose nonfermenter Salmonella enteritidis grew as colorless colonies. Salmonella enteritidis mixed with Escherichia coli is able to utilize the acid products as energy source, resulting in an insufficient acid buildup to precipitate out the Eosin Methylene Blue and no green metallic sheen is produced by Escherichia coli. (Jerry Keplinger, East Tennessee State University, Johnson City, TN)

Figure 10: Clostridium perfringens on EMB (Enlarged view). FIG. 10. EMB agar plate inoculated with Clostridium perfringens (a gram-positive bacterium) maintained under anaerobic conditions and showing no growth. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 11: Bacillus subtilis on EMB (Enlarged view). FIG. 11. EMB agar plate inoculated with Bacillus subtilis (a gram-positive bacterium) showing poor growth. (Naowarat Cheeptham,Thompson Rivers University, Kamloops, BC, Canada)

Figure 12: Micrococcus luteus on EMB (Enlarged view). FIG. 12. EMB agar plate inoculated with Micrococcus luteus (a gram-positive bacterium) showing no growth. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 13: Staphylococcus aureus on EMB (Enlarged view). FIG. 13. EMB agar plate inoculated with Staphylococcus aureus (a gram-positive bacterium) showing no growth. (Archana Lal, Independence Community College, Independence, KS)

Figure 14: Streptococcus group B on EMB (Enlarged view). FIG. 14. EMB agar plate inoculated with Streptococcus group B (a gram-positive bacterium) showing no growth. (Archana Lal, Independence Community College, Independence, KS)

Figure 15: Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus on EMB (Enlarged view). FIG. 15. EMB agar plate inoculated with (A) Escherichia coli, (B) Bacillus subtilis, (C) Pseudomonas aeruginosa, and (D) Staphylococcus aureus. The two gram-positive bacteria Bacillus subtilis and Staphylococcus aureus did not grow, while the two gram-negative bacteria Escherischia coli and Pseudomonas aeruginosa grew with typical lactose fermenter (colonies with green metallic sheen) and nonfermenter (pink colonies) morphology. (Archana Lal, Independence Community College, Independence, KS)

Figure 16: Salmonella enteritidis on EMB(Enlarged view). FIG. 16. EMB agar plate inoculated with Salmonella enteritidis (a gram-negative coliform bacterium) showing good growth of grey mucoid colonies with no fermentation of lactose or acid production. (Naowarat Cheeptham and Carolynne Fardy, Thompson Rivers University, Kamloops, BC, Canada)

Figure 17: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes on EMB (Enlarged view). FIG. 17. EMB agar plate inoculated with (A) Escherichia coli, (B) Pseudomonas aeruginosa, (C) Klebsiella pneumoinae, and (D) Enterobacter aerogenes. All four gram negative bacteria grew exhibiting different morphology. Escherischia coli grew with typical lactose fermenter morphology with excessive acid production and precipitation of green metallic pigment (colonies with green metallic sheen). Pseudomonas aeruginosa grew exhibiting the nonfermenter morphology (pinkish colonies), both Klebsiella pneumoniae and Enterobacter aerogenes grew with lactose fermentation and acid production morphology (with purple dark centered mucoid colonies). (Naowarat Cheeptham and Carolynne Fardy, Thompson Rivers University, Kamloops, BC, Canada)

Figure 18: Lactose Fermentation FIG. 18. Eosin-methylene blue agar plate inoculated with Escherichia coli. This plate shows a metallic green sheen on the colonies, which indicates strong acid production from lactose and/or sucrose fermentation. (Jesus Antonio Romo, University of Texas at San Antonio, San Antonio, TX)

Источник: https://asm.org/Image-Gallery/Eosin-Methylene-Blue

Eosin methylene blue

Eosin methylene blue (EMB, also known as "Levine's formulation") is a selective stain for Gram-negative bacteria.[1] EMB contains dyes that are toxic to Gram-positive bacteria. EMB is the selective and differential medium for coliforms. It is a blend of two stains, eosin and methylene blue in the ratio of 6:1. EMB is a differential microbiological medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella).[2] Organisms that ferment lactose display "nucleated colonies"—colonies with dark centers.[3]

This medium is important in medical laboratories by distinguishing pathogenic microbes in a short period of time.[4]

  • Rapid lactose fermentation produces acids, which lower the pH. This encourages dye absorption by the colonies, which are now colored purple-black.
  • Lactose non-fermenters may increase the pH by deamination of proteins. This ensures that the dye is not absorbed. The colonies will be colorless.

On EMB if E. coli is grown it will give a distinctive metallic green sheen (due to the metachromatic properties of the dyes, E. coli movement using flagella, and strong acid end-products of fermentation). Some species of Citrobacter and Enterobacter will also react this way to EMB.[5] This medium has been specifically designed to discourage the growth of Gram-positive bacteria.[6]

EMB contains the following ingredients: peptone, lactose, dipotassium phosphate, eosin Y (dye), methylene blue (dye), and agar.

There are also EMB agars that do not contain lactose.

References[edit]

External links[edit]

Источник: https://en.wikipedia.org/wiki/Eosin_methylene_blue

Eosin Methylene Blue Agar


Eosin Methylene Blue (EMB) agar is both selective and differential.  It contains the dyes eosin and methylene blue, which inhibit the growth of gram-positive bacteria and therefore select for gram-negative bacteria.  It also contains the carbohydrate lactose, which allows differentiation of gram-negative bacteria based on their ability to ferment lactose.

Quadrant 1:  Growth on the plate indicates the organism, Escherichia coli, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The green metallic sheen indicates E. coli is able to ferment lactose to produce strong acid end-products.

Quadrant 2:  Growth on the plate indicates the organism, Pseudomonas aeruginosa, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The absence of color in the bacterial growth indicates P. aeruginosa is unable to ferment lactose.

Quadrant 3: Growth on the plate indicates the organism, Enterobacter aerogenes, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The pink color of the bacterial growth indicates E. aerogenes is able to ferment lactose to produce weak acid end-products.

Quadrant 4:  Absence of growth indicates the organism, Staphylococcus aureus, is inhibited by eosin and methylene blue and is a gram-positive bacterium.

 

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Источник: http://faculty.collin.edu/dcain/CCCCD%20Micro/embagar.htm

Emb agar e coli results -

Learning Objectives

  • Define the term culture medium
  • Give examples of the following types of media:: complex, chemically defined, selective, and differential

The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive under laboratory conditions. Culturing many microorganisms is challenging because of highly specific nutritional and environmental requirements and the diversity of these requirements among different species.

Nutritional Requirements

Culture medium is defined as a medium of nutrients that supports microbial growth. The number of available media to grow bacteria is considerable.  When the complete chemical composition of a medium is known, it is called a chemically defined medium.  In contrast for a complex medium, the precise chemical composition is not known because they contain extracts and digests of yeasts, meat, or plants, .  Nutrient broth, tryptic soy broth, and brain heart infusion, are all examples of complex media.

Differential and selective media are commonly used in microbiology laboratories. A differential medium support the growth of any microbe but distinguishes them based on how they metabolize or change the medium.  One example of differential medium is blood agar.  Blood agar distinguishes microbes based on their ability to lyse red blood cells (RBCs), a property known as hemolysis.  There are three types of hemolysis (Figure 1):

  • Beta hemolysis is complete breakdown of RBCs.  A clear area develops around the colonies.
  • Alpha hemolysis is partial breakdown of RBCs.  A green grey color develops around the colonies on blood agar.
  • Gamma hemolysis is no hemolyis; the microbe does not change the appearance of the blood agar.
hemolysis on blood agar

Figure 1. The three types of hemolysis on a blood agar plate

Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest  are called selective media. Selective medium contain particular ingredients that inhibit the growth of certain microbes.  An example of a selective medium is MacConkey agar. It contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria.  MacConkey agar is also a differential medium.   The lactose fermenters produce acid, which turns the medium and the colonies of strong fermenters hot pink. The medium is supplemented with the pH indicator neutral red, which turns to hot pink at low pH. Selective and differential media can be combined and play an important role in the identification of bacteria by biochemical methods.

A light brown agar plate. Two streaks on the plate are bright pink and two streaks are beige.

Figure 2. On this MacConkey agar plate, the lactose-fermenter E. coli colonies are bright pink. Serratia marcescens, which does not ferment lactose, forms a cream-colored streak on the tan medium. (credit: American Society for Microbiology)

Another commonly used medium that is both selective and differential is eosin-methylene blue (EMB) agar.  EMB contains the dyes eosin and methylene blue that inhibit the growth of gram-positve 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.  When bacterial cells ferment lactose, acid is produced that precipitates the dyes in the medium and the colonies develop a green metallic sheen (Figure 3).

Selective and Differential properties of EMB with E.coli

Figure 3 E.coli growing on eosin-methylene-blue agar. The gram-negative bacterium grows and ferments lactose, giving the colonies a green metallic sheen

Think about It

  • Distinguish complex and chemically defined media.
  • Distinguish selective and enrichment media.

Compare the compositions of EZ medium and sheep blood agar.

The End-of-Year Picnic

The microbiology department is celebrating the end of the school year in May by holding its traditional picnic on the green. The speeches drag on for a couple of hours, but finally all the faculty and students can dig into the food: chicken salad, tomatoes, onions, salad, and custard pie. By evening, the whole department, except for two vegetarian students who did not eat the chicken salad, is stricken with nausea, vomiting, retching, and abdominal cramping. Several individuals complain of diarrhea. One patient shows signs of shock (low blood pressure). Blood and stool samples are collected from patients, and an analysis of all foods served at the meal is conducted.

Bacteria can cause gastroenteritis (inflammation of the stomach and intestinal tract) either by colonizing and replicating in the host, which is considered an infection, or by secreting toxins, which is considered intoxication. Signs and symptoms of infections are typically delayed, whereas intoxication manifests within hours, as happened after the picnic.

Blood samples from the patients showed no signs of bacterial infection, which further suggests that this was a case of intoxication. Since intoxication is due to secreted toxins, bacteria are not usually detected in blood or stool samples. MacConkey agar and sorbitol-MacConkey agar plates and xylose-lysine-deoxycholate (XLD) plates were inoculated with stool samples and did not reveal any unusually colored colonies, and no black colonies or white colonies were observed on XLD. All lactose fermenters on MacConkey agar also ferment sorbitol. These results ruled out common agents of food-borne illnesses: E. coli, Salmonella spp., and Shigella spp.

A micrograph of clusters of purple spheres.

Figure 2. Gram-positive cocci in clusters. (credit: Centers for Disease Control and Prevention)

Analysis of the chicken salad revealed an abnormal number of gram-positive cocci arranged in clusters (Figure 2). A culture of the gram-positive cocci releases bubbles when mixed with hydrogen peroxide. The culture turned mannitol salt agar yellow after a 24-hour incubation.

All the tests point to Staphylococcus aureus as the organism that secreted the toxin. Samples from the salad showed the presence of gram-positive cocci bacteria in clusters. The colonies were positive for catalase. The bacteria grew on mannitol salt agar fermenting mannitol, as shown by the change to yellow of the medium. The pH indicator in mannitol salt agar is phenol red, which turns to yellow when the medium is acidified by the products of fermentation.

The toxin secreted by S. aureus is known to cause severe gastroenteritis. The organism was probably introduced into the salad during preparation by the food handler and multiplied while the salad was kept in the warm ambient temperature during the speeches.

  • What are some other factors that might have contributed to rapid growth of S. aureus in the chicken salad?
  • Why would S. aureus not be inhibited by the presence of salt in the chicken salad?

Key Concepts and Summary

  • Chemically defined media have a a known quantities of each chemical component
  • Selective media favor the growth of some microorganisms while inhibiting others.
  • Differential media help distinguish bacteria by the color of the colonies or the change in the medium.

Fill in the Blank

Blood agar contains many unspecified nutrients, supports the growth of a large number of bacteria, and allows differentiation of bacteria according to hemolysis (breakdown of blood). The medium is ________ and ________.

Show Answer

Blood agar contains many unspecified nutrients, supports the growth of a large number of bacteria, and allows differentiation of bacteria according to hemolysis (breakdown of blood). The medium is complex and differential.

Rogosa agar contains yeast extract. The pH is adjusted to 5.2 and discourages the growth of many microorganisms; however, all the colonies look similar. The medium is ________ and ________.

Show Answer

Rogosa agar contains yeast extract. The pH is adjusted to 5.2 and discourages the growth of many microorganisms; however, all the colonies look similar. The medium is complex and selective.

Источник: https://courses.lumenlearning.com/cuny-kbcc-microbiologyhd/chapter/media-used-for-bacterial-growth/

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

Tryptic Soy Agar - uninoculated

Type: General

Purpose: Cultivation of non-fastidious bacteria

Interpretation: Growth indicates non-fastidious bacteria present

S. aureus

S. aureus

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

E. coli

E. coli

Tryptic Soy Agar - Escherichia coli

P. aeruginosa

trypticsoy_ps.aeruginosa

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

Chocolate Agar

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

S. aureus

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

N. gonorrhoeae

N. gonorrhoeae

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

E. coli

E. coli

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

Thayer-Martin Agar

Chocolate 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

no growth

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

N. gonorrhoeae

N. gonorrhoeae

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

E. coli

no growth

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

MacConkey (lactose) Agar

MacConkey 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

E. coli

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

N. gonorrhoeae

N. gonorrhroeae

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

S. enteritidis

Salmonella

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

N. gonorrhroeae

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

Eosin-methylene Blue Agar (EMB)

Eosin Methylene Blue

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

Salmonella

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

E. coli

E. coli

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

K. pneumoniae

E. coli

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

Hektoen Agar

Hektoen

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

E. Coli

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

S. enteritidis

Salmonella

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

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

S. epidermidis

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

S. aureus

S. aureus

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

E coli

Mannitol Salt Agar

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

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

E. coli

Triple Sugar Iron Agar - Escherichia coli

Salmonella

Salmonella

Triple Sugar Iron Agar - Salmonella

Shigella

Shigella

Triple Sugar Iron Agar - Shigella

P. aeruginosa

Ps. 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

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

Alpha haemolysis

Sheep Blood Agar - Streptococcus pneumoniae, alpha hemolytic

Beta hemolysis

Beta haemolysis

Sheep Blood Agar - Streptococcus pyogenes, beta hemolytic

Gamma hemolysis

Gamma haemolysis

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

No growth

N. gonorrhoeae

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

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Faculty: Cindy Arvidson
Culture preparation: Poorna Viswanathan
Producer: Jiatyan Chen
Scripting: Jiatyan Chen, Matt Guibord
Photography: Deon Foster, Jiatyan Chen
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Источник: https://learn.chm.msu.edu/vibl/content/differential/

MacConkey agar (MAC) was the first solid differential media to be formulated which was developed at 20th century by Alfred Theodore MacConkey. MacConkey agar is a selective and differential media used for the isolation and differentiation of non-fastidious gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas.

Composition of MacConkey Agar

IngredientsAmount
Peptone (Pancreatic digest of gelatin)  17 gm
Proteose peptone (meat and casein) 3 gm
Lactose monohydrate  10 gm
Bile salts 1.5 gm
Sodium chloride 5 gm
Neutral red 0.03 gm
Crystal Violet 0.001 g
Agar 13.5 gm
Distilled WaterAdd to make 1 Liter

Final pH 7.1 +/- 0.2 at 25 degrees C.

Principle of MacConkey Agar

MacConkey agar is used for the isolation of gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria. Pancreatic digest of gelatin and peptones (meat and casein) provide the essential nutrients, vitamins and nitrogenous factors required for growth of microorganisms. Lactose monohydrate is the fermentable source of carbohydrate. The selective action of this medium is attributed to crystal violet and bile salts, which are inhibitory to most species of gram-positive bacteria. Sodium chloride maintains the osmotic balance in the medium. Neutral red is a pH indicator that turns red at a pH below 6.8 and is colorless at any pH greater than 6.8. Agar is the solidifying agent.

Uses of MacConkey Agar

  1. MacConkey agar is used for the isolation of gram-negative enteric bacteria.
  2. It is used in the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria.
  3. It is used for the isolation of coliforms and intestinal pathogens in water, dairy products and biological specimens.

Preparation of MacConkey Agar

  1. Suspend 49.53 grams of dehydrated medium in 1000 ml purified/distilled water.
  2. Heat to boiling to dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. 
  4. Cool to 45-50°C.
  5. Mix well before pouring into sterile Petri plates.

Result Interpretation on MacConkey Agar

Lactose fermenting strains grow as red or pink and may be surrounded by a zone of acid precipitated bile. The red colour is due to production of acid from lactose, absorption of neutral red and a subsequent colour change of the dye when the pH of medium falls below 6.8.

Lactose non-fermenting strains, such as Shigella and Salmonella are colourless and transparent and typically do not alter appearance of the medium. Yersinia enterocolitica may appear as small, non-lactose fermenting colonies after incubation at room temperature.

Colony Morphology on MacConkey Agar

Colony Morphology on MacConkey Agar

Organism

Colour

Remarks

Escherichia coli

red/pink

non-mucoid

Aerobacter aerogenes

pink

mucoid

Enterococcus species

red

minute, round

Staphylococcus species

pale pink

opaque

Pseudomonas aeruginosa

green-brown

fluorescent growth

Limitations of MacConkey Agar

  1. The colonial characteristics described give presumptive identification only of the isolated organisms. It is necessary to subculture and carry out confirmation tests for final identification.
  2. Some strains may be encountered that grow poorly or fail to grow on this medium.
  3. Incubation of MacConkey Agar plates under increased CO2 has been reported to reduce growth and recovery of a number of strains of Gram-negative bacilli.
  4. Some strains of Proteus may swarm on this medium.

References

  1. Austin Community College, 5930 Middle Fiskville Rd., Austin, Texas
  2. ASM Microbe Library: MacConkey Agar Plates Protocols
  3. Thermo Fisher Scientific Inc., Dehydrated Culture Media: MacConkey Agar
  4. Acumedia Manufacturers: MacConkey Agar
  5. HiMedia Laboratories Pvt. Ltd, Technical data: MacConkey Agar
  6. Hardy Diagnostics: MacConkey Agar
  7. Science Prof Online (SPO): MacConkey Agar
  8. Bacteriological Analytical Manual, 8th Edition, Revision A, 1998.
  9. Collin County Community College District.
  10. Microbe Online
  11. Wikipedia
Categories Culture MediaИсточник: https://microbiologyinfo.com/macconkey-agar-composition-principle-uses-preparation-and-colony-morphology/

EOSIN METHYLENE BLUE AGAR (MODIFIED) LEVINE

Code: CM0069

An isolation medium for the differentiation of the Enterobacteriaceae.

Typical Formula*

gm/litre

Peptone

10.0

Lactose

10.0

Dipotassium hydrogen phosphate

2.0

Eosin Y

0.4

Methylene blue

0.065

Agar

15.0

pH 6.8 ± 0.2

 
* Adjusted as required to meet performance standards

Directions
Suspend 37.5g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 60°C and shake the medium in order to oxidise the methylene blue (i.e. restore its blue colour) and to suspend the precipitate which is an essential part of the medium.

Description
This versatile medium, modified by Levine1,2, is used for the differentiation of Escherichia coli and Enterobacteria aerogenes, for the rapid identification of Candida albicans, and for the identification of coagulase-positive staphylococci.

The medium is prepared to the formula specified by the APHA3,4,5,6 for the detection and differentiation of the coliform group of organisms7,8.
Weld 9,10 proposed the use of Levine eosin methylene blue agar, with added chlortetracycline hydrochloride for the rapid identification of Candida albicans in clinical materials. A positive identification of Candida albicans could be made after 24 to 48 hours incubation at 37°C in 10% carbon dioxide from faeces, oral and vaginal secretions, and nail or skin scrapings. Vogel and Moses 11 confirmed the reliability of Weld’s method for the relatively rapid identification of Candida albicans in sputum. They found that use of eosin methylene blue agar was just as reliable as more conventional methods for the identification of this organism in sputum. In addition, the medium provided a means for the identification of several Gram- negative genera. Doupagne12 also investigated the use of the Levine medium under tropical conditions.

Haley and Stonerod 13 found that Weld’s method was variable so that Walker and Huppert 14 advocated the use of corn meal agar and a rapid fermentation test in addition to the Levine medium. Using the combined rapid technique they were able to obtain results within 48 to 72 hours.
Subsequent to the findings of Vogel and Moses 11, Menolasino et al.15 used Levine eosin methylene blue agar for the identification of coagulase-positive staphylococci which grew as characteristic colourless, pin-point colonies. The Levine medium was more efficient than tellurite glycine agar and showed good correlation with the plasma coagulase test.

Colonial Characteristics
Escherichia coli- isolated colonies, 2-3mm diameter, with little tendency to confluent growth, exhibiting a greenish metallic sheen by reflected light and dark purple centres by transmitted light.
Enterobacter aerogenes - 4-6mm diameter, raised and mucoid colonies, tending to become confluent, metallic sheen usually absent, grey-brown centres by transmitted light.
Non-lactose fermenting intestinal pathogens - translucent and colourless
Candida albicans - after 24 to 48 hours at 35°C in 10% carbon dioxide `spidery’ or `feathery’ colonies. Other Candida species produce smooth yeast-like colonies. Since a typical appearance is variable it is advisable to use a combined method such as that of Walker and Huppert 14.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates at 2-8°C away from light.

Appearance
Dehydrated medium: purple coloured, free-flowing powder
Prepared medium: dark purple gel

Quality control

Positive controls:

Expected results

Escherichia coli ATCC® 25922 *

Good growth; purple coloured colonies with green metallic sheen.

Enterobacter aerogenes ATCC® 13048 *

Good growth; purple mucoid colonies.

Negative control:

 

Uninoculated medium.

No change

* This organism is available as a Culti-Loop®

Precautions
Further tests are required to confirm the presumptive identity of organisms isolated on this medium. Some strains of Salmonella and Shigella species will not grow in the presence of eosin and methylene blue. Store the medium away from light to prevent photo-oxidation.

References
1. Levine M. (1918) J. Infect. Dis. 23. 43-47.
2. Levine M. (1921) `Bacteria Fermenting Lactose and the Significance in Water Analysis’ Bull. 62. Iowa State College Engr. Exp. Station.
3. American Public Health Association (1980) Standard Methods for the Examination of Water and Wastewater. 15th Edn. APHA Inc. Washington DC.
4. American Public Health Association (1978) Standard Methods for the Examination of Dairy Products. 14th Edn. APHA Inc. Washington DC.
5. American Public Health Association (1992) Compendium of Methods for the Microbiological Examination of Foods 3rd Edn. APHA Inc. Washington DC.
6. American Public Health Association (1970) `Diagnostic Procedures’. 5th Edn. APHA Inc. Washington DC.
7. American Society for Microbiology (1974) Manual of Clinical Microbiology 2nd Edn. ASM Washington DC.
8. Windle Taylor E. (1958) `The Examination of Waters and Water Supplies’ 7th Ed., Churchill Ltd., London.
9. Weld Julia T. (1952) Arch. Dermat. Syph. 66. 691-694.
10. Weld Julia T. (1953) Arch. Dermat. Syph. 67(5). 473-478.
11. Vogel R. A. and Moses Mary R. (1957) Am. J. Clin. Path. 28. 103-106.
12. Doupagne P. (1960) Ann. Soc. Belge de Med. Trop. 40(6). 893-897.
13. Haley L. D. and Stonerod M. H. (1955) Am. J. Med. Tech. 21. 304-308.
14. Walker Leila and Huppert M. (1959) Am. J. Clin. Path. 31. 551-558.
15. Menolasino N. J., Grieves Barbara, Payne Pearl (1960) J. Lab. Clin. Med. 56. 908-910.

Источник: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0069&c=UK&lang=EN

American Society for Microbiology

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PowerPoint Contents

Figure 1: Escherichia coli on EMB (Enlarged view). FIG. 1. Eosin-methylene blue (EMB) agar plate inoculated with Escherichia coli (a gram-negative coliform bacterium) showing good growth of dark blue-black colonies with metallic green sheen indicating vigorous fermentation of lactose and acid production which precipitates the green metallic pigment. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 2: Enterobacter aerogenes on EMB (Enlarged view). FIG. 2. EMB agar plate inoculated with Enterobacter aerogenes (a gram-negative coliform bacterium) showing good growth of brown, dark-centered, mucoid colonies indicating lactose fermentation and acid production. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 3: Klebsiella pneumoniae on EMB (Enlarged view). FIG. 3. EMB agar plate inoculated with Klebsiella pneumoniae (a gram-negative coliform bacterium) showing good growth of brown, dark-centered, mucoid colonies (smaller than Enterobacter) indicating lactose fermentation and acid production. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 4: Pseudomonas aeruginosa on EMB (Enlarged view). FIG. 4. EMB agar plate inoculated with Pseudomonas aeruginosa (a gram-negative noncoliform bacterium) showing good growth but no fermentation of sugars or acid production. (Naowarat Cheeptham and Carolynne Fardy, Thompson Rivers University, Kamloops, BC, Canada)

Figure 5: Proteus vulgaris on EMB (Enlarged view). FIG. 5. EMB agar plate inoculated with Proteus vulgaris (a gram-negative coliform bacterium) showing growth of pink colonies indicating non-lactose fermentation and some acid production. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 6: Escherichia coli and Pseudomonas aeruginosa on EMB (Enlarged view) FIG. 6. EMB agar plate inoculated with a mixed culture of Escherichia coli and Pseudomonas aeruginosa. Note the metallic green sheen of the strong lactose-fermenting Escherichia coli and the pinkish colonies of nonfermenter Pseudomonas aeruginosa. (Archana Lal, Independence Community College, Independence, KS)

Figure 7: Acinetobacter baumannii on EMB (Enlarged view). FIG. 7. EMB agar plate inoculated with Acinetobacter baumannii (a gram-negative non-glucose-fermenting bacillus) showing a colony with a classic blue-grey center. This should not be mistaken for evidence of lactose fermentation on EMB agar. (Bobbi Pritt, Mayo Clinic, Rochester, MN)

Figure 8: Stenotrophomonas maltophilia on EMB (Enlarged view). FIG. 8. EMB agar plate inoculated with Stenotrophomonas maltophilia (a gram-negative non-glucose-fermenting bacillus) showing good growth and non-lactose-fermenting morphology. (Bobbi Pritt, Mayo Clinic, Rochester, MN)

Figure 9: Escherichia coli and Salmonella enteritidis on EMB (Enlarged view). FIG. 9. EMB agar plate inoculated with mixed enteric flora. The lactose fermenter Escherichia coli grew with purple-centered colonies while the lactose nonfermenter Salmonella enteritidis grew as colorless colonies. Salmonella enteritidis mixed with Escherichia coli is able to utilize the acid products as energy source, resulting in an insufficient acid buildup to precipitate out the Eosin Methylene Blue and no green metallic sheen is produced by Escherichia coli. (Jerry Keplinger, East Tennessee State University, Johnson City, TN)

Figure 10: Clostridium perfringens on EMB (Enlarged view). FIG. 10. EMB agar plate inoculated with Clostridium perfringens (a gram-positive bacterium) maintained under anaerobic conditions and showing no growth. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 11: Bacillus subtilis on EMB (Enlarged view). FIG. 11. EMB agar plate inoculated with Bacillus subtilis (a gram-positive bacterium) showing poor growth. (Naowarat Cheeptham,Thompson Rivers University, Kamloops, BC, Canada)

Figure 12: Micrococcus luteus on EMB (Enlarged view). FIG. 12. EMB agar plate inoculated with Micrococcus luteus (a gram-positive bacterium) showing no growth. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Figure 13: Staphylococcus aureus on EMB (Enlarged view). FIG. 13. EMB agar plate inoculated with Staphylococcus aureus (a gram-positive bacterium) showing no growth. (Archana Lal, Independence Community College, Independence, KS)

Figure 14: Streptococcus group B on EMB (Enlarged view). FIG. 14. EMB agar plate inoculated with Streptococcus group B (a gram-positive bacterium) showing no growth. (Archana Lal, Independence Community College, Independence, KS)

Figure 15: Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus on EMB (Enlarged view). FIG. 15. EMB agar plate inoculated with (A) Escherichia coli, (B) Bacillus subtilis, (C) Pseudomonas aeruginosa, and (D) Staphylococcus aureus. The two gram-positive bacteria Bacillus subtilis and Staphylococcus aureus did not grow, while the two gram-negative bacteria Escherischia coli and Pseudomonas aeruginosa grew with typical lactose fermenter (colonies with green metallic sheen) and nonfermenter (pink colonies) morphology. (Archana Lal, Independence Community College, Independence, KS)

Figure 16: Salmonella enteritidis on EMB(Enlarged view). FIG. 16. EMB agar plate inoculated with Salmonella enteritidis (a gram-negative coliform bacterium) showing good growth of grey mucoid colonies with no fermentation of lactose or acid production. (Naowarat Cheeptham and Carolynne Fardy, Thompson Rivers University, Kamloops, BC, Canada)

Figure 17: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes on EMB (Enlarged view). FIG. 17. EMB agar plate inoculated with (A) Escherichia coli, (B) Pseudomonas aeruginosa, (C) Klebsiella pneumoinae, and (D) Enterobacter aerogenes. All four gram negative bacteria grew exhibiting different morphology. Escherischia coli grew with typical lactose fermenter morphology with excessive acid production and precipitation of green metallic pigment (colonies with green metallic sheen). Pseudomonas aeruginosa grew exhibiting the nonfermenter morphology (pinkish colonies), both Klebsiella pneumoniae and Enterobacter aerogenes grew with lactose fermentation and acid production morphology (with purple dark centered mucoid colonies). (Naowarat Cheeptham and Carolynne Fardy, Thompson Rivers University, Kamloops, BC, Canada)

Figure 18: Lactose Fermentation FIG. 18. Eosin-methylene blue agar plate inoculated with Escherichia coli. This plate shows a metallic green sheen on the colonies, which indicates strong acid production from lactose and/or sucrose fermentation. (Jesus Antonio Romo, University of Texas at San Antonio, San Antonio, TX)

Источник: https://asm.org/Image-Gallery/Eosin-Methylene-Blue

Eosin Methylene Blue Agar


Eosin Methylene Blue (EMB) agar is both selective and differential.  It contains the dyes eosin and methylene blue, which inhibit the growth of gram-positive bacteria and therefore select for gram-negative bacteria.  It also contains the carbohydrate lactose, which allows differentiation of gram-negative bacteria based on their ability to ferment lactose.

Quadrant 1:  Growth on the plate indicates the organism, Escherichia coli, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The green metallic sheen indicates E. coli is able to ferment lactose to produce strong acid end-products.

Quadrant 2:  Growth on the plate indicates the organism, Pseudomonas aeruginosa, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The absence of color in the bacterial growth indicates P. aeruginosa is unable to ferment lactose.

Quadrant 3: Growth on the plate indicates the organism, Enterobacter aerogenes, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The pink color of the bacterial growth indicates E. aerogenes is able to ferment lactose to produce weak acid end-products.

Quadrant 4:  Absence of growth indicates the organism, Staphylococcus aureus, is inhibited by eosin and methylene blue and is a gram-positive bacterium.

 

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Источник: http://faculty.collin.edu/dcain/CCCCD%20Micro/embagar.htm

EMB Plate Results:


Staphylococcus epidermidis (no growth), E. coli (dark purple), Pseudomonas aeruginosa (bottom), Enterobacter aerogenes (left)


Enterobacter aerogenes


E. coli


P. aeruginosa


E. coli - note metallic sheen = lactose fermentor


E. coli - note metallic sheen = lactose fermentor


P. aeruginosa (lactose nonfermentor)

Источник: https://www.uwyo.edu/virtual_edge/results/emb_results.htm
emb agar e coli results
emb agar e coli results

emb agar e coli results 23: Eosin Methylene Blue Agar (EMB)

EMB contents

EMB contains lactose and sucrose sugars, but it is the lactose that is the key to the medium. Lactose-fermenting bacteria (E. coli and other coliforms) produce acid from lactose use, and the combination of the dyes (which serve as pH indicators in this medium) produces color variations in the colonies because of the acidity. Strong acidity produces a deep purple colony with a green metallic sheen, whereas less acidity may produce a brown-pink coloration of colony. Nonlactose fermenters appear as translucent or pink.

Lactose-fermenter (E. coli) emb agar e coli results emb agar e coli results alt="" width="300px" height="252px" src="https://bio.libretexts.org/@api/deki/files/4930/emb3.png?revision=1&size=bestfit&width=300&height=252">

plate at left enlarged

Various stages of bacterial formation in EMB

Colonies of lactose fermenters will appear very dark purple, or have dark purple centers.

SOME bacteria gram + bacteria may grow--although not well--particularly if you let cultures sit for more than a couple of days. Usually those species will show as pinpoint colonies.

Источник: https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_I/23%3A_Eosin_Methylene_Blue_Agar_(EMB)

Learning Objectives

  • Define the term culture medium
  • Give examples of the following types of media:: complex, chemically defined, selective, and differential

The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive under laboratory conditions. Culturing many microorganisms is challenging because of highly specific nutritional and environmental requirements and the diversity of these requirements among different species.

Nutritional Requirements

Culture medium is defined as a medium of nutrients that supports microbial growth. The number of available media to grow bacteria is considerable.  When the complete chemical composition of a medium is known, it is called a chemically defined medium.  In contrast for a complex medium, the precise chemical composition is not known because they contain extracts and digests of yeasts, meat, or plants.  Nutrient broth, tryptic soy broth, and brain heart infusion, are all examples of complex media.

Differential and selective media are commonly used in microbiology laboratories. A differential medium support the growth of any microbe but distinguishes them based on how they metabolize or change the medium.  One example of differential medium is blood agar.  Blood agar distinguishes microbes based on their ability to lyse red blood cells (RBCs), a property known as hemolysis.  There are three types of hemolysis (Figure 1):

  • Beta hemolysis is complete breakdown of RBCs.  A clear area develops around the colonies.
  • Alpha hemolysis is partial breakdown of RBCs.  A green grey color develops around the colonies on blood agar.
  • Gamma hemolysis is no hemolyis; the microbe does not change the appearance of the blood agar.
hemolysis on blood agar

Figure 1. The three types of hemolysis on a blood agar plate

Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest  are called selective media. Selective medium contain particular ingredients that inhibit the growth of certain microbes.  An example of a selective medium is MacConkey agar. It contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria.  MacConkey agar is also a differential medium.   The lactose fermenters produce acid, which turns the medium and the colonies of strong fermenters hot pink. The medium is supplemented with the pH indicator neutral red, which turns to hot pink at low pH. Selective and differential media can be combined and play an important role in the identification of bacteria by biochemical methods.

A light brown agar plate. Two streaks on the plate are bright pink and two streaks are beige.

Figure 2. On this MacConkey agar plate, the lactose-fermenter E. coli colonies are bright pink. Serratia marcescens, which does not ferment lactose, forms a cream-colored streak on the tan medium. (credit: American Society for Microbiology)

Another commonly used medium that is both selective and differential is eosin-methylene blue (EMB) agar.  EMB contains the dyes eosin and methylene blue that inhibit the growth of gram-positve 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.  When bacterial cells ferment lactose, acid is produced that precipitates the dyes in the medium and the colonies develop a green metallic sheen emb agar e coli results 3).

Selective and Differential properties of EMB with E.coli

Figure 3 E.coli growing on eosin-methylene-blue agar. The gram-negative bacterium grows and ferments lactose, giving the colonies a green metallic sheen

Think about It

  • Distinguish complex and chemically defined media.
  • Distinguish selective and enrichment media.

Compare the compositions of EZ medium and sheep blood agar.

The End-of-Year Picnic

The microbiology department is celebrating the end of the school year in May by holding its traditional picnic on the green. The speeches drag on for a couple of hours, but finally all the faculty and students can dig into the food: chicken emb agar e coli results, tomatoes, onions, salad, and custard pie. By evening, the whole department, except for two vegetarian students who did not eat the chicken salad, is stricken with nausea, vomiting, retching, and abdominal cramping. Several individuals complain of diarrhea. One patient shows signs of shock (low blood pressure). Blood and stool samples are collected from patients, and an analysis of all foods served at the meal is conducted.

Bacteria can cause gastroenteritis (inflammation of the stomach and intestinal tract) either by colonizing and replicating in the host, which is considered an infection, or by secreting toxins, which is considered intoxication. Signs and symptoms of infections are typically delayed, whereas intoxication manifests within hours, as happened after the picnic.

Blood samples from the patients showed no signs of bacterial infection, which further suggests that this was a case of intoxication. Since intoxication is due to secreted toxins, bacteria are not usually detected in blood or stool samples. MacConkey agar and sorbitol-MacConkey agar plates and xylose-lysine-deoxycholate (XLD) plates were inoculated with stool samples and did not reveal any unusually colored colonies, and no black colonies or white colonies were observed on XLD. All lactose fermenters on MacConkey agar also ferment sorbitol. These results ruled out common agents of food-borne illnesses: E. coli, Salmonella spp., and Shigella spp.

A micrograph of clusters of purple spheres.

Figure 2. Gram-positive cocci in clusters. (credit: Centers for Disease Control and Prevention)

Analysis of the chicken salad revealed an abnormal number of gram-positive cocci arranged in clusters (Figure 2). A culture of the gram-positive cocci releases bubbles when mixed with hydrogen peroxide. The culture turned mannitol salt agar yellow after a 24-hour incubation.

All the tests point to Staphylococcus aureus as the organism that secreted the toxin. Samples from the salad showed the presence of gram-positive cocci bacteria in clusters. The colonies were positive for catalase. The bacteria grew on mannitol salt agar fermenting mannitol, as shown by the change to yellow of the medium. The pH indicator in mannitol salt agar is phenol red, which turns to yellow when the medium is acidified by the products of fermentation.

The toxin secreted by S. aureus is known to cause severe gastroenteritis. The organism was probably introduced into the salad during preparation by the food handler and multiplied while the salad was kept in the warm ambient temperature during the speeches.

  • What are some other factors that might have contributed to rapid growth of S. aureus in the chicken salad?
  • Why would S. aureus not be inhibited by the presence of salt in the chicken salad?

Key Concepts and Emb agar e coli results defined media have a a known quantities of each chemical component
  • Selective media favor the growth of some microorganisms while inhibiting others.
  • Differential media help distinguish bacteria by the color of the colonies or the change in the medium.
  • Fill in the Blank

    Blood agar contains many unspecified nutrients, supports the growth of a large number of bacteria, and allows differentiation of bacteria according to hemolysis (breakdown of blood). The medium is ________ and ________.

    Show Answer

    Blood agar contains many unspecified nutrients, supports the growth of a large number of bacteria, and allows differentiation of bacteria according to hemolysis (breakdown of blood). The medium is complex and differential.

    Rogosa agar contains yeast extract. The pH is adjusted to 5.2 and discourages the growth of many microorganisms; however, all the colonies look similar. The medium is ________ and ________.

    Show Answer

    Rogosa agar contains yeast extract. The pH is adjusted to 5.2 and discourages the growth of many microorganisms; however, all the colonies look similar. The medium is complex and selective.

    Источник: https://courses.lumenlearning.com/cuny-kbcc-microbiologyhd/chapter/media-used-for-bacterial-growth/

    Determination of the Sensitivity of a Rapid Escherichia coli O157:H7 Assay for Testing 375-Gram Composite Samples

    REFERENCES

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    Источник: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92276/

    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

    Tryptic Soy Agar - uninoculated

    Type: General

    Purpose: Cultivation of non-fastidious bacteria

    Interpretation: Growth indicates non-fastidious bacteria present

    S. aureus

    S. aureusally financial dealer services phone number Tryptic Soy Agar - Staphylococcus aureus
    Note the carotenoid pigment typical of S. aureus.

    E. coli

    E. coli

    Tryptic Soy Agar - Escherichia coli

    P. aeruginosa

    trypticsoy_ps.aeruginosa

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

    Chocolate Agar

    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

    S. aureus

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

    N. gonorrhoeae

    N. gonorrhoeae

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

    E. coli

    E. coli

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

    Thayer-Martin Agar

    Chocolate 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

    no growth

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

    N. gonorrhoeae

    N. gonorrhoeae

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

    E. coli

    no growth

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

    MacConkey (lactose) Agar

    MacConkey 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

    E. coli

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

    N. gonorrhoeae

    N. gonorrhroeae

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

    S. enteritidis

    Salmonella

    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

    N. gonorrhroeae

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

    Eosin-methylene Blue Agar (EMB)

    Eosin Methylene Blue

    Eosin Methylene Blue Agar - uninoculated

    Type: Emb agar e coli results (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

    Salmonella

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

    E. coli

    E. coli

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

    K. pneumoniae

    E. coli

    Eosin Methylene Blue Agar - Klebsiella pneumoniae
    what kind of cranberry juice is good for you Note: Mucoid colonies with dark centers due to capsule production and emb agar e coli results fermentation respectively.

    Hektoen Agar

    Hektoen

    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

    E. Coli

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

    S. enteritidis

    Salmonella

    Hektoen - Salmonella enteritidis
    emb agar e coli results 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

    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

    S. epidermidis

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

    S. aureus

    S. aureus

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

    E coli

    Mannitol Salt Agar

    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

    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

    E. coli

    Triple Sugar Iron Agar - Escherichia coli

    Salmonella

    Salmonella

    Triple Sugar Iron Agar - Salmonella

    Shigella

    Shigella

    Triple Sugar Iron Agar - Shigella

    P. aeruginosa

    Ps. 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

    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

    Alpha haemolysis

    Sheep Blood Agar - Streptococcus pneumoniae, alpha hemolytic

    Beta hemolysis

    Beta haemolysis

    Sheep Blood Agar - Streptococcus pyogenes, beta hemolytic

    Gamma hemolysis

    Gamma haemolysis

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

    No growth

    N. gonorrhoeae

    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
    Graphic Art: Deon Foster, Elizabeth Barney
    Highslide JS thumbnail viewer script
    Red Icon set by Ken Saunders twiddy realty obx nc MouseRunner.com

    Источник: https://learn.chm.msu.edu/vibl/content/differential/

    Eosin Methylene Blue Agar


    Eosin Methylene Blue (EMB) agar is both selective and differential.  It contains the dyes eosin and methylene blue, which inhibit the growth of gram-positive bacteria and therefore select for gram-negative bacteria.  It also contains the carbohydrate lactose, which allows differentiation of gram-negative bacteria based on their ability to ferment lactose.

    Quadrant 1:  Growth on the plate indicates the organism, Escherichia coli, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The green metallic sheen indicates E. coli is able to ferment lactose to produce strong acid end-products.

    Quadrant 2:  Growth on the plate indicates the organism, Pseudomonas best mortgage refinance rates in texas, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The absence of color in the bacterial growth indicates P. aeruginosa is unable to ferment lactose.

    Quadrant 3: Growth on the plate indicates the organism, Enterobacter aerogenes, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The pink color of the bacterial growth indicates E. aerogenes is able to ferment lactose to produce weak acid end-products.

    Quadrant 4:  Absence of growth indicates the organism, Staphylococcus aureus, is inhibited by eosin and methylene blue and is a gram-positive bacterium.

     

    Back to Tutorial

    Источник: http://faculty.collin.edu/dcain/CCCCD%20Micro/embagar.htm

    American Society for Microbiology

    Download the PowerPoint

    PowerPoint Contents

    Figure 1: Escherichia coli on EMB (Enlarged view). FIG. 1. Eosin-methylene blue (EMB) agar plate inoculated with Escherichia coli (a gram-negative coliform bacterium) showing good growth of dark blue-black colonies with metallic green sheen indicating vigorous fermentation of lactose and acid production which precipitates the green metallic pigment. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

    Figure 2: Enterobacter aerogenes on EMB (Enlarged view). FIG. 2. EMB agar plate inoculated with Enterobacter aerogenes (a gram-negative coliform bacterium) showing good growth of brown, dark-centered, mucoid colonies indicating lactose fermentation and acid production. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

    Figure 3: Klebsiella pneumoniae on EMB (Enlarged view). FIG. 3. EMB agar plate emb agar e coli results with Klebsiella pneumoniae (a gram-negative coliform bacterium) showing good growth of brown, dark-centered, mucoid colonies (smaller than Enterobacter) indicating lactose fermentation and acid production. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

    Figure 4: Pseudomonas aeruginosa on EMB (Enlarged view). FIG. 4. EMB agar plate inoculated with Pseudomonas aeruginosa (a gram-negative noncoliform bacterium) showing good growth but no fermentation of sugars or acid production. (Naowarat Cheeptham and Carolynne Fardy, Thompson Rivers University, Kamloops, BC, Canada)

    Figure 5: Proteus vulgaris on EMB (Enlarged view). FIG. 5. EMB agar plate inoculated with Proteus vulgaris (a gram-negative coliform bacterium) showing growth of pink colonies indicating non-lactose fermentation and some acid production. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

    Figure 6: Escherichia coli and Pseudomonas aeruginosa on EMB (Enlarged view) FIG. 6. EMB agar plate inoculated with a mixed culture of Escherichia coli and Pseudomonas aeruginosa. Note the metallic green sheen of the strong lactose-fermenting Escherichia coli and the pinkish colonies of nonfermenter Pseudomonas aeruginosa. (Archana Lal, Independence Community College, Independence, KS)

    Figure 7: Acinetobacter baumannii on EMB (Enlarged view). FIG. 7. EMB agar plate inoculated with Acinetobacter baumannii (a gram-negative non-glucose-fermenting bacillus) showing a colony with a classic blue-grey center. This should not be mistaken for evidence of lactose fermentation on EMB agar. (Bobbi Pritt, Mayo Clinic, Rochester, MN)

    Figure 8: Stenotrophomonas maltophilia on EMB (Enlarged view). FIG. 8. EMB agar plate inoculated with Stenotrophomonas maltophilia (a gram-negative non-glucose-fermenting bacillus) showing good growth and non-lactose-fermenting morphology. (Bobbi Pritt, Mayo Clinic, Rochester, MN)

    Figure 9: Escherichia coli and Salmonella enteritidis on EMB (Enlarged view). FIG. 9. EMB agar plate inoculated with mixed enteric flora. The lactose fermenter Escherichia coli grew with purple-centered colonies while the lactose nonfermenter Salmonella enteritidis grew as colorless colonies. Salmonella enteritidis mixed with Escherichia coli is able to utilize the acid products as energy source, resulting in an insufficient acid buildup to precipitate out the Eosin Methylene Blue and no green metallic sheen is produced by Escherichia coli. (Jerry Keplinger, East Tennessee State University, Johnson City, TN)

    Figure 10: Clostridium perfringens on EMB (Enlarged view). FIG. 10. EMB agar plate inoculated with Clostridium perfringens (a gram-positive bacterium) maintained under anaerobic conditions and showing no growth. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

    Figure 11: Bacillus subtilis on EMB (Enlarged view). FIG. 11. EMB agar plate inoculated with Bacillus subtilis (a gram-positive bacterium) showing poor growth. (Naowarat Cheeptham,Thompson Rivers University, Kamloops, BC, Canada)

    Figure 12: Micrococcus luteus on EMB (Enlarged view). FIG. 12. EMB agar plate inoculated with Micrococcus luteus (a gram-positive bacterium) showing no growth. (Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

    Figure 13: Staphylococcus aureus on EMB (Enlarged view). FIG. 13. EMB agar plate inoculated with Staphylococcus aureus (a gram-positive bacterium) showing no growth. (Archana Lal, Independence Community College, Independence, KS)

    Figure 14: Streptococcus group B on EMB (Enlarged view). FIG. 14. EMB agar plate inoculated with Streptococcus group B (a gram-positive bacterium) showing no growth. (Archana Lal, Independence Community College, Independence, KS)

    Figure 15: Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus on EMB (Enlarged view). FIG. 15. EMB agar plate inoculated with (A) Escherichia coli, (B) Bacillus subtilis, (C) Pseudomonas aeruginosa, and (D) Staphylococcus aureus. The two gram-positive bacteria Bacillus subtilis and Staphylococcus aureus did not grow, while the two gram-negative bacteria Escherischia coli and Pseudomonas aeruginosa grew with typical lactose fermenter (colonies with green metallic sheen) and nonfermenter (pink colonies) morphology. (Archana Lal, Independence Community College, Independence, KS)

    Figure 16: Salmonella enteritidis on EMB(Enlarged view). FIG. 16. EMB agar plate inoculated with Salmonella enteritidis (a gram-negative coliform bacterium) showing good growth of grey mucoid colonies with no fermentation of lactose or acid production. (Naowarat Cheeptham and Carolynne Fardy, Thompson Rivers University, Kamloops, BC, Canada)

    Figure 17: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes on EMB (Enlarged view). FIG. 17. EMB agar plate inoculated with (A) Escherichia coli, (B) Pseudomonas aeruginosa, (C) Klebsiella pneumoinae, and (D) Enterobacter aerogenes. All four gram negative bacteria metro vacuum vnb 83ba exhibiting different morphology. Escherischia coli grew with typical lactose fermenter morphology with excessive acid production and precipitation of green metallic pigment (colonies with green metallic sheen). Pseudomonas aeruginosa grew exhibiting the nonfermenter morphology (pinkish colonies), both Klebsiella pneumoniae and Enterobacter aerogenes grew with lactose fermentation and acid production morphology (with purple dark centered mucoid why is pickle juice good for you. (Naowarat Cheeptham and Carolynne Fardy, Thompson Rivers University, Kamloops, BC, Canada)

    Figure 18: Lactose Fermentation FIG. 18. Simple bible com blue agar plate inoculated with Escherichia coli. This plate shows a metallic green sheen on the colonies, which indicates strong acid production from lactose and/or sucrose fermentation. (Jesus Antonio Romo, University of Texas at San Antonio, San Antonio, TX)

    Источник: https://asm.org/Image-Gallery/Eosin-Methylene-Blue

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