emb agar test results

Results obtained with Levine EMB Agar without Lactose are dependent upon the substituted carbohydrate. *Store at 2-8°C. Free Shipping within the Continental USA. Eosin Methylene Blue Agar [EMB] is a differential plating medium recommended for TEST ORGANISM. RESULTS. ATCC# 25922 Escherichia coli. coliforms, it is necessary to inoculate EMB (eosin methylene blue) agar plates Parallel testing has shown that results obtained from the membrane filter.

Emb agar test results -

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/

Bacteriological Analytical Manual (BAM) Main Page

Authors: Peter Feng (ret.), Stephen D. Weagant (ret.), Michael A. Grant (dec.), William Burkhardt

Revision History

  • October 2020 - Section I A.3 modified to reflect that enrichment should take place at 35 ± 0.5⁰C and not at 35 ± 1⁰C.
  • July 2017 - Chap. 4 Sec. I. E. For the completed phase of testing for E. coli, the incubation temperature of EC tubes has been changed from 45.5 ± 0.2°C to 44.5 ± 0.2°C. The change was made in part due to the poor ability of the control strain ATCC25922 to grown and ferment lactose to produce acid and gas at 45.5 ± 0.2°C. The use of 44.5 ± 0.2°C would also make it consistent with that used for fecal Coliform analysis in shellfish and shellfish meats (Sec. VI) as well as conditions used for E. coli testing by other International organizations.
  • February 2013 - Shellfish analysis method revised to be consistent with the APHA Examination of seawater and shellfish, 4th ed.
  • February 2013 - Membrane filter methods added to water analysis.

Chapter Contents


Escherichia coli, originally known as Bacterium coli commune, was identified in 1885 by the German pediatrician, Theodor Escherich (14, 29). E. coli is widely distributed in the intestine of humans and warm-blooded animals and is the predominant facultative anaerobe in the bowel and part of the essential intestinal flora that maintains the physiology of the healthy host (9, 29). E. coli is a member of the family Enterobacteriaceae (15), which includes many genera, including known pathogens such as Salmonella, Shigella, and Yersinia. Although most strains of E. coli are not regarded as pathogens, they can be opportunistic pathogens that cause infections in immunocompromised hosts. There are also pathogenic strains of E. coli that when ingested, causes gastrointestinal illness in healthy humans (see Chap. 4A).

In 1892, Shardinger proposed the use of E. coli as an indicator of fecal contamination. This was based on the premise that E. coli is abundant in human and animal feces and not usually found in other niches. Furthermore, since E. coli could be easily detected by its ability to ferment glucose (later changed to lactose), it was easier to isolate than known gastrointestinal pathogens. Hence, the presence of E. coli in food or water became accepted as indicative of recent fecal contamination and the possible presence of frank pathogens. Although the concept of using E. coli as an indirect indicator of health risk was sound, it was complicated in practice, due to the presence of other enteric bacteria like Citrobacter, Klebsiella and Enterobacter that can also ferment lactose and are similar to E. coli in phenotypic characteristics, so that they are not easily distinguished. As a result, the term "coliform" was coined to describe this group of enteric bacteria. Coliform is not a taxonomic classification but rather a working definition used to describe a group of Gram-negative, facultative anaerobic rod-shaped bacteria that ferments lactose to produce acid and gas within 48 h at 35°C. In 1914, the U.S. Public Health Service adopted the enumeration of coliforms as a more convenient standard of sanitary significance.

Although coliforms were easy to detect, their association with fecal contamination was questionable because some coliforms are found naturally in environmental samples (6). This led to the introduction of the fecal coliforms as an indicator of contamination. Fecal coliform, first defined based on the works of Eijkman (12) is a subset of total coliforms that grows and ferments lactose at elevated incubation temperature, hence also referred to as thermotolerant coliforms. Fecal coliform analyses are done at 45.5°C for food testing, except for water, shellfish and shellfish harvest water analyses, which use 44.5°C (1, 3, 30). The fecal coliform group consists mostly of E. coli but some other enterics such as Klebsiella can also ferment lactose at these temperatures and therefore, be considered as fecal coliforms. The inclusion of Klebsiella spp in the working definition of fecal coliforms diminished the correlation of this group with fecal contamination. As a result, E. coli has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli.

Currently, all 3 groups are used as indicators but in different applications. Detection of coliforms is used as an indicator of sanitary quality of water or as a general indicator of sanitary condition in the food-processing environment. Fecal coliforms remain the standard indicator of choice for shellfish and shellfish harvest waters; and E. coli is used to indicate recent fecal contamination or unsanitary processing. Almost all the methods used to detect E. coli, total coliforms or fecal coliforms are enumeration methods that are based on lactose fermentation (4). The Most Probable Number (MPN) method is a statistical, multi-step assay consisting of presumptive, confirmed and completed phases. In the assay, serial dilutions of a sample are inoculated into broth media. Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other 2 phases of the assay are performed, and then uses the combinations of positive results to consult a statistical table (Appendix 2), to estimate the number of organisms present. Typically only the first 2 phases are performed in coliform and fecal coliform analysis, while all 3 phases are done for E. coli. The 3-tube MPN test is used for testing most foods. Analysis of seawater using a multiple dilution series should not use less than 3 tubes per dilution (5 tubes are recommended); in certain instances a single dilution series using no less than 12 tubes may also be acceptable. (For additional details, see: FDA. National Shellfish Sanitation Program, Manual of Operations. 2009 Revision. DHHS/PHS/FDA, Washington DC). Likewise,  analysis of bivalve molluscan shellfish should be performed using a multiple dilution MPN series whereby no fewer than 5- tubes per dilution should be used, see section IV. There is also a 10-tube MPN method that is used to test bottled water or samples that are not expected to be highly contaminated (3).  Analysis of citrus juice for E. coli is performed as an absence/presence method, see section V.

Also, there is a solid medium plating method for coliforms that uses Violet Red Bile Agar, which contains neutral red pH indicator, so that lactose fermentation results in formation of pink colonies. There are also membrane filtration tests for coliform and fecal coliform that measure aldehyde formation due to fermentation of lactose. This chapter also includes variations of above tests that use fluorogenic substrates to detect E. coli (18), special tests for shellfish analysis, a brief consideration of bottled water testing and a method for testing large volumes of citrus juices for presence of E. coli in conjunction with the Juice HACCP rule.


I. Conventional Method for coliforms, fecal coliforms and E. coli

  1. Equipment and materials

    1. Covered water bath, with circulating system to maintain temperature of 44.5 ± 0.2°C.  The temperature for water baths for the shellfish program is 44.5°C ± 0.2°C. Water level should be above the medium in immersed tubes.
    2. Immersion-type thermometer, 1-55°C, about 55 cm long, with 0.1°C subdivisions, certified by National Institute of Standards and Technology (NIST), or equivalent Incubator, 35 ± 0.5°C.
    3. Balance with capacity of >2 kg and sensitivity of 0.1 g
    4. Blender and blender jar (see Chapter 1)
    5. Sterile graduated pipets, 1.0 and 10.0 mL
    6. Sterile utensils for sample handling (see Chapter 1)
    7. Dilution bottles made of borosilicate glass, with polyethylene screw caps equipped with Teflon liners. Commercially prepared dilution bottles containing sterile Butterfield's phosphate buffer can also be used.
    8. Quebec colony counter, or equivalent, with magnifying lens
    9. Longwave UV light [~365 nm], not to exceed 6 W.
    10. pH meter
  2. Media and Reagents

    1. Brilliant green lactose bile (BGLB) broth, 2% (M25)
    2. Lauryl tryptose (LST) broth (M76)
    3. Lactose Broth (M74)
    4. EC broth (M49)
    5. Levine's eosin-methylene blue (L-EMB) agar (M80)
    6. Tryptone (tryptophane) broth (M164)
    7. MR-VP broth (M104)
    8. Koser's citrate broth (M72)
    9. Plate count agar (PCA) (standard methods) (M124)
    10. Butterfield's phosphate-buffered water (R11) or equivalent diluent

      (Note: This same formulation is referred to as Buffered Dilution Water in American Public Health Association. 1970. Recommended Procedures for the Examination of Seawater and Shellfish, 4th ed. APHA, Washington, DC., p14-15)

    11. Kovacs' reagent (R38)
    12. Voges-Proskauer (VP) reagents (R89)
    13. Gram stain reagents (R32)
    14. Methyl red indicator (R44)
    15. Violet red bile agar (VRBA) (M174)
    16. VRBA-MUG agar (M175)
    17. EC-MUG medium (M50)
    18. Lauryl tryptose MUG (LST-MUG) broth (M77)
    19. Peptone Diluent, 0.5% (R97)
  3. MPN - Presumptive test for coliforms, fecal coliforms and E. coli

    Weigh 50 g of food into sterile high-speed blender jar (see Chapter 1 and current FDA compliance programs for instructions on sample size and compositing) Frozen samples can be softened by storing  for <18 h at 2-5°c, but do not thaw. Add 450 mL of Butterfield's phosphate-buffered water and blend for 2 min. If <50 g of sample are available, weigh portion that is equivalent to half of the sample and add sufficient volume of sterile diluent to make a 1:10 dilution. The total volume in the blender jar should completely cover the blades.

    Prepare decimal dilutions with sterile Butterfield's phosphate diluent or equivalent. Number of dilutions to be prepared depends on anticipated coliform density. Shake all suspensions 25 times in 30 cm arc or vortex mix for 7 s. Using at least 3 consecutive dilutions, inoculate 1 mL aliquots from each dilution into 3 LST tubes for a 3 tube MPN analysis (other analysis may require the use of 5 tubes for each dilution; See IV). Lactose Broth may also be used. For better accuracy, use a 1 mL or 5 mL pipet for inoculation. Do not use pipets to deliver<10% of their total volume; eg. a 10 mL pipet to deliver 0.5 mL. Hold pipet at angle so that its lower edge rests against the tube.  Not more than 15  min  should  elapse  from  time  the  sample is  blended  until  all  dilutions  are  inoculated  in appropriate media.

    Incubate LST tubes at 35°C± 0.5°C . Examine tubes and record reactions at 24 ± 2 h for gas, i.e., displacement of medium in fermentation vial or effervescence when tubes are gently agitated. Re-incubate gas-negative tubes for an additional 24 h and examine and record reactions again at 48 ± 3 h. Perform confirmed test on all presumptive positive (gas) tubes.

  4. MPN - Confirmed test for coliforms

    From each gassing LST or lactose broth tube, transfer a loopful of suspension to a tube of BGLB broth, avoiding pellicle if present. (a sterile wooden applicator stick may also be used for these transfers). Incubate BGLB tubes at 35°C ± 0.5°C and examine for gas production at 48 ± 3 h. Calculate most probable number (MPN) (see Appendix 2) of coliforms based on proportion of confirmed gassing LST tubes for 3 consecutive dilutions.

  5. MPN - Confirmed test for fecal coliforms and E. coli

    From each gassing LST or Lactose broth tube from the Presumptive test, transfer a loopful of each suspension to a tube of EC broth (a sterile wooden applicator stick may also be used for these transfers). Incubate EC tubes 24 ± 2 h at 44.5°C and examine for gas production. If negative, reincubate and examine again at 48 ± 2 h. Use results of this test to calculate fecal coliform MPN. To continue with E. coli analysis, proceed to Section F below. The EC broth MPN method may be used for seawater and shellfish since it conforms to recommended procedures (1).

  6. MPN - Completed test for E. coli.

    To perform the completed test for E. coli, gently agitate each gassing EC tube, remove a loopful of broth   and streak for isolation on a L-EMB agar plate and incubate for 18-24 h at 35°C ± 0.5°C . Examine plates for suspicious E. coli colonies, i.e., dark centered and flat, with or without metallic sheen. Transfer up to 5 suspicious colonies from each L-EMB plate to PCA slants, incubate them for 18-24 h at 35°C ± 0.5°C and use for further testing.

    NOTE: Identification of any 1 of the 5 colonies as E. coli is sufficient to regard that EC tube as positive; hence, not all 5 isolates may need to be tested.

    Perform Gram stain. All cultures appearing as Gram-negative, short rods should be tested for the IMViC reactions below and also re-inoculated back into LST to confirm gas production.

    Indole production. Inoculate tube of tryptone broth and incubate 24 ± 2 h at 35°C ± 0.5°C . Test for indole by adding 0.2-0.3 mL of Kovacs' reagent. Appearance of distinct red color in upper layer is positive test.

    Voges-Proskauer (VP)-reactive compounds. Inoculate tube of MR-VP broth and incubate 48 ± 2 h at 35°C± 0.5°C . Transfer 1 mL to 13 × 100 mm tube. Add 0.6 mL α-naphthol solution and 0.2 mL 40% KOH, and shake. Add a few crystals of creatine. Shake and let stand 2 h. Test is positive if eosin pink color develops.

    Methyl red-reactive compounds. After VP test, incubate MR-VP tube additional 48 ± 2 h at 35°C± 0.5°C . Add 5 drops of methyl red solution to each tube. Distinct red color is positive test. Yellow is negative reaction.

    Citrate. Lightly inoculate tube of Koser's citrate broth; avoid detectable turbidity. Incubate for 96 h at 35°C ± 0.5°C . Development of distinct turbidity is positive reaction.

    Gas from lactose. Inoculate a tube of LST and incubate 48 ± 2 h at 35°C ± 0.5°C . Gas production (displacement of medium from inner vial) or effervescence after gentle agitation is positive reaction.

    Interpretation: All cultures that (a) ferment lactose with gas production within 48 h at 35°C, (b) appear as Gram-negative nonsporeforming rods and (c) give IMViC patterns of ++-- (biotype 1) or -+-- (biotype 2) are considered to be E. coli. Calculate MPN (see Appendix 2) of E. coli based on proportion of EC tubes in 3 successive dilutions that contain E. coli.

    NOTE: Alternatively, instead of performing the IMViC test, use API20E or the automated VITEK biochemical assay to identify the organism as E. coli. Use growth from the PCA slants and perform these assays as described by the manufacturer.

  7. Solid medium method - Coliforms

    Prepare violet red bile agar (VRBA) according to manufacturer's instructions. Cool to 48°C before use. Prepare, homogenize, and decimally dilute sample as described in section I. C above so that isolated colonies will be obtained when plated. Transfer two 1 mL aliquots of each dilution to petri dishes, and use either of the following two pour plating methods, depending on whether injured or stressed cells are suspected to be present (1).

    Pour 10 mL VRBA tempered to 48°C into plates, swirl plates to mix, and let solidify. To prevent surface growth and spreading of colonies, overlay with 5 mL VRBA, and let solidify. If resuscitation is necessary, pour a basal layer of 8-10 mL of tryptic soy agar tempered to 48°C. Swirl plates to mix, and incubate at room temperature for 2 ± 0.5 h. Then overlay with 8-10 mL of melted, cooled VRBA and let solidify.

    Invert solidified plates and incubate 18-24 h at 35°C. Incubate dairy products at 32°C (2). Examine plates under magnifying lens and with illumination. Count purple-red colonies that are 0.5 mm or larger in diameter and surrounded by zone of precipitated bile acids. Plates should have 25-250 colonies. To confirm that the colonies are coliforms, pick at least 10 representative colonies and transfer each to a tube of BGLB broth. Incubate tubes at 35°C. Examine at 24 and 48 h for gas production.

    NOTE: If gas-positive BGLB tube shows a pellicle, perform Gram stain to ensure that gas production was not due to Gram-positive, lactose-fermenting bacilli.

    Determine the number of coliforms per gram by multiplying the number of suspect colonies by percent confirmed in BGLB by dilution factor.

    Alternatively, E. coli colonies can be distinguished among the coliform colonies on VRBA by adding 100 µg of 4-methyl-umbelliferyl-β-D-glucuronide (MUG) per mL in the VRBA overlay. After incubation, observe for bluish fluorescence around colonies under longwave UV light. (see LST-MUG section II for theory and applicability.)

  8. Membrane Filtration (MF) Method - coliforms: see Section III. Bottled Water.

    Food homogenates will easily clog filters, hence MF are most suitable for analysis of water samples; however, MF may be used in the analysis of liquid foods that do not contain high levels of particulate matter such as bottled water (see Section III for application of MF).

II. LST-MUG Method for Detecting E. coli in Chilled or Frozen Foods Exclusive of Bivalve Molluscan Shellfish

The LST-MUG assay is based on the enzymatic activity of β-glucuronidase (GUD), which cleaves the substrate 4-methylumbelliferyl β-D-glucuronide (MUG), to release 4-methylumbelliferone (MU). When exposed to longwave (365 nm) UV light, MU exhibits a bluish fluorescence that is easily visualized in the medium or around the colonies. Over 95% of E. coli produces GUD, including anaerogenic (non-gas-producing) strains. One exception is enterohemorrhagic E. coli (EHEC) of serotype O157:H7, which is consistently GUD negative (11, 17). The lack of GUD phenotype in O157:H7 is often used to differentiate this serotype from other E. coli, although GUD positive variants of O157:H7 do exist (24, 26). The production of GUD by other members of the family Enterobacteriaceae is rare, except for some shigellae (44 -58%) and salmonellae (20-29%) (18, 27). However, the inadvertent detection of these pathogens by GUD-based assays is not considered a drawback from a public health perspective. Expression of GUD activity is affected by catabolite repression (8) so on occasion, some E. coli are GUD-negative, even though they carry the uidA gene (gusA) that encodes for the enzyme (19). In most analyses however, about 96% of E. coli isolates tested are GUD-positive without the need for enzyme induction (27).

MUG can be incorporated into almost any medium for use in detecting E. coli. But some media such as EMB, which contain fluorescent components, are not suitable, as they will mask the fluorescence of MU. When MUG is incorporated into LST medium, coliforms can be enumerated on the basis of gas production from lactose and E. coli are presumptively identified by fluorescence in the medium under longwave UV light, thus it is capable of providing a presumptive identification of E. coli within 24 h (18, 28). The LST-MUG method described below has been adopted as Official Final Action by the AOAC for testing for E. coli in chilled or frozen foods, exclusive of shellfish (28). See Sec. IV.4. D. for precautions in using MUG in testing shellfish. For information on MUG assay contact, Dr. Bill Burkhardt III (email [email protected] ), FDA, CFSAN, Dauphin Island, AL, 36528; 251-406-8125

CAUTION: To observe for fluorescence, examine inoculated LST-MUG tubes under longwave (365 nm) UV light in the dark. A 6-watt hand-held UV lamp is adequate and safe. When using a more powerful UV source, such as a 15-watt fluorescent lamp, wear protective glasses or goggles. Also, prior to use in MUG assays, examine all glass tubes for auto fluorescence. Cerium oxide, which is sometimes added to glass as a quality control measure, will fluoresce under UV light and interfere with the MUG test (25). The use of positive and negative control strains for MUG reaction is essential.

  1. Equipment and material:see section I.A above and in addition,
    1. New, disposable borosilicate glass tubes (100 × 16 mm)
    2. New, disposable borosilicate glass Durham vials (50 × 9 mm) for gas collection
    3. Longwave UV lamp, not to exceed 6-watt
  2. Media and reagents:see section I.B above
  3. Presumptive LST-MUG test for E. coli.

Prepare food samples and perform the MPN Presumptive test as described in section I.C. above, except use LST-MUG tubes instead of LST. Be sure to inoculate one tube of LST-MUG with a known GUD-positive E. coli isolate as positive control (ATCC 25922). In addition, inoculate another tube with a culture of Enterobacter aerogenes (ATCC 13048) culture of Enterobacter aerogenes (ATCC 13048) or a Klebsiella pneumoniae strain as negative control, to facilitate differentiation of sample tubes that show only growth from those showing both growth and fluorescence. Incubate tubes for 24 to 48 ± 2 h at 35°C. Examine each tube for growth (turbidity, gas) then examine tubes in the dark under longwave UV lamp (365 nm). A bluish fluorescence is a positive presumptive test for E. coli. Studies by Moberg et al. (28) show that a 24 h fluorescence reading is an accurate predictor of E. coli and can identify 83-95% of the E. coli-positive tubes. After 48 h of incubation, 96-100% of E. coli-positive tubes can be identified (28). Perform a confirmed test on all presumptive positive tubes by streaking a loopful of suspension from each fluorescing tube to L-EMB agar and incubate 24 ± 2 h at 35°C. Follow protocols outlined in I. F, above, for Completed test for E. coli. Calculate MPN of E. coli based on combination of confirmed fluorescing tubes in 3 successive dilutions.

III. Examination of Bottled Water

Consumption of bottled water is increasing rapidly worldwide. In the U.S. alone, over 3.6 billion gallons of bottled water were consumed in 1998 (International Bottled Water Association, Alexandria, VA). Unlike potable water, which is regulated by the U.S. EPA, bottled water is legally classified as food in the U.S. and regulated by the FDA (Federal Register. 1995. 21 CFR Part 103 et al. beverages: bottled water; final rule. 60(218) 57076-57130). FDA defines bottled water as "water that is intended for human consumption and that is sealed in bottles or other containers with no added ingredients except that it may contain safe and suitable antimicrobial agents" and, within limitations, some added fluoride. Bottled water may be used as a beverage by itself or as an ingredient in other beverages. These regulations do not apply to soft drinks or similar beverages. In addition to "bottled water" or "drinking water", in 21 CFR Part 103 FDA also defines various types of bottled water that meet certain criteria. These identities include "artesian or artesian well water", "ground water", mineral water", "purified or demineralized water", "sparkling bottled water", "spring water" and "well water". Additionally "sterile water" is defined as water that meets the requirements under the "Sterility Test" in the United States Pharmacopeia.

Coliform organisms are not necessarily pathogens and are rarely found in bottled water, however, they serve as an indicator of insanitation or possible contamination. Surveys have shown that coliforms are useful indicators of bottled water quality, but some countries also monitor additional microbial populations as indicators of bottle water quality (10, 33). Under the current bottled water quality standard, FDA has established a microbiological quality requirement that is based on coliform detection levels. These levels may be obtained by membrane filtration (MF) or by 10-tube MPN analysis of ten 10-mL analytical units. For information on bottled water methods contact Dr. Bill Burkhardt III (email [email protected] ), FDA, CFSAN, Dauphin Island, AL, 36528; 251-406-8125

  1. Equipment and Materials.

    1. Incubator at 35° ± 0.5°C.
    2. Membrane filtration units (filter base and funnels): glass, plastic, or stainless steel; wrapped in foil or paper and sterilized.
    3. Ultraviolet sterilization chamber for sterilizing filter base and funnels (optional).
    4. Filter manifold or vacuum flask to hold filter funnels.
    5. Vacuum source (line vacuum, electric vacuum pump or water aspirator).
    6. Membrane filters; sterile, white, gridded, 47 mm diameter, 0.45 µm pore size (or equivalent, as specified by the manufacturer) for enumeration of bacteria.
    7. Petri dishes, sterile, plastic, 50 × 12 mm, with tight fitting lids.
    8. Forceps designed to transfer membranes without damage.
  2. Culture media.

    1. Lauryl sulfate tryptose (LST) broth (M-76).
    2. Brilliant green lactose bile broth (BGLB) (M-25).
    3. M-Endo Medium (BD#274930) (M-196).
    4. LES-Endo Agar (BD#273620) (M-197).
  3. Ten tube MPN coliform test - Presumptive and Confirmed procedures.

    For routine examination of bottled water, take 100 mL of sample and inoculate 10 tubes of 2X LST (10 mL of medium) with 10 mL of undiluted sample each. Incubate tubes at 35°C. Examine tubes at 24 ± 2 h for growth and gas formation as evidenced by displacement of medium in fermentation vial or effervescence when tubes are gently agitated. If negative at 24 h, reincubate tubes for an additional 24 h and examine again for gas. Perform a confirmed test on all presumptive positive (gassing) tubes as follows: gently agitate each positive LST tube and, using a 3.0 - 3.5 mm sterile loop, transfer one or more loopfuls of suspension to a tube of BGLB broth. Sterile wooden applicator sticks may also be used for transfer by inserting it at least 2.5 cm into the broth culture. Incubate BGLB tubes for 48 ± 2 h at 35°C. Examine for gas production and record. Calculate MPN using 10 tube MPN Table (9221.III), p. 9-52, Standard Methods for the Examination of Water and Wastewater (3).

    NOTE: if a sample is found to contain coliforms (at any level) follow procedure outlined in Sec. I. F. above to determine if it is E. coli. Bottled water is not permitted to contain E. coli.

  4. Membrane filter method for coliforms.

    Filter 100 mL of test sample and transfer the filter to M-Endo medium (M-196) or LES Endo Agar (M-197) and incubate at 35 °C± 0.5°C for 22-24 h. Count colonies that are pink to dark red with a green metallic surface sheen. The sheen may vary from pinpoint to complete coverage of the colony. Use of a low power, dissecting-type microscope to examine filters is recommended.

    Confirmation - If there are 5 to 10 sheen colonies on the filter, confirm all by inoculating growth from each sheen colony into tubes of LST and incubate at 35 °C± 0.5°C for 48 h. If the number of sheen colonies exceeds 10, randomly select and confirm 10 colonies that are representative of all sheen colonies. Any gas positive LST tubes should be sub cultured to BGLB and incubated at 35°C± 0.5°C for 48 hr. Gas production in BGLB within 48 h is a confirmed coliform test. Report results as number of coliform colonies per 100 mL. NOTE: Standard Method, 1998, 20th ed, p. 9-60 (3), allows for simultaneous inoculation of LST and BGLB during verification. However, BGLB is somewhat inhibitory so the method described above, where samples are sub cultured from LST into BGLB is regarded as a more sensitive verification assay and therefore, recommended.

    NOTE: if a sample is found to contain coliforms (at any level) follow procedure outlined in Sec. I. F. above to determine if it is E. coli. Bottled water is not permitted to contain E. coli.

IV. Examination of Shellfish and Shellfish Meats

The official FDA procedure for bacteriological analysis of domestic and imported bivalve molluscan shellfish is fully and properly described in the APHA's Recommended Procedures for the Examination of Sea Water and Shellfish, 4th ed. 1970 (1). The methods, including the conventional 5-tube MPN for coliform, fecal coliform and standard total plate count for bacteria (see Part III, APHA's Recommended Procedures the Examination of Sea Water and Shellfish, 4th ed. 1970 (1), are described below for examining shell stock, fresh-shucked meats, fresh-shucked frozen shellfish, and shellfish frozen on the half shell. These procedures do not apply to the examination of crustaceans (crabs, lobsters, and shrimp) or to processed shellfish meats such as breaded, shucked, pre-cooked, and heat-processed products (see section I. C. this chapter). Also, there are many methods that are used for testing for shellfish harvest and environmental water for fecal coliforms. One example, the mTEC agar (M-198) is a suitable membrane filter medium for enumerating fecal coliforms in marine and estuarine waters. Briefly, following the filtration of 100 ml of water, the filter funnels should be rinsed twice with approx. 20 ml of PBS. The filter is then transferred onto mTEC agar and incubated for 22-24 h at 44.5°C in Ethyfoam. All yellow, yellow-green or yellow-brown colonies are counted as fecal coliforms. Only plates having fewer than 80 colonies are counted. However, analysis of environmental waters will not be covered in detail here, as environmental water analyses are done by the U.S. EPA (3) and the quality of shellfish harvest waters are mainly the responsibilities of each State's Shellfish Control Authorities (20).

  1. Sample Preparation

    Using 10-12 shellfish, obtain 200 g of shellfish liquor and meat. Blend 2 min, with 200 mL sterile phosphate buffered dilution water or 0.5% peptone water (R97) to yield a 1:2 dilution of sample. Analysis of the ground sample must begin within 2 min after blending. Make serial dilutions in 0.5% sterile peptone water or sterile phosphate buffered dilution water.

  2. MPN - Presumptive and Confirmed Test for Coliform

    Use Lactose Broth (M74) or Lauryl Tryptose Broth (M76), at single strength in 10 ml volumes. For 5-tube MPN analysis, inoculate the 5 tubes at each dilution as follows:

    To each of 5 tubes, add 2 mL of the blended homogenate (equivalent to 1 g of shellfish).

    To each of 5 tubes, add 1 mL of 1:10 dilution of homogenate (0.1 g shellfish).

    To each of 5 tubes, add 1 mL of 1:100 dilution of homogenate (0.01 g shellfish).

    To each of 5 tubes, add 1 mL of 1:1000 dilution of homogenate (0.001 g shellfish).

    Further dilutions may be necessary to avoid indeterminate results. Incubate tubes at 35°C ± 0.5°C then follow instructions in section 1.C and perform Confirmed test as in 1.D above, under "Conventional Method for Coliforms, fecal coliforms and E. coli". Calculate MPN as described in section 1.D above, except that shellfish analysis specifies that the coliform density be expressed as MPN per 100 g of sample rather than per g.

  3. MPN - Presumptive and Confirmed Test for Fecal Coliforms in Shellfish

     Perform presumptive test as described in section II above. To confirm positive tubes, transfer one loopful from gas positive LST tubes to EC broth and incubate in a covered circulating waterbath at 44.5°±0.2°C for 24 ± 2 hr. Gas production in EC is a positive confirmed test for fecal coliforms. Calculate the MPN per 100 g for fecal coliforms as described above for coliform.

  4. MPN - EC-MUG Method for Determining E. coli in Shellfish Meats

    The MUG assay for β-glucuronidase (GUD) described above for detecting E. coli in chilled and frozen food can also be used for testing for E. coli in shellfish meats; but with slight modifications. This is due to the fact that foods such as shellfish meats contain natural GUD activity (32). As a result, oyster homogenate inoculated directly into LST-MUG tubes in the Presumptive phase of the MPN test can cause false positive fluorescence reactions. Hence, in the analysis of E. coli in shellfish meats, the MUG reagent is added to the EC medium and used in the confirmatory phase of the assay. The EC-MUG tubes, incubated at 44.5°C + 0.2°C, can be used in the confirmatory phase of a conventional 5-tube MPN assay to determine fecal coliform levels in shellfish meats, then by examining tubes for fluorescence under longwave UV, an E. coli MPN can also be readily obtained (32).

    See section 1.A and 1.B above for materials and reagents required. Use commercially prepared dehydrated EC-MUG, or prepare medium by adding MUG to EC broth (0.05 g/L) (M50). Several sources of MUG compound are suitable: Marcor Development Corp., Carlstadt, NJ; Biosynth International, Itasca, IL; Sigma Chemical Co., St. Louis, MO and Hach Chemical, Loveland, CO. Dispense 5 mL into new disposable borosilicate glass tubes (100 × 16 mm) containing, new disposable borosilicate glass Durham vials (50 × 9 mm) for gas collection. Sterilize EC-MUG broth tubes at 121°C for 15 min; store up to 1 week at room temperature or up to a month under refrigeration.

    Perform the 5-tube MPN Presumptive and Confirmed Test for Fecal Coliforms in Shellfish as described above in Section 3, except use EC-MUG tubes instead of EC for the confirmed test. Determination of fluorescence in EC-MUG broth requires the use of 3 control tubes, one inoculated with E. coli as positive control; one with Enterobacter aerogenes (ATCC 13048) or K. pneumoniae as negative control; and an uninoculated tube as EC-MUG medium batch control. Inoculate the positive and negative controls at the time when Confirmed test is being performed and incubate all tubes at 44.5°C ± 0.2°C for 24 h.

    Read fluorescence as described above under LST-MUG assay. Note that some (<10%)>E. coli are anaerogenic (gas-negative), but should be MUG-positive. Include all fluorescence positive tubes in the E. coli MPN calculations. Determine E. coli MPN/100g from the tables in the BAM (Appendix 2) using combination of fluorescence positive tubes at each dilution.

    NOTE: If analysis is to determine compliance with established E. coli limits, it will be necessary to confirm the presence of E. coli in MUG positive tubes.

V. Analysis for E. coli in citrus juices

Analysis for E. coli has been implemented to identify potentially contaminated juices or for verifying the effectiveness of HACCP during processing of unpasteurized juices (21 CFR Part 120, Vol. 66, No. 13, January 19, 2001). The standard method commonly used for testing for E. coli is the MPN however, it does not seem adequate for juice testing because of the acidity (pH 3.6 to 4.3) of juices, which can interfere with the test, plus it only allows for testing 3.33 mL of sample. Unlike most E. coli methods, which are enumeration assays, the following method is a simple Presence/Absence test that can examine 10-mL volume of juices (34, 35). This assay, designated as modified ColiComplete (CC) Method, is a modification of AOAC Official Method 992.30, which uses MUG for detection of E. coli (see Section on LST-MUG Method for details).

  1. Equipment and materials

    1. Covered water bath, with circulating system to maintain temperature of 44.5 ± 0.2°C.
      Water level should be above the medium in immersed tubes.
    2. Incubator, 35 ± 0.5°C
    3. Longwave UV light [~365 nm], not to exceed 6 W.
  2. Media and reagents:

    1. Universal Preenrichment Broth (UPEB) (M188) or can be purchased from BD(#223510)
    2. EC medium (M49)
    3. ColiComplete (CC) discs (#10800) - BioControl, Bellevue, WA
  3. Sample preparation, enrichment and analysis

    Perform assay in duplicate. Aseptically, inoculate 10-mL portion of juice into 90 mL of UPEB and incubate at 35°C ± 0.5°C for 24 h. After enrichment, mix and transfer 1-mL from each UPEB enrichment broth into 9 mL of EC broth containing a CC disc. Incubate EC/CC broth tubes at 44.5± 0.2°C in a circulating water bath for 24 ± 2 h. Include a tube inoculated with a MUG (+) E. coli strain as positive control and another with K. pneumoniae or Enterobacter aerogenes (ATCC 13048) as negative control. Examine tubes in the dark and under long wave UV light. The presence of blue fluorescence in either tube is indicative that E. coli is present in the sample. Note: The CC discs also contain X-gal, which when cleaved by β-galactosidase will yield blue color on or around the disc. This reaction is analogous to measuring acid/gas production from fermentation of lactose hence, the presence of blue color is indicative of coliforms.

VI. Other Methods for Enumerating Coliforms and E. coli

There are many other methods for enumerating coliforms and E. coli , including several that uses fluorogenic reagents like MUG or other chromogenic substrates for presumptive detection and identification of coliform and E. coli in foods. Many of these tests, such as the Petrifilm dry rehydratable film, the hydrophobic grid membrane filter/MUG (HGMF/MUG) method (13), ColiComplete disc (16), Colilert (AOAC 991.15), have been evaluated by collaborative studies and adopted as official first or final action by the AOAC. There are also many modifications of the membrane filtration assays that have been developed for testing for coliform, fecal coliform and E. coli and some of these may be useful in testing foods such as milk and beverages, but they are used mostly for water, environmental waters, and shellfish harvest waters analysis (5, 7, 20, 22, 23, 31).


References

  1. American Public Health Association. 1970. Recommended Procedures for the Examination of Seawater and Shellfish, 4th ed. APHA, Washington, DC.
  2. American Public Health Association. 1992. In: Marshall, R.T. (ed). Standard Methods for the Examination of Dairy Products, 16th ed. APHA. Washington, DC.
  3. American Public Health Association. 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. APHA, Washington, DC.
  4. American Public Health Association. 1992. Compendium of Methods for the Microbiological Examination of Foods, 3rd ed. APHA, Washington, DC.
  5. Brenner, K. P., C. C. Rankin, M. Sivaganesan, and P.V. Scarpino. 1996. Comparison of the recoveries of Escherichia coli and total coliforms from drinking water by the MI agar method and the U.S. Environmental protection agency-approved membrane filter method. Appl. Environ. Microbiol.62:203-208.
  6. Caplenas, N.R. and M.S. Kanarek. 1984. Thermotolerant non-fecal source Klebsiella pneumoniae: validity of the fecal coliform test in recreational waters. Am. J. Public Health.74:1273-1275
  7. Ciebin, B.W., M.H. Brodsky, R. Eddington, G. Horsnell, A. Choney, G. Palmateer, A. Ley, R. Joshi, and G. Shears. 1995. Comparative evaluation of modified m-FC and m-TEC media for membrane filter enumeration of Escherichia coli in water. Appl. Environ. Microbiol.61:3940-3942.
  8. Chang, G.W., J. Brill, and R. Lum. 1989. Proportion of beta-glucuronidase-negative Escherichia coli in human fecal samples. Appl. Environ. Microbiol.55:335-339.
  9. Conway, P.L. 1995. Microbial ecology of the human large intestine. In: G.R. Gibson and G.T. Macfarlane, eds. p.1-24. Human colonic bacteria: role in nutrition, physiology, and pathology. CRC Press, Boca Raton, FL.
  10. Dege, N.J. 1998. Categories of bottled water. Chapter 3, In: D.A.G. Senior and P. R. Ashurst (ed). Technology of Bottled Water. CRC Press, Boca Raton, Florida.
  11. Doyle, M.P. and J.L. Schoeni. 1987. Isolation of Escherichia coli O157:H7 from retail meats and poultry. Appl. Environ. Microbiol.53:2394-2396.
  12. Eijkman, C. 1904. Die garungsprobe bei 46° als hilfsmittel bei der trinkwasseruntersuchung. Zentr. Bakteriol. Parasitenk. Abt. I. Orig.37:742.
  13. Entis, P. 1989. Hydrophobic grid membrane filter/MUG method for total coliform and Escherichia coli enumeration in foods: collaborative study. J. Assoc. Off. Anal. Chem.72:936-950.
  14. Escherich, T. 1885. Die darmbakterien des neugeborenen und sauglings. Fortshr. Med.3:5-15-522, 547-554.
  15. Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Elsevier, New York.
  16. Feldsine, P.T., M.T. Falbo-Nelson, and D.L. Hustead. 1994. ColiComplete Substrate-supporting disc method for confirmed detection of total coliforms and Escherichia coli in all foods: comparative study. J. Assoc. Off. Anal.Chem.77:58-63.
  17. Feng, P. 1995. Escherichia coli serotype O157:H7: Novel vehicles of infection and emergence phenotypic variants. Emerging Infectious Dis.1:16-21.
  18. Feng, P.C.S. and P.A. Hartman. 1982. Fluorogenic assays for immediate confirmation of Escherichia coli. Appl. Environ. Microbiol.43:1320-1329.
  19. Feng, P., R. Lum, and G. Chang. 1991. Identification of uidA gene sequences in beta-D-glucuronidase (-) Escherichia coli. Appl. Environ. Microbiol.57:320-323.
  20. FDA. 1998. Fish and Fisheries Products Hazards and Control Guide. 2 nd ed. Office of Seafood, CFSAN, U.S. FDA, Public Health Service, Dept. Health and Human Services, Washington DC.
  21. Frampton, E.W. and L. Restaino. 1993. Methods for E. coli identification in food, water and clinical samples based on beta-glucuronidase detection. J. Appl. Bacteriol.74:223-233.
  22. Geissler, K., M. Manafi, I. Amoros, and J.L. Alonso. 2000. Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media. J. Appl. Microbiol.88:280-285.
  23. Grant, M.A. 1997. A new membrane filtration medium for simultaneous detection and enumeration of Escherichia coli and total coliforms. Appl. Environ. Microbiol.63:3526-4530.
  24. Gunzer, F., H. Bohm, H. Russmann, M. Bitzan, S. Aleksic, and H. Karch. 1992. Molecular detection of sorbitol fermenting Escherichia coli O157 in patients with hemolytic uremic syndrome. J. Clin. Microbiol.30:1807-10.
  25. Hartman, P.A. 1989. The MUG (glucuronidase) test for Escherichia coli in food and water, pp. 290-308. In: Rapid Methods and Automation in Microbiology and Immunology. A. Balows, R.C. Tilton, and A. Turano (eds). Brixia Academic Press, Brescia, Italy.
  26. Hayes, P.S., K. Blom, P. Feng, J. Lewis, N.A. Strockbine, and B. Swaminathan. 1995. Isolation and characterization of a β-D-glucuronidase-producing strain of Escherichia coli O157:H7 in the United States. J. Clin. Microbiol.33:3347-3348.
  27. Manafi, M. 1996. Fluorogenic and chromogenic enzyme substrates in culture media and identification tests. Int. J. Food Microbiol.31:45-58.
  28. Moberg, L.J., M.K. Wagner, and L.A. Kellen. 1988. Fluorogenic assay for rapid detection of Escherichia coli in chilled and frozen foods: collaborative study. J. Assoc. Off. Anal. Chem. 71:589-602.
  29. Neill, M. A., P. I. Tarr, D. N. Taylor, and A. F. Trofa. 1994. Escherichia coli. In Foodborne Disease Handbook, Y. H. Hui, J. R. Gorham, K. D. Murell, and D. O. Cliver, eds. Marcel Decker, Inc. New York. pp. 169-213.
  30. Neufeld, N. 1984. Procedures for the bacteriological examination of seawater and shellfish. In: Greenberg, A.E. and D.A. Hunt (eds). 1984. Laboratory Procedures for the Examination of Seawater and Shellfish, 5th ed. American Public Health Association. Washington, DC.
  31. Rippey, S.R., W.N. Adams, and W.D. Watkins. 1987. Enumeration of fecal coliforms and E. coli in marine andestuarine waters: an alternative to the APHA-MPN approach. J. Water Pollut. Control Fed.59:795-798.
  32. Rippey, S.R., L.A. Chandler, and W.D. Watkins. 1987. Fluorometric method for enumeration of Escherichia coli in molluscan shellfish. J. Food Prot.50:685-690, 710.
  33. Warburton, D.W. 2000. Methodology for screening bottled water for the presence of indicator and pathogenic bacteria. Food Microbiol.17:3-12.
  34. Weagant, S.D. and P. Feng. 2001. Comparative evaluation of a rapid method for detecting Escherichia coli in artificially contaminated orange juice. FDA Laboratory Information Bulletin #4239, 17:1-6.
  35. Weagant, S.D. and P. Feng. 2002. Comparative Analysis of a Modified Rapid Presence-Absence Test and the standard MPN Method for Detecting Escherichia coli in Orange Juice. Food Microbiol.19:111-115.

Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 4.

  • Content current as of:

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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.

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What kind of medium is MacConkey Agar?

Undefined, selective, and differential. Selects for Gram-Negative bacteria

What are the components of MacConkey Agar and what does each do?

Lactose - decreases the pH and differentiates organisms that ferment lactose from those that don't.
Bile salts and crystal violet - inhibit growth of Gram-positive bacteria.
Neutral red dye - a pH indicator that is colorless above a pH of 6.8 and red at a pH less than 6.8

What ingredient makes MacConkey Agar selective?

Bile salts and crystal violet select for Gram-negative bacteria

What ingredient makes MacConkey Agar differential?

Lactose differentiates organisms that ferment lactose from those that don't

What is MacConkey agar typically used for?

to isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose. (Gram-negative gut bacteria)

What would poor growth or no growth of a bacteria on a plate of MacConkey agar mean?

The organism is inhibited by crystal violet and/or bile and is Gram positive

What would good growth of a bacteria on a plate of MacConkey agar mean?

The organism is not inhibited by crystal violet or bile and is Gram negative

What would pink to red growth with or without bile precipitate of a bacteria on a plate of MacConkey agar mean?

The organism produces acid from lactose fermentation and is most likely a Gram negative coliform

What would colorless growth (not red or pink) of a bacteria on a plate of MacConkey agar mean?

The organism does not ferment lactose and is noncoliform

What organisms did we test on the MacConkey plate in lab and what were the results?

Escherichia coli - red growth
Staphylococcus epidermidis - no growth
Providencia stuartii - no growth

What kind of medium is Eosin Methylene Blue Agar?

undefined, selective, and differential. Selects for Gram-negative bacteria.

What are the components of Eosin Methylene Blue Agar and what does each do?

Digests of gelatin (peptone, sucrose, and lactose) - provides nitrogen and organic carbon; provide fermentable carbohydrates for fecal coliforms.
Eosin Y and Methylene blue dyes - inhibit the growth of most Gram-positive organisms and they react with vigorous lactose fermenters whose acid end products turn the growth dark purple or black.

What is Eosin Methylene Blue Agar typically used for?

It is used for the isolation of fecal coliforms - it encourages the growth of Gram-negative Enterobacteria ("gut" bacteria)

What component makes Eosin Methylene Blue Agar selective?

Eosin Y and Methylene blue dyes

What component makes Eosin Methylene Blue Agar differential?

What microbes did we test on the Eosin Methylene Blue Agar in lab and what were the results?

Escherichia coli - dark growth with metallic green
Enterobacter aerogenes - pink growth
Staphylococcus epidermidis - very light growth
Proteus vulgaris - grayish growth

Bacteria that results in poor growth or no growth on EMB Agar means what?

The organism is inhibited and is Gram-positive

Bacteria that results in good growth on EMB Agar means what?

The organism is not inhibited and is Gram-negative

Bacteria that results in growth that is pink and mucoid on EMB Agar means what?

The organism ferments lactose with little acid production and is a possible coliform

Bacteria that results in growth that is "dark" (purple to black, with or without green metallic sheen) on EMB Agar means what?

The organism ferments lactose with acid production and is a coliform

Bacteria that results in "colorless" growth (not purple, green, red, or pink) on EMB Agar means what?

The organism does not ferment lactose and is noncoliform.

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Last updated on June 21st, 2021

Eosin Methylene Blue (EMB) agar is both a selective and differential culture medium. It selectively promotes the growth of Gram-negative bacteria (inhibits Gram-positive bacteria) and aids in the differentiation of lactose fermenter and non-lactose fermenting colonies.

EMB Agar

EMB agar, first described by Holt-Harris and Teague, contained lactose and sucrose as source of carbohydrates. Levine further modified the medium by adding peptone and phosphate and removed sucrose from the formula and increased the lactose content. This aided in the differentiation of fecal and non-fecal types of the coliforms and also salmonellae and other non-lactose fermenters from the coliforms.

Another commonly used media for selective isolation of Gram-negative rods and differentiation of the member of Enterobacteriaceaeas lactose fermenter and non-lactose fermenter is MacConkey Agar. 

Contents

Principle

EMB agar contains sucrose and lactose, utilized as fermentable carbohydrates substrates, which encourage the growth of some gram-negative bacteria, especially fecal and non-fecal coliforms. Differentiation of enteric bacteria is possible due to the presence of the sugars lactose and sucrose in the EMB agar and the ability of certain bacteria to ferment the lactose in the medium.

  • Lactose-fermenting gram-negative bacteria acidify the medium, which reduces the pH, and the dye produces a dark purple complex usually associated with a green metallic sheen. This metallic green sheen is an indicator of vigorous lactose and/or sucrose fermentation ability typical of fecal coliforms.
  • Organisms that are slow lactose-fermenters, produce less acid, and the colonies appear brown-pink.
  • Non-lactose fermenters, increase the pH of the medium by deamination of proteins and produce colorless or light pink colonies.

Eosin Y and methylene blue are pH indicator dyes that combine to form a dark purple precipitate at low pH; they also serve to inhibit the growth of most Gram-positive organisms. Peptic digest of animal tissue serves as a source of carbon, nitrogen, and other essential growth nutrients. Phosphate buffers the medium.

Composition of EMB Agar

The composition of EMB agar and modified EMB agar (Levine EMB) agar differs slightly. Levine modification contains 10g of lactose (twice as in EMB agar) and contains no sucrose.

IngredientsEMB agar (gm/L)Levine EMB agar (gm/L)
Peptone10 g10g
Lactose5 g10g
Sucrose5g
Dipottasium,PO42g2g
Agar13.5g13.5g
Eosin Y0.4g0.4g
Methylene blue0.065g0.065g

Preparation of EMB agar

  1. Weigh and suspend 35.96 grams of dehydrated media in 1000 ml distilled water.
  2. Mix until the suspension is uniform and 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, and with frequent gentle swirling, pour the media into sterile Petri plates.
    Note: frequent swirling is recommended to restore the blue color o methylene blue and to suspend the flocculent precipitate if any.
  5. Label with initials of the name of the medium, date of preparation, and store the plates upside down (lids below) in the refrigerator until use.

Colony morphology

OrganismColonial appearance on EMB agar
Escherichia coliColonies are 2-3 mm in diameter, and have greenish metallic sheen in reflected light, dark or even black centre in transmitted light
Enterobacter aerogenesColonies are 4-6mm in diameter, raised and mucoid, tending to become confluent.
No metallic sheen, grey-brown centers by transmitted light
Salmonella and Shigella sppTranslucent and colorless colonies

Pseudomonas sppColorless irregular colonies
Proteus sppColorless colonies
Gram positive cocciPartially inhibited or no growth
Coagulase-positive staphylococciColorless, “pin-point” colonies on modified EMB

Quality control of EMB agar

Sterility testing can be performed by incubating 3-5% uninoculated plates from each batch at 37°C for 18-24 hours. Any growth on the media should be regarded as contamination and the whole lot should be discarded.

Performance testing of prepared EMB agar plates can be done by inoculating known strains of bacteria into the medium and observing growth and colonial characteristics.

OrganismGrowth and colony characteristics
E.coli ATCC 25922Good growth, blue-black colonies with a green metallic sheen
Salmonella choleraesuis subsp. Choleraesuis serotype Typhimurium ATCC 14028Luxuriant growth, colorless to amber colonies
Enterococcus faecalis ATCC 29212Inhibition (partial)
Shigella flexneri ATCC 12022Moderate to heavy growth, colorless to amber colonies

Uses of EMB agar

Isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli.

  1. EMB agar is used in water quality tests to distinguish coliforms and fecal coliforms that signal possible pathogenic microorganism contamination in water samples (presence of E.coli in the river/water sample indicates the possibility of fecal contamination of water so does the presence of other pathogenic enterics).
  2. EMB media assists in the visual distinction ofEscherichia coli, other nonpathogenic lactose-fermenting enteric gram-negative rods, and the Salmonella and Shigella genera. Escherichia coli colonies grow with a metallic sheen with a dark center. Aerobacter aerogenes colonies have a brown center, and non-lactose-fermenting gram-negative bacteria appear pink.
  3. EMB agar is also used to differentiate the organisms in the colon-typhoid-dysentery group. For culture of Salmonella and Shigella, selective medium such as MacConkey agar and EMB agar is commonly used.
  4. Levine EMB Agar can be used for the isolation and identification of Candida albicans from clinical specimens. Addition of 0.1g/L of chlortetracycline hydrochloride after autoclaving makes the medium selective by inhibiting the accompanying bacterial flora. The culture medium then is blue in color. Colonies of Candida albicans appear `spidery’ or `feathery’ after 24 to 48 hours of incubation at 35°C in 10% carbon dioxide. Other Candida species produce smooth yeast-like colonies.

References and further readings

  • ASMscience

    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) nonfermenter

    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)

    Bacteriological Profile and Antimicrobial Susceptibility Patterns of Bacteria Isolated from Pus/Wound Swab Samples from Children Attending a Tertiary Care Hospital in Kathmandu, Nepal


    Yellow colony on mannitol salt agar, Gram-positive cocci, catalase positive, slide and tube coagulase positive, gelatin hydrolysis positive, beta-hemolysis in blood agar, methyl red test and Voges-Proskauer test positive, nitrate reduction positiveS. aureus

    Gram-positive cocci, catalase negative, beta-hemolysis in blood agar, bacitracin sensitive, bile-esculin test negative, L-pyrrolidonyl-β-naphthylamide (PYR) test positiveS. pyogenes

    Gram-negative bacilli, oxidase positive, catalase positive, greenish yellow colonies on nutrient agar, citrate positive, motile, indole negative, urease negative, lactose non-fermenting, hydrogen sulphide negative, gas negativeP. aeruginosa

    Gram-negative bacilli, catalase emb agar test results, oxidase negative, mucoid colony, nonmotile, urease positive, citrate positive, indole negative, lactose fermenting, gas positive, hydrogen sulphide negativeK. pneumoniae

    Gram-negative bacilli, catalase positive, oxidase negative, motile, urease negative, citrate negative, indole positive, lactose fermenting, gas positive, hydrogen sulphide negativeE. coli

    Gram-negative bacilli, catalase positive, oxidase negative, citrate positive, lactose non-fermenting, indole negative, nonmotile, gas negative, hydrogen sulphide negativeAcinetobacter spp.

    Gram-negative bacilli, oxidase negative, catalase positive, lactose fermenting, indole negative, motile, urease differential, hydrogen sulphide positive, gas positive, citrate positiveC. freundii

    Gram-negative bacilli, oxidase negative, catalase positive, lactose emb agar test results, indole positive, motile, urease differential, hydrogen sulphide negative, gas positive, citrate positiveC. koseri

    Gram-negative bacilli, oxidase negative, catalase positive, lactose non-fermenting, indole negative, motile, urease positive, hydrogen sulphide positive, gas positive, citrate positiveP. mirabilis

    Источник: https://www.hindawi.com/journals/ijmicro/2017/2529085/tab1/

    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 pg county maryland 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 emb agar test results 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 Emb agar test results 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 emb agar test results, 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, emb agar test results 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/

    Eosin Methylene Blue (EMB) Agar : Principle ,purpose and colonies characteristics

    EMB Agar culture plates

    Article available in PDF 




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

    in medical laboratories,This medium is important by distinguishing pathogenic microbes in a short period of time.
    It emb agar test results it encourages some bacteria to grow while inhibiting others.  Eosin Y and Methylene Blue generally inhibits Gram positive bacteria from growing (a few will grow) and generally allow Gram negative organism to grow (some will not grow).
    • If good growth, generally you have a Gram negative bacteria.  
    • If no growth or poor growth, you most likely have a Gram positive.

    PRINCIPLE OF EMB

    The Rapid lactose fermentation by these bacteria 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. This medium has been specifically designed to discourage the growth of gram positive bacteria.Hence EMB is highly differentiale and can be use as a substitute to MacConket Agar.

    Purpose:  to select for and isolate Gram negative organisms, to select for and isolate coliforms, and to differentiate among the family of Zions bank lobby hours Its main use is to isolate fecal coliforms and to detect for fecal contamination.  Both the Gram negative selection and the detection of coliforms is imperfect, a small percentage of strains do not act as expected.

    Also Read:

    EMB contains the following ingredients for it Composition

    composition of EMB agar
    1. Enzymatic Digest of gelatin
    2. Lactose: Sugar, helps to differentiate lactose fermenter from non lactose fermenter
    3. Dipotassium Phosphate
    4. Eosin Y: Indicator
    5. Methylene Blue: pH indicator
    6. Agar

    Procedure:  

    1. Appropriately label you plates.  Mark the dish bottom into thirds and label what species will be in each section.  It is best to label the side of the bottom plate or write on the bottom in tiny letters so that you will be able to observe the growth clearly.
    2. Inoculate one third with your unknown streak for isolated colonies if possible.  Isolated colonies work best for this test but it is not essential.
    3. Inoculate one third with E. coli, and the other third with S. epidermidis.  Generally it is best to keep E. coli well away from the other cultures because it may overgrow them.  (Streak only one species/third, don't mix the bacteria!  See later for how to do this quickly.)
    4. You will want to compare the growth on these plates to growth inoculated on a general purpose media such as NA. or TSA.
    5. Incubate your plates upside down in the incubator at 35-37 C for 1-2 days.  If possible, exam plates at both 1 and 2 days.
    6. Slow growing species may require a day or two of additional emb agar test results your growth on these plates to growth on a general purpose media.  (If no growth on the general purpose media, discard your results and repeat the test.)  Look for the presence or absence of growth, and if there is growth if it is reduced from normal.  
    7. If there is growth, check to see the color of the growth.  Is the growth essentially colorless, bright pink, purplish, or greenish with a metallic sheen?  To obtain your results, use the darkest color areas which may be only in the centers of the colonies.
    8. One predicts that if an organism grows well on EMB Agar, it is most likely Gram negative, otherwise it is most likely Gram positive.  And if the growth is good and pink or darker, it is most likely a coliform, otherwise it is likely a noncoliform.  If the colonies are darker than pink (purplish, greenish with a metallic sheen, or even blackish), the organisms highly utilize the sugars lactose/sucrose and have highly lowered the pH.

    Differentiation between gram negative bacilli  base on colony growth on EMB AGAR

    Growing on EMB agar

    These plates differentiate between species and their ability to lower the pH.

    • If growth is colorless, off-white or a very light and dull pink, the pH has largely been unaffected:  is probably a Gram negative noncoliform.
    • If good growth that is pink, sugar utilization has slightly lowered the pH:  is probably a Gram negative coliform.
    • If good growth that is purplish or has a purple center, sugar utilization has lowered the pH:  is probably a Gram negative coliform.
    • If good growth with a greenish metallic sheen, strong sugar utilization has greatly lowered the pH:  is probably a Gram negative coliform.
    • (If poor or no growth, recall it is probably a Gram positive organism.

    Quality Control 

    Inorder to confirm the tentative conclusions from this culture media,  performe a Gram stain and testing for lactose and sucrose utilization.  Other species of bacteria may be used as positive and negative controls.  

    • E. coli will have good purplish or greenish or blackish growth (with or without a metallic sheen),
    • Enterbacter aerogenes will have good pink or purplish growth, 
    • S. epidermidis will grow poorly if at all. 

     And yes, the same species can produce different colors depending on how far the pH shifts. 

    EMB agar is emb agar test results moderately inhibitive to many Gram positives and yeasts while some strains of Salmonella and Shigella may not grow well. 

    The greenish metallic sheen does not always appear on strains that produce it. 

     Sterilization of this media reduces the methylene blue and creates a precipitate which may be oxidized back and dispersed respectively by mixing the media.

    Article available in PDF 




    Source http://spot.pcc.edu/



    Источник: https://mltexpo.blogspot.com/2018/04/eosin-methylene-blue-emb-agar-principle.html

    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 bank of america wire transfer routing number california 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 blue ridge bank and trust co raytown mo 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 meredith village savings bank alton nh. 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

    What kind of medium is MacConkey Agar?

    Undefined, selective, and differential. Selects for Gram-Negative bacteria

    What are the components of MacConkey Agar and what does each do?

    Emb agar test results - decreases the pH and differentiates organisms that ferment lactose from those that don't.
    Bile salts and crystal violet - inhibit growth of Gram-positive bacteria.
    Neutral red dye - a pH indicator that is colorless above a pH of 6.8 and red at a pH less than 6.8

    What ingredient makes MacConkey Agar selective?

    Bile salts and crystal violet select for Gram-negative bacteria

    What ingredient makes MacConkey Agar differential?

    Lactose differentiates organisms that ferment lactose from those that don't

    What is MacConkey agar typically used for?

    to isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose. (Gram-negative gut bacteria)

    What would poor growth or no growth of a bacteria on a plate of MacConkey agar mean?

    The organism is inhibited by crystal violet and/or bile and is Gram positive

    What would good growth of a bacteria on a plate of MacConkey agar mean?

    The organism is not inhibited by crystal violet or bile and is Gram negative

    What would pink to red growth with or without bile precipitate of a bacteria on a plate of MacConkey agar mean?

    The organism produces acid from lactose fermentation and is most likely a Gram negative coliform

    What would colorless growth (not red or pink) of a bacteria on a plate of MacConkey agar mean?

    The organism does not ferment lactose and is noncoliform

    What organisms did we test on the MacConkey plate in lab and what were the results?

    Escherichia coli - red growth
    Staphylococcus epidermidis - no growth
    Providencia stuartii - no growth

    What kind of medium is Eosin Methylene Blue Agar?

    undefined, selective, and differential. Selects for Gram-negative bacteria.

    What are the components of Eosin Methylene Blue Agar and what does each do?

    Digests of gelatin (peptone, sucrose, and lactose) - provides nitrogen and organic carbon; provide fermentable carbohydrates for the giving keys inc coliforms.
    Eosin Y and Methylene blue dyes - inhibit the growth of most Gram-positive organisms and they react with vigorous lactose fermenters whose acid end products turn the growth dark purple or black.

    What is Eosin Methylene Blue Agar typically used for?

    It is used for the isolation of fecal coliforms - it encourages the growth of Gram-negative Enterobacteria ("gut" bacteria)

    What component makes Eosin Methylene Blue Emb agar test results selective? alliance bank housing loan calculator Eosin Y and Methylene blue dyes

    What component makes Eosin Methylene Blue Agar differential?

    What microbes did we test on the Eosin Methylene Blue Agar in lab and what were the results?

    Escherichia coli - dark growth with metallic green
    Enterobacter aerogenes - pink growth
    Staphylococcus epidermidis - very light growth
    Proteus vulgaris - grayish growth

    Bacteria that results in poor growth or no growth on EMB Agar means what?

    The organism is inhibited and is Gram-positive

    Bacteria that results in good growth on EMB Agar means what?

    The organism is not inhibited and is Gram-negative

    Bacteria that results in growth that is pink and mucoid on EMB Agar means what?

    The organism ferments lactose with little acid production and is a possible coliform

    Bacteria that results in growth that is "dark" (purple to black, with or without green metallic sheen) state bank of cross plains EMB Agar means what?

    The organism ferments lactose with acid production and emb agar test results a coliform

    Bacteria that results in "colorless" growth (not purple, green, red, or pink) on EMB Agar means what? volunteer state bank mobile app The organism does not ferment lactose and is noncoliform.

    Источник: https://www.brainscape.com/flashcards/lab-exam-2-ex-4-4-4-5-5520632/packs/7983568
    Eosin-Methylene Blue Agar Plates Protocol
    . Retrieved June 12, 2020, from https://www.asmscience.org/content/education/protocol/protocol.2869
  • Welcome to Microbugz—Eosin Methylene Blue Agar. Retrieved June 12, 2020, from https://www.austincc.edu/microbugz/eosin_methylene_blue_agar.php
  • Eosin Methylene Blue Agar (EMB). (2016, April 12). Biology LibreTexts.

Related

Источник: https://microbeonline.com/eosin-methylene-blue-emb-agar-composition-uses-colony-characteristics/

Emb agar test results -

Bacteriological Profile and Antimicrobial Susceptibility Patterns of Bacteria Isolated from Pus/Wound Swab Samples from Children Attending a Tertiary Care Hospital in Kathmandu, Nepal


Yellow colony on mannitol salt agar, Gram-positive cocci, catalase positive, slide and tube coagulase positive, gelatin hydrolysis positive, beta-hemolysis in blood agar, methyl red test and Voges-Proskauer test positive, nitrate reduction positiveS. aureus

Gram-positive cocci, catalase negative, beta-hemolysis in blood agar, bacitracin sensitive, bile-esculin test negative, L-pyrrolidonyl-β-naphthylamide (PYR) test positiveS. pyogenes

Gram-negative bacilli, oxidase positive, catalase positive, greenish yellow colonies on nutrient agar, citrate positive, motile, indole negative, urease negative, lactose non-fermenting, hydrogen sulphide negative, gas negativeP. aeruginosa

Gram-negative bacilli, catalase positive, oxidase negative, mucoid colony, nonmotile, urease positive, citrate positive, indole negative, lactose fermenting, gas positive, hydrogen sulphide negativeK. pneumoniae

Gram-negative bacilli, catalase positive, oxidase negative, motile, urease negative, citrate negative, indole positive, lactose fermenting, gas positive, hydrogen sulphide negativeE. coli

Gram-negative bacilli, catalase positive, oxidase negative, citrate positive, lactose non-fermenting, indole negative, nonmotile, gas negative, hydrogen sulphide negativeAcinetobacter spp.

Gram-negative bacilli, oxidase negative, catalase positive, lactose fermenting, indole negative, motile, urease differential, hydrogen sulphide positive, gas positive, citrate positiveC. freundii

Gram-negative bacilli, oxidase negative, catalase positive, lactose non-fermenting, indole positive, motile, urease differential, hydrogen sulphide negative, gas positive, citrate positiveC. koseri

Gram-negative bacilli, oxidase negative, catalase positive, lactose non-fermenting, indole negative, motile, urease positive, hydrogen sulphide positive, gas positive, citrate positiveP. mirabilis

Источник: https://www.hindawi.com/journals/ijmicro/2017/2529085/tab1/

What kind of medium is MacConkey Agar?

Undefined, selective, and differential. Selects for Gram-Negative bacteria

What are the components of MacConkey Agar and what does each do?

Lactose - decreases the pH and differentiates organisms that ferment lactose from those that don't.
Bile salts and crystal violet - inhibit growth of Gram-positive bacteria.
Neutral red dye - a pH indicator that is colorless above a pH of 6.8 and red at a pH less than 6.8

What ingredient makes MacConkey Agar selective?

Bile salts and crystal violet select for Gram-negative bacteria

What ingredient makes MacConkey Agar differential?

Lactose differentiates organisms that ferment lactose from those that don't

What is MacConkey agar typically used for?

to isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose. (Gram-negative gut bacteria)

What would poor growth or no growth of a bacteria on a plate of MacConkey agar mean?

The organism is inhibited by crystal violet and/or bile and is Gram positive

What would good growth of a bacteria on a plate of MacConkey agar mean?

The organism is not inhibited by crystal violet or bile and is Gram negative

What would pink to red growth with or without bile precipitate of a bacteria on a plate of MacConkey agar mean?

The organism produces acid from lactose fermentation and is most likely a Gram negative coliform

What would colorless growth (not red or pink) of a bacteria on a plate of MacConkey agar mean?

The organism does not ferment lactose and is noncoliform

What organisms did we test on the MacConkey plate in lab and what were the results?

Escherichia coli - red growth
Staphylococcus epidermidis - no growth
Providencia stuartii - no growth

What kind of medium is Eosin Methylene Blue Agar?

undefined, selective, and differential. Selects for Gram-negative bacteria.

What are the components of Eosin Methylene Blue Agar and what does each do?

Digests of gelatin (peptone, sucrose, and lactose) - provides nitrogen and organic carbon; provide fermentable carbohydrates for fecal coliforms.
Eosin Y and Methylene blue dyes - inhibit the growth of most Gram-positive organisms and they react with vigorous lactose fermenters whose acid end products turn the growth dark purple or black.

What is Eosin Methylene Blue Agar typically used for?

It is used for the isolation of fecal coliforms - it encourages the growth of Gram-negative Enterobacteria ("gut" bacteria)

What component makes Eosin Methylene Blue Agar selective?

Eosin Y and Methylene blue dyes

What component makes Eosin Methylene Blue Agar differential?

What microbes did we test on the Eosin Methylene Blue Agar in lab and what were the results?

Escherichia coli - dark growth with metallic green
Enterobacter aerogenes - pink growth
Staphylococcus epidermidis - very light growth
Proteus vulgaris - grayish growth

Bacteria that results in poor growth or no growth on EMB Agar means what?

The organism is inhibited and is Gram-positive

Bacteria that results in good growth on EMB Agar means what?

The organism is not inhibited and is Gram-negative

Bacteria that results in growth that is pink and mucoid on EMB Agar means what?

The organism ferments lactose with little acid production and is a possible coliform

Bacteria that results in growth that is "dark" (purple to black, with or without green metallic sheen) on EMB Agar means what?

The organism ferments lactose with acid production and is a coliform

Bacteria that results in "colorless" growth (not purple, green, red, or pink) on EMB Agar means what?

The organism does not ferment lactose and is noncoliform.

Источник: https://www.brainscape.com/flashcards/lab-exam-2-ex-4-4-4-5-5520632/packs/7983568

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

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/

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) nonfermenter

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)

Bacteriological Analytical Manual (BAM) Main Page

Authors: Peter Feng (ret.), Stephen D. Weagant (ret.), Michael A. Grant (dec.), William Burkhardt

Revision History

  • October 2020 - Section I A.3 modified to reflect that enrichment should take place at 35 ± 0.5⁰C and not at 35 ± 1⁰C.
  • July 2017 - Chap. 4 Sec. I. E. For the completed phase of testing for E. coli, the incubation temperature of EC tubes has been changed from 45.5 ± 0.2°C to 44.5 ± 0.2°C. The change was made in part due to the poor ability of the control strain ATCC25922 to grown and ferment lactose to produce acid and gas at 45.5 ± 0.2°C. The use of 44.5 ± 0.2°C would also make it consistent with that used for fecal Coliform analysis in shellfish and shellfish meats (Sec. VI) as well as conditions used for E. coli testing by other International organizations.
  • February 2013 - Shellfish analysis method revised to be consistent with the APHA Examination of seawater and shellfish, 4th ed.
  • February 2013 - Membrane filter methods added to water analysis.

Chapter Contents


Escherichia coli, originally known as Bacterium coli commune, was identified in 1885 by the German pediatrician, Theodor Escherich (14, 29). E. coli is widely distributed in the intestine of humans and warm-blooded animals and is the predominant facultative anaerobe in the bowel and part of the essential intestinal flora that maintains the physiology of the healthy host (9, 29). E. coli is a member of the family Enterobacteriaceae (15), which includes many genera, including known pathogens such as Salmonella, Shigella, and Yersinia. Although most strains of E. coli are not regarded as pathogens, they can be opportunistic pathogens that cause infections in immunocompromised hosts. There are also pathogenic strains of E. coli that when ingested, causes gastrointestinal illness in healthy humans (see Chap. 4A).

In 1892, Shardinger proposed the use of E. coli as an indicator of fecal contamination. This was based on the premise that E. coli is abundant in human and animal feces and not usually found in other niches. Furthermore, since E. coli could be easily detected by its ability to ferment glucose (later changed to lactose), it was easier to isolate than known gastrointestinal pathogens. Hence, the presence of E. coli in food or water became accepted as indicative of recent fecal contamination and the possible presence of frank pathogens. Although the concept of using E. coli as an indirect indicator of health risk was sound, it was complicated in practice, due to the presence of other enteric bacteria like Citrobacter, Klebsiella and Enterobacter that can also ferment lactose and are similar to E. coli in phenotypic characteristics, so that they are not easily distinguished. As a result, the term "coliform" was coined to describe this group of enteric bacteria. Coliform is not a taxonomic classification but rather a working definition used to describe a group of Gram-negative, facultative anaerobic rod-shaped bacteria that ferments lactose to produce acid and gas within 48 h at 35°C. In 1914, the U.S. Public Health Service adopted the enumeration of coliforms as a more convenient standard of sanitary significance.

Although coliforms were easy to detect, their association with fecal contamination was questionable because some coliforms are found naturally in environmental samples (6). This led to the introduction of the fecal coliforms as an indicator of contamination. Fecal coliform, first defined based on the works of Eijkman (12) is a subset of total coliforms that grows and ferments lactose at elevated incubation temperature, hence also referred to as thermotolerant coliforms. Fecal coliform analyses are done at 45.5°C for food testing, except for water, shellfish and shellfish harvest water analyses, which use 44.5°C (1, 3, 30). The fecal coliform group consists mostly of E. coli but some other enterics such as Klebsiella can also ferment lactose at these temperatures and therefore, be considered as fecal coliforms. The inclusion of Klebsiella spp in the working definition of fecal coliforms diminished the correlation of this group with fecal contamination. As a result, E. coli has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli.

Currently, all 3 groups are used as indicators but in different applications. Detection of coliforms is used as an indicator of sanitary quality of water or as a general indicator of sanitary condition in the food-processing environment. Fecal coliforms remain the standard indicator of choice for shellfish and shellfish harvest waters; and E. coli is used to indicate recent fecal contamination or unsanitary processing. Almost all the methods used to detect E. coli, total coliforms or fecal coliforms are enumeration methods that are based on lactose fermentation (4). The Most Probable Number (MPN) method is a statistical, multi-step assay consisting of presumptive, confirmed and completed phases. In the assay, serial dilutions of a sample are inoculated into broth media. Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other 2 phases of the assay are performed, and then uses the combinations of positive results to consult a statistical table (Appendix 2), to estimate the number of organisms present. Typically only the first 2 phases are performed in coliform and fecal coliform analysis, while all 3 phases are done for E. coli. The 3-tube MPN test is used for testing most foods. Analysis of seawater using a multiple dilution series should not use less than 3 tubes per dilution (5 tubes are recommended); in certain instances a single dilution series using no less than 12 tubes may also be acceptable. (For additional details, see: FDA. National Shellfish Sanitation Program, Manual of Operations. 2009 Revision. DHHS/PHS/FDA, Washington DC). Likewise,  analysis of bivalve molluscan shellfish should be performed using a multiple dilution MPN series whereby no fewer than 5- tubes per dilution should be used, see section IV. There is also a 10-tube MPN method that is used to test bottled water or samples that are not expected to be highly contaminated (3).  Analysis of citrus juice for E. coli is performed as an absence/presence method, see section V.

Also, there is a solid medium plating method for coliforms that uses Violet Red Bile Agar, which contains neutral red pH indicator, so that lactose fermentation results in formation of pink colonies. There are also membrane filtration tests for coliform and fecal coliform that measure aldehyde formation due to fermentation of lactose. This chapter also includes variations of above tests that use fluorogenic substrates to detect E. coli (18), special tests for shellfish analysis, a brief consideration of bottled water testing and a method for testing large volumes of citrus juices for presence of E. coli in conjunction with the Juice HACCP rule.


I. Conventional Method for coliforms, fecal coliforms and E. coli

  1. Equipment and materials

    1. Covered water bath, with circulating system to maintain temperature of 44.5 ± 0.2°C.  The temperature for water baths for the shellfish program is 44.5°C ± 0.2°C. Water level should be above the medium in immersed tubes.
    2. Immersion-type thermometer, 1-55°C, about 55 cm long, with 0.1°C subdivisions, certified by National Institute of Standards and Technology (NIST), or equivalent Incubator, 35 ± 0.5°C.
    3. Balance with capacity of >2 kg and sensitivity of 0.1 g
    4. Blender and blender jar (see Chapter 1)
    5. Sterile graduated pipets, 1.0 and 10.0 mL
    6. Sterile utensils for sample handling (see Chapter 1)
    7. Dilution bottles made of borosilicate glass, with polyethylene screw caps equipped with Teflon liners. Commercially prepared dilution bottles containing sterile Butterfield's phosphate buffer can also be used.
    8. Quebec colony counter, or equivalent, with magnifying lens
    9. Longwave UV light [~365 nm], not to exceed 6 W.
    10. pH meter
  2. Media and Reagents

    1. Brilliant green lactose bile (BGLB) broth, 2% (M25)
    2. Lauryl tryptose (LST) broth (M76)
    3. Lactose Broth (M74)
    4. EC broth (M49)
    5. Levine's eosin-methylene blue (L-EMB) agar (M80)
    6. Tryptone (tryptophane) broth (M164)
    7. MR-VP broth (M104)
    8. Koser's citrate broth (M72)
    9. Plate count agar (PCA) (standard methods) (M124)
    10. Butterfield's phosphate-buffered water (R11) or equivalent diluent

      (Note: This same formulation is referred to as Buffered Dilution Water in American Public Health Association. 1970. Recommended Procedures for the Examination of Seawater and Shellfish, 4th ed. APHA, Washington, DC., p14-15)

    11. Kovacs' reagent (R38)
    12. Voges-Proskauer (VP) reagents (R89)
    13. Gram stain reagents (R32)
    14. Methyl red indicator (R44)
    15. Violet red bile agar (VRBA) (M174)
    16. VRBA-MUG agar (M175)
    17. EC-MUG medium (M50)
    18. Lauryl tryptose MUG (LST-MUG) broth (M77)
    19. Peptone Diluent, 0.5% (R97)
  3. MPN - Presumptive test for coliforms, fecal coliforms and E. coli

    Weigh 50 g of food into sterile high-speed blender jar (see Chapter 1 and current FDA compliance programs for instructions on sample size and compositing) Frozen samples can be softened by storing  for <18 h at 2-5°c, but do not thaw. Add 450 mL of Butterfield's phosphate-buffered water and blend for 2 min. If <50 g of sample are available, weigh portion that is equivalent to half of the sample and add sufficient volume of sterile diluent to make a 1:10 dilution. The total volume in the blender jar should completely cover the blades.

    Prepare decimal dilutions with sterile Butterfield's phosphate diluent or equivalent. Number of dilutions to be prepared depends on anticipated coliform density. Shake all suspensions 25 times in 30 cm arc or vortex mix for 7 s. Using at least 3 consecutive dilutions, inoculate 1 mL aliquots from each dilution into 3 LST tubes for a 3 tube MPN analysis (other analysis may require the use of 5 tubes for each dilution; See IV). Lactose Broth may also be used. For better accuracy, use a 1 mL or 5 mL pipet for inoculation. Do not use pipets to deliver<10% of their total volume; eg. a 10 mL pipet to deliver 0.5 mL. Hold pipet at angle so that its lower edge rests against the tube.  Not more than 15  min  should  elapse  from  time  the  sample is  blended  until  all  dilutions  are  inoculated  in appropriate media.

    Incubate LST tubes at 35°C± 0.5°C . Examine tubes and record reactions at 24 ± 2 h for gas, i.e., displacement of medium in fermentation vial or effervescence when tubes are gently agitated. Re-incubate gas-negative tubes for an additional 24 h and examine and record reactions again at 48 ± 3 h. Perform confirmed test on all presumptive positive (gas) tubes.

  4. MPN - Confirmed test for coliforms

    From each gassing LST or lactose broth tube, transfer a loopful of suspension to a tube of BGLB broth, avoiding pellicle if present. (a sterile wooden applicator stick may also be used for these transfers). Incubate BGLB tubes at 35°C ± 0.5°C and examine for gas production at 48 ± 3 h. Calculate most probable number (MPN) (see Appendix 2) of coliforms based on proportion of confirmed gassing LST tubes for 3 consecutive dilutions.

  5. MPN - Confirmed test for fecal coliforms and E. coli

    From each gassing LST or Lactose broth tube from the Presumptive test, transfer a loopful of each suspension to a tube of EC broth (a sterile wooden applicator stick may also be used for these transfers). Incubate EC tubes 24 ± 2 h at 44.5°C and examine for gas production. If negative, reincubate and examine again at 48 ± 2 h. Use results of this test to calculate fecal coliform MPN. To continue with E. coli analysis, proceed to Section F below. The EC broth MPN method may be used for seawater and shellfish since it conforms to recommended procedures (1).

  6. MPN - Completed test for E. coli.

    To perform the completed test for E. coli, gently agitate each gassing EC tube, remove a loopful of broth   and streak for isolation on a L-EMB agar plate and incubate for 18-24 h at 35°C ± 0.5°C . Examine plates for suspicious E. coli colonies, i.e., dark centered and flat, with or without metallic sheen. Transfer up to 5 suspicious colonies from each L-EMB plate to PCA slants, incubate them for 18-24 h at 35°C ± 0.5°C and use for further testing.

    NOTE: Identification of any 1 of the 5 colonies as E. coli is sufficient to regard that EC tube as positive; hence, not all 5 isolates may need to be tested.

    Perform Gram stain. All cultures appearing as Gram-negative, short rods should be tested for the IMViC reactions below and also re-inoculated back into LST to confirm gas production.

    Indole production. Inoculate tube of tryptone broth and incubate 24 ± 2 h at 35°C ± 0.5°C . Test for indole by adding 0.2-0.3 mL of Kovacs' reagent. Appearance of distinct red color in upper layer is positive test.

    Voges-Proskauer (VP)-reactive compounds. Inoculate tube of MR-VP broth and incubate 48 ± 2 h at 35°C± 0.5°C . Transfer 1 mL to 13 × 100 mm tube. Add 0.6 mL α-naphthol solution and 0.2 mL 40% KOH, and shake. Add a few crystals of creatine. Shake and let stand 2 h. Test is positive if eosin pink color develops.

    Methyl red-reactive compounds. After VP test, incubate MR-VP tube additional 48 ± 2 h at 35°C± 0.5°C . Add 5 drops of methyl red solution to each tube. Distinct red color is positive test. Yellow is negative reaction.

    Citrate. Lightly inoculate tube of Koser's citrate broth; avoid detectable turbidity. Incubate for 96 h at 35°C ± 0.5°C . Development of distinct turbidity is positive reaction.

    Gas from lactose. Inoculate a tube of LST and incubate 48 ± 2 h at 35°C ± 0.5°C . Gas production (displacement of medium from inner vial) or effervescence after gentle agitation is positive reaction.

    Interpretation: All cultures that (a) ferment lactose with gas production within 48 h at 35°C, (b) appear as Gram-negative nonsporeforming rods and (c) give IMViC patterns of ++-- (biotype 1) or -+-- (biotype 2) are considered to be E. coli. Calculate MPN (see Appendix 2) of E. coli based on proportion of EC tubes in 3 successive dilutions that contain E. coli.

    NOTE: Alternatively, instead of performing the IMViC test, use API20E or the automated VITEK biochemical assay to identify the organism as E. coli. Use growth from the PCA slants and perform these assays as described by the manufacturer.

  7. Solid medium method - Coliforms

    Prepare violet red bile agar (VRBA) according to manufacturer's instructions. Cool to 48°C before use. Prepare, homogenize, and decimally dilute sample as described in section I. C above so that isolated colonies will be obtained when plated. Transfer two 1 mL aliquots of each dilution to petri dishes, and use either of the following two pour plating methods, depending on whether injured or stressed cells are suspected to be present (1).

    Pour 10 mL VRBA tempered to 48°C into plates, swirl plates to mix, and let solidify. To prevent surface growth and spreading of colonies, overlay with 5 mL VRBA, and let solidify. If resuscitation is necessary, pour a basal layer of 8-10 mL of tryptic soy agar tempered to 48°C. Swirl plates to mix, and incubate at room temperature for 2 ± 0.5 h. Then overlay with 8-10 mL of melted, cooled VRBA and let solidify.

    Invert solidified plates and incubate 18-24 h at 35°C. Incubate dairy products at 32°C (2). Examine plates under magnifying lens and with illumination. Count purple-red colonies that are 0.5 mm or larger in diameter and surrounded by zone of precipitated bile acids. Plates should have 25-250 colonies. To confirm that the colonies are coliforms, pick at least 10 representative colonies and transfer each to a tube of BGLB broth. Incubate tubes at 35°C. Examine at 24 and 48 h for gas production.

    NOTE: If gas-positive BGLB tube shows a pellicle, perform Gram stain to ensure that gas production was not due to Gram-positive, lactose-fermenting bacilli.

    Determine the number of coliforms per gram by multiplying the number of suspect colonies by percent confirmed in BGLB by dilution factor.

    Alternatively, E. coli colonies can be distinguished among the coliform colonies on VRBA by adding 100 µg of 4-methyl-umbelliferyl-β-D-glucuronide (MUG) per mL in the VRBA overlay. After incubation, observe for bluish fluorescence around colonies under longwave UV light. (see LST-MUG section II for theory and applicability.)

  8. Membrane Filtration (MF) Method - coliforms: see Section III. Bottled Water.

    Food homogenates will easily clog filters, hence MF are most suitable for analysis of water samples; however, MF may be used in the analysis of liquid foods that do not contain high levels of particulate matter such as bottled water (see Section III for application of MF).

II. LST-MUG Method for Detecting E. coli in Chilled or Frozen Foods Exclusive of Bivalve Molluscan Shellfish

The LST-MUG assay is based on the enzymatic activity of β-glucuronidase (GUD), which cleaves the substrate 4-methylumbelliferyl β-D-glucuronide (MUG), to release 4-methylumbelliferone (MU). When exposed to longwave (365 nm) UV light, MU exhibits a bluish fluorescence that is easily visualized in the medium or around the colonies. Over 95% of E. coli produces GUD, including anaerogenic (non-gas-producing) strains. One exception is enterohemorrhagic E. coli (EHEC) of serotype O157:H7, which is consistently GUD negative (11, 17). The lack of GUD phenotype in O157:H7 is often used to differentiate this serotype from other E. coli, although GUD positive variants of O157:H7 do exist (24, 26). The production of GUD by other members of the family Enterobacteriaceae is rare, except for some shigellae (44 -58%) and salmonellae (20-29%) (18, 27). However, the inadvertent detection of these pathogens by GUD-based assays is not considered a drawback from a public health perspective. Expression of GUD activity is affected by catabolite repression (8) so on occasion, some E. coli are GUD-negative, even though they carry the uidA gene (gusA) that encodes for the enzyme (19). In most analyses however, about 96% of E. coli isolates tested are GUD-positive without the need for enzyme induction (27).

MUG can be incorporated into almost any medium for use in detecting E. coli. But some media such as EMB, which contain fluorescent components, are not suitable, as they will mask the fluorescence of MU. When MUG is incorporated into LST medium, coliforms can be enumerated on the basis of gas production from lactose and E. coli are presumptively identified by fluorescence in the medium under longwave UV light, thus it is capable of providing a presumptive identification of E. coli within 24 h (18, 28). The LST-MUG method described below has been adopted as Official Final Action by the AOAC for testing for E. coli in chilled or frozen foods, exclusive of shellfish (28). See Sec. IV.4. D. for precautions in using MUG in testing shellfish. For information on MUG assay contact, Dr. Bill Burkhardt III (email [email protected] ), FDA, CFSAN, Dauphin Island, AL, 36528; 251-406-8125

CAUTION: To observe for fluorescence, examine inoculated LST-MUG tubes under longwave (365 nm) UV light in the dark. A 6-watt hand-held UV lamp is adequate and safe. When using a more powerful UV source, such as a 15-watt fluorescent lamp, wear protective glasses or goggles. Also, prior to use in MUG assays, examine all glass tubes for auto fluorescence. Cerium oxide, which is sometimes added to glass as a quality control measure, will fluoresce under UV light and interfere with the MUG test (25). The use of positive and negative control strains for MUG reaction is essential.

  1. Equipment and material:see section I.A above and in addition,
    1. New, disposable borosilicate glass tubes (100 × 16 mm)
    2. New, disposable borosilicate glass Durham vials (50 × 9 mm) for gas collection
    3. Longwave UV lamp, not to exceed 6-watt
  2. Media and reagents:see section I.B above
  3. Presumptive LST-MUG test for E. coli.

Prepare food samples and perform the MPN Presumptive test as described in section I.C. above, except use LST-MUG tubes instead of LST. Be sure to inoculate one tube of LST-MUG with a known GUD-positive E. coli isolate as positive control (ATCC 25922). In addition, inoculate another tube with a culture of Enterobacter aerogenes (ATCC 13048) culture of Enterobacter aerogenes (ATCC 13048) or a Klebsiella pneumoniae strain as negative control, to facilitate differentiation of sample tubes that show only growth from those showing both growth and fluorescence. Incubate tubes for 24 to 48 ± 2 h at 35°C. Examine each tube for growth (turbidity, gas) then examine tubes in the dark under longwave UV lamp (365 nm). A bluish fluorescence is a positive presumptive test for E. coli. Studies by Moberg et al. (28) show that a 24 h fluorescence reading is an accurate predictor of E. coli and can identify 83-95% of the E. coli-positive tubes. After 48 h of incubation, 96-100% of E. coli-positive tubes can be identified (28). Perform a confirmed test on all presumptive positive tubes by streaking a loopful of suspension from each fluorescing tube to L-EMB agar and incubate 24 ± 2 h at 35°C. Follow protocols outlined in I. F, above, for Completed test for E. coli. Calculate MPN of E. coli based on combination of confirmed fluorescing tubes in 3 successive dilutions.

III. Examination of Bottled Water

Consumption of bottled water is increasing rapidly worldwide. In the U.S. alone, over 3.6 billion gallons of bottled water were consumed in 1998 (International Bottled Water Association, Alexandria, VA). Unlike potable water, which is regulated by the U.S. EPA, bottled water is legally classified as food in the U.S. and regulated by the FDA (Federal Register. 1995. 21 CFR Part 103 et al. beverages: bottled water; final rule. 60(218) 57076-57130). FDA defines bottled water as "water that is intended for human consumption and that is sealed in bottles or other containers with no added ingredients except that it may contain safe and suitable antimicrobial agents" and, within limitations, some added fluoride. Bottled water may be used as a beverage by itself or as an ingredient in other beverages. These regulations do not apply to soft drinks or similar beverages. In addition to "bottled water" or "drinking water", in 21 CFR Part 103 FDA also defines various types of bottled water that meet certain criteria. These identities include "artesian or artesian well water", "ground water", mineral water", "purified or demineralized water", "sparkling bottled water", "spring water" and "well water". Additionally "sterile water" is defined as water that meets the requirements under the "Sterility Test" in the United States Pharmacopeia.

Coliform organisms are not necessarily pathogens and are rarely found in bottled water, however, they serve as an indicator of insanitation or possible contamination. Surveys have shown that coliforms are useful indicators of bottled water quality, but some countries also monitor additional microbial populations as indicators of bottle water quality (10, 33). Under the current bottled water quality standard, FDA has established a microbiological quality requirement that is based on coliform detection levels. These levels may be obtained by membrane filtration (MF) or by 10-tube MPN analysis of ten 10-mL analytical units. For information on bottled water methods contact Dr. Bill Burkhardt III (email [email protected] ), FDA, CFSAN, Dauphin Island, AL, 36528; 251-406-8125

  1. Equipment and Materials.

    1. Incubator at 35° ± 0.5°C.
    2. Membrane filtration units (filter base and funnels): glass, plastic, or stainless steel; wrapped in foil or paper and sterilized.
    3. Ultraviolet sterilization chamber for sterilizing filter base and funnels (optional).
    4. Filter manifold or vacuum flask to hold filter funnels.
    5. Vacuum source (line vacuum, electric vacuum pump or water aspirator).
    6. Membrane filters; sterile, white, gridded, 47 mm diameter, 0.45 µm pore size (or equivalent, as specified by the manufacturer) for enumeration of bacteria.
    7. Petri dishes, sterile, plastic, 50 × 12 mm, with tight fitting lids.
    8. Forceps designed to transfer membranes without damage.
  2. Culture media.

    1. Lauryl sulfate tryptose (LST) broth (M-76).
    2. Brilliant green lactose bile broth (BGLB) (M-25).
    3. M-Endo Medium (BD#274930) (M-196).
    4. LES-Endo Agar (BD#273620) (M-197).
  3. Ten tube MPN coliform test - Presumptive and Confirmed procedures.

    For routine examination of bottled water, take 100 mL of sample and inoculate 10 tubes of 2X LST (10 mL of medium) with 10 mL of undiluted sample each. Incubate tubes at 35°C. Examine tubes at 24 ± 2 h for growth and gas formation as evidenced by displacement of medium in fermentation vial or effervescence when tubes are gently agitated. If negative at 24 h, reincubate tubes for an additional 24 h and examine again for gas. Perform a confirmed test on all presumptive positive (gassing) tubes as follows: gently agitate each positive LST tube and, using a 3.0 - 3.5 mm sterile loop, transfer one or more loopfuls of suspension to a tube of BGLB broth. Sterile wooden applicator sticks may also be used for transfer by inserting it at least 2.5 cm into the broth culture. Incubate BGLB tubes for 48 ± 2 h at 35°C. Examine for gas production and record. Calculate MPN using 10 tube MPN Table (9221.III), p. 9-52, Standard Methods for the Examination of Water and Wastewater (3).

    NOTE: if a sample is found to contain coliforms (at any level) follow procedure outlined in Sec. I. F. above to determine if it is E. coli. Bottled water is not permitted to contain E. coli.

  4. Membrane filter method for coliforms.

    Filter 100 mL of test sample and transfer the filter to M-Endo medium (M-196) or LES Endo Agar (M-197) and incubate at 35 °C± 0.5°C for 22-24 h. Count colonies that are pink to dark red with a green metallic surface sheen. The sheen may vary from pinpoint to complete coverage of the colony. Use of a low power, dissecting-type microscope to examine filters is recommended.

    Confirmation - If there are 5 to 10 sheen colonies on the filter, confirm all by inoculating growth from each sheen colony into tubes of LST and incubate at 35 °C± 0.5°C for 48 h. If the number of sheen colonies exceeds 10, randomly select and confirm 10 colonies that are representative of all sheen colonies. Any gas positive LST tubes should be sub cultured to BGLB and incubated at 35°C± 0.5°C for 48 hr. Gas production in BGLB within 48 h is a confirmed coliform test. Report results as number of coliform colonies per 100 mL. NOTE: Standard Method, 1998, 20th ed, p. 9-60 (3), allows for simultaneous inoculation of LST and BGLB during verification. However, BGLB is somewhat inhibitory so the method described above, where samples are sub cultured from LST into BGLB is regarded as a more sensitive verification assay and therefore, recommended.

    NOTE: if a sample is found to contain coliforms (at any level) follow procedure outlined in Sec. I. F. above to determine if it is E. coli. Bottled water is not permitted to contain E. coli.

IV. Examination of Shellfish and Shellfish Meats

The official FDA procedure for bacteriological analysis of domestic and imported bivalve molluscan shellfish is fully and properly described in the APHA's Recommended Procedures for the Examination of Sea Water and Shellfish, 4th ed. 1970 (1). The methods, including the conventional 5-tube MPN for coliform, fecal coliform and standard total plate count for bacteria (see Part III, APHA's Recommended Procedures the Examination of Sea Water and Shellfish, 4th ed. 1970 (1), are described below for examining shell stock, fresh-shucked meats, fresh-shucked frozen shellfish, and shellfish frozen on the half shell. These procedures do not apply to the examination of crustaceans (crabs, lobsters, and shrimp) or to processed shellfish meats such as breaded, shucked, pre-cooked, and heat-processed products (see section I. C. this chapter). Also, there are many methods that are used for testing for shellfish harvest and environmental water for fecal coliforms. One example, the mTEC agar (M-198) is a suitable membrane filter medium for enumerating fecal coliforms in marine and estuarine waters. Briefly, following the filtration of 100 ml of water, the filter funnels should be rinsed twice with approx. 20 ml of PBS. The filter is then transferred onto mTEC agar and incubated for 22-24 h at 44.5°C in Ethyfoam. All yellow, yellow-green or yellow-brown colonies are counted as fecal coliforms. Only plates having fewer than 80 colonies are counted. However, analysis of environmental waters will not be covered in detail here, as environmental water analyses are done by the U.S. EPA (3) and the quality of shellfish harvest waters are mainly the responsibilities of each State's Shellfish Control Authorities (20).

  1. Sample Preparation

    Using 10-12 shellfish, obtain 200 g of shellfish liquor and meat. Blend 2 min, with 200 mL sterile phosphate buffered dilution water or 0.5% peptone water (R97) to yield a 1:2 dilution of sample. Analysis of the ground sample must begin within 2 min after blending. Make serial dilutions in 0.5% sterile peptone water or sterile phosphate buffered dilution water.

  2. MPN - Presumptive and Confirmed Test for Coliform

    Use Lactose Broth (M74) or Lauryl Tryptose Broth (M76), at single strength in 10 ml volumes. For 5-tube MPN analysis, inoculate the 5 tubes at each dilution as follows:

    To each of 5 tubes, add 2 mL of the blended homogenate (equivalent to 1 g of shellfish).

    To each of 5 tubes, add 1 mL of 1:10 dilution of homogenate (0.1 g shellfish).

    To each of 5 tubes, add 1 mL of 1:100 dilution of homogenate (0.01 g shellfish).

    To each of 5 tubes, add 1 mL of 1:1000 dilution of homogenate (0.001 g shellfish).

    Further dilutions may be necessary to avoid indeterminate results. Incubate tubes at 35°C ± 0.5°C then follow instructions in section 1.C and perform Confirmed test as in 1.D above, under "Conventional Method for Coliforms, fecal coliforms and E. coli". Calculate MPN as described in section 1.D above, except that shellfish analysis specifies that the coliform density be expressed as MPN per 100 g of sample rather than per g.

  3. MPN - Presumptive and Confirmed Test for Fecal Coliforms in Shellfish

     Perform presumptive test as described in section II above. To confirm positive tubes, transfer one loopful from gas positive LST tubes to EC broth and incubate in a covered circulating waterbath at 44.5°±0.2°C for 24 ± 2 hr. Gas production in EC is a positive confirmed test for fecal coliforms. Calculate the MPN per 100 g for fecal coliforms as described above for coliform.

  4. MPN - EC-MUG Method for Determining E. coli in Shellfish Meats

    The MUG assay for β-glucuronidase (GUD) described above for detecting E. coli in chilled and frozen food can also be used for testing for E. coli in shellfish meats; but with slight modifications. This is due to the fact that foods such as shellfish meats contain natural GUD activity (32). As a result, oyster homogenate inoculated directly into LST-MUG tubes in the Presumptive phase of the MPN test can cause false positive fluorescence reactions. Hence, in the analysis of E. coli in shellfish meats, the MUG reagent is added to the EC medium and used in the confirmatory phase of the assay. The EC-MUG tubes, incubated at 44.5°C + 0.2°C, can be used in the confirmatory phase of a conventional 5-tube MPN assay to determine fecal coliform levels in shellfish meats, then by examining tubes for fluorescence under longwave UV, an E. coli MPN can also be readily obtained (32).

    See section 1.A and 1.B above for materials and reagents required. Use commercially prepared dehydrated EC-MUG, or prepare medium by adding MUG to EC broth (0.05 g/L) (M50). Several sources of MUG compound are suitable: Marcor Development Corp., Carlstadt, NJ; Biosynth International, Itasca, IL; Sigma Chemical Co., St. Louis, MO and Hach Chemical, Loveland, CO. Dispense 5 mL into new disposable borosilicate glass tubes (100 × 16 mm) containing, new disposable borosilicate glass Durham vials (50 × 9 mm) for gas collection. Sterilize EC-MUG broth tubes at 121°C for 15 min; store up to 1 week at room temperature or up to a month under refrigeration.

    Perform the 5-tube MPN Presumptive and Confirmed Test for Fecal Coliforms in Shellfish as described above in Section 3, except use EC-MUG tubes instead of EC for the confirmed test. Determination of fluorescence in EC-MUG broth requires the use of 3 control tubes, one inoculated with E. coli as positive control; one with Enterobacter aerogenes (ATCC 13048) or K. pneumoniae as negative control; and an uninoculated tube as EC-MUG medium batch control. Inoculate the positive and negative controls at the time when Confirmed test is being performed and incubate all tubes at 44.5°C ± 0.2°C for 24 h.

    Read fluorescence as described above under LST-MUG assay. Note that some (<10%)>E. coli are anaerogenic (gas-negative), but should be MUG-positive. Include all fluorescence positive tubes in the E. coli MPN calculations. Determine E. coli MPN/100g from the tables in the BAM (Appendix 2) using combination of fluorescence positive tubes at each dilution.

    NOTE: If analysis is to determine compliance with established E. coli limits, it will be necessary to confirm the presence of E. coli in MUG positive tubes.

V. Analysis for E. coli in citrus juices

Analysis for E. coli has been implemented to identify potentially contaminated juices or for verifying the effectiveness of HACCP during processing of unpasteurized juices (21 CFR Part 120, Vol. 66, No. 13, January 19, 2001). The standard method commonly used for testing for E. coli is the MPN however, it does not seem adequate for juice testing because of the acidity (pH 3.6 to 4.3) of juices, which can interfere with the test, plus it only allows for testing 3.33 mL of sample. Unlike most E. coli methods, which are enumeration assays, the following method is a simple Presence/Absence test that can examine 10-mL volume of juices (34, 35). This assay, designated as modified ColiComplete (CC) Method, is a modification of AOAC Official Method 992.30, which uses MUG for detection of E. coli (see Section on LST-MUG Method for details).

  1. Equipment and materials

    1. Covered water bath, with circulating system to maintain temperature of 44.5 ± 0.2°C.
      Water level should be above the medium in immersed tubes.
    2. Incubator, 35 ± 0.5°C
    3. Longwave UV light [~365 nm], not to exceed 6 W.
  2. Media and reagents:

    1. Universal Preenrichment Broth (UPEB) (M188) or can be purchased from BD(#223510)
    2. EC medium (M49)
    3. ColiComplete (CC) discs (#10800) - BioControl, Bellevue, WA
  3. Sample preparation, enrichment and analysis

    Perform assay in duplicate. Aseptically, inoculate 10-mL portion of juice into 90 mL of UPEB and incubate at 35°C ± 0.5°C for 24 h. After enrichment, mix and transfer 1-mL from each UPEB enrichment broth into 9 mL of EC broth containing a CC disc. Incubate EC/CC broth tubes at 44.5± 0.2°C in a circulating water bath for 24 ± 2 h. Include a tube inoculated with a MUG (+) E. coli strain as positive control and another with K. pneumoniae or Enterobacter aerogenes (ATCC 13048) as negative control. Examine tubes in the dark and under long wave UV light. The presence of blue fluorescence in either tube is indicative that E. coli is present in the sample. Note: The CC discs also contain X-gal, which when cleaved by β-galactosidase will yield blue color on or around the disc. This reaction is analogous to measuring acid/gas production from fermentation of lactose hence, the presence of blue color is indicative of coliforms.

VI. Other Methods for Enumerating Coliforms and E. coli

There are many other methods for enumerating coliforms and E. coli , including several that uses fluorogenic reagents like MUG or other chromogenic substrates for presumptive detection and identification of coliform and E. coli in foods. Many of these tests, such as the Petrifilm dry rehydratable film, the hydrophobic grid membrane filter/MUG (HGMF/MUG) method (13), ColiComplete disc (16), Colilert (AOAC 991.15), have been evaluated by collaborative studies and adopted as official first or final action by the AOAC. There are also many modifications of the membrane filtration assays that have been developed for testing for coliform, fecal coliform and E. coli and some of these may be useful in testing foods such as milk and beverages, but they are used mostly for water, environmental waters, and shellfish harvest waters analysis (5, 7, 20, 22, 23, 31).


References

  1. American Public Health Association. 1970. Recommended Procedures for the Examination of Seawater and Shellfish, 4th ed. APHA, Washington, DC.
  2. American Public Health Association. 1992. In: Marshall, R.T. (ed). Standard Methods for the Examination of Dairy Products, 16th ed. APHA. Washington, DC.
  3. American Public Health Association. 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. APHA, Washington, DC.
  4. American Public Health Association. 1992. Compendium of Methods for the Microbiological Examination of Foods, 3rd ed. APHA, Washington, DC.
  5. Brenner, K. P., C. C. Rankin, M. Sivaganesan, and P.V. Scarpino. 1996. Comparison of the recoveries of Escherichia coli and total coliforms from drinking water by the MI agar method and the U.S. Environmental protection agency-approved membrane filter method. Appl. Environ. Microbiol.62:203-208.
  6. Caplenas, N.R. and M.S. Kanarek. 1984. Thermotolerant non-fecal source Klebsiella pneumoniae: validity of the fecal coliform test in recreational waters. Am. J. Public Health.74:1273-1275
  7. Ciebin, B.W., M.H. Brodsky, R. Eddington, G. Horsnell, A. Choney, G. Palmateer, A. Ley, R. Joshi, and G. Shears. 1995. Comparative evaluation of modified m-FC and m-TEC media for membrane filter enumeration of Escherichia coli in water. Appl. Environ. Microbiol.61:3940-3942.
  8. Chang, G.W., J. Brill, and R. Lum. 1989. Proportion of beta-glucuronidase-negative Escherichia coli in human fecal samples. Appl. Environ. Microbiol.55:335-339.
  9. Conway, P.L. 1995. Microbial ecology of the human large intestine. In: G.R. Gibson and G.T. Macfarlane, eds. p.1-24. Human colonic bacteria: role in nutrition, physiology, and pathology. CRC Press, Boca Raton, FL.
  10. Dege, N.J. 1998. Categories of bottled water. Chapter 3, In: D.A.G. Senior and P. R. Ashurst (ed). Technology of Bottled Water. CRC Press, Boca Raton, Florida.
  11. Doyle, M.P. and J.L. Schoeni. 1987. Isolation of Escherichia coli O157:H7 from retail meats and poultry. Appl. Environ. Microbiol.53:2394-2396.
  12. Eijkman, C. 1904. Die garungsprobe bei 46° als hilfsmittel bei der trinkwasseruntersuchung. Zentr. Bakteriol. Parasitenk. Abt. I. Orig.37:742.
  13. Entis, P. 1989. Hydrophobic grid membrane filter/MUG method for total coliform and Escherichia coli enumeration in foods: collaborative study. J. Assoc. Off. Anal. Chem.72:936-950.
  14. Escherich, T. 1885. Die darmbakterien des neugeborenen und sauglings. Fortshr. Med.3:5-15-522, 547-554.
  15. Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Elsevier, New York.
  16. Feldsine, P.T., M.T. Falbo-Nelson, and D.L. Hustead. 1994. ColiComplete Substrate-supporting disc method for confirmed detection of total coliforms and Escherichia coli in all foods: comparative study. J. Assoc. Off. Anal.Chem.77:58-63.
  17. Feng, P. 1995. Escherichia coli serotype O157:H7: Novel vehicles of infection and emergence phenotypic variants. Emerging Infectious Dis.1:16-21.
  18. Feng, P.C.S. and P.A. Hartman. 1982. Fluorogenic assays for immediate confirmation of Escherichia coli. Appl. Environ. Microbiol.43:1320-1329.
  19. Feng, P., R. Lum, and G. Chang. 1991. Identification of uidA gene sequences in beta-D-glucuronidase (-) Escherichia coli. Appl. Environ. Microbiol.57:320-323.
  20. FDA. 1998. Fish and Fisheries Products Hazards and Control Guide. 2 nd ed. Office of Seafood, CFSAN, U.S. FDA, Public Health Service, Dept. Health and Human Services, Washington DC.
  21. Frampton, E.W. and L. Restaino. 1993. Methods for E. coli identification in food, water and clinical samples based on beta-glucuronidase detection. J. Appl. Bacteriol.74:223-233.
  22. Geissler, K., M. Manafi, I. Amoros, and J.L. Alonso. 2000. Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media. J. Appl. Microbiol.88:280-285.
  23. Grant, M.A. 1997. A new membrane filtration medium for simultaneous detection and enumeration of Escherichia coli and total coliforms. Appl. Environ. Microbiol.63:3526-4530.
  24. Gunzer, F., H. Bohm, H. Russmann, M. Bitzan, S. Aleksic, and H. Karch. 1992. Molecular detection of sorbitol fermenting Escherichia coli O157 in patients with hemolytic uremic syndrome. J. Clin. Microbiol.30:1807-10.
  25. Hartman, P.A. 1989. The MUG (glucuronidase) test for Escherichia coli in food and water, pp. 290-308. In: Rapid Methods and Automation in Microbiology and Immunology. A. Balows, R.C. Tilton, and A. Turano (eds). Brixia Academic Press, Brescia, Italy.
  26. Hayes, P.S., K. Blom, P. Feng, J. Lewis, N.A. Strockbine, and B. Swaminathan. 1995. Isolation and characterization of a β-D-glucuronidase-producing strain of Escherichia coli O157:H7 in the United States. J. Clin. Microbiol.33:3347-3348.
  27. Manafi, M. 1996. Fluorogenic and chromogenic enzyme substrates in culture media and identification tests. Int. J. Food Microbiol.31:45-58.
  28. Moberg, L.J., M.K. Wagner, and L.A. Kellen. 1988. Fluorogenic assay for rapid detection of Escherichia coli in chilled and frozen foods: collaborative study. J. Assoc. Off. Anal. Chem. 71:589-602.
  29. Neill, M. A., P. I. Tarr, D. N. Taylor, and A. F. Trofa. 1994. Escherichia coli. In Foodborne Disease Handbook, Y. H. Hui, J. R. Gorham, K. D. Murell, and D. O. Cliver, eds. Marcel Decker, Inc. New York. pp. 169-213.
  30. Neufeld, N. 1984. Procedures for the bacteriological examination of seawater and shellfish. In: Greenberg, A.E. and D.A. Hunt (eds). 1984. Laboratory Procedures for the Examination of Seawater and Shellfish, 5th ed. American Public Health Association. Washington, DC.
  31. Rippey, S.R., W.N. Adams, and W.D. Watkins. 1987. Enumeration of fecal coliforms and E. coli in marine andestuarine waters: an alternative to the APHA-MPN approach. J. Water Pollut. Control Fed.59:795-798.
  32. Rippey, S.R., L.A. Chandler, and W.D. Watkins. 1987. Fluorometric method for enumeration of Escherichia coli in molluscan shellfish. J. Food Prot.50:685-690, 710.
  33. Warburton, D.W. 2000. Methodology for screening bottled water for the presence of indicator and pathogenic bacteria. Food Microbiol.17:3-12.
  34. Weagant, S.D. and P. Feng. 2001. Comparative evaluation of a rapid method for detecting Escherichia coli in artificially contaminated orange juice. FDA Laboratory Information Bulletin #4239, 17:1-6.
  35. Weagant, S.D. and P. Feng. 2002. Comparative Analysis of a Modified Rapid Presence-Absence Test and the standard MPN Method for Detecting Escherichia coli in Orange Juice. Food Microbiol.19:111-115.

Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 4.

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Источник: https://www.fda.gov/food/laboratory-methods-food/bam-chapter-4-enumeration-escherichia-coli-and-coliform-bacteria

Last updated on June 21st, 2021

Eosin Methylene Blue (EMB) agar is both a selective and differential culture medium. It selectively promotes the growth of Gram-negative bacteria (inhibits Gram-positive bacteria) and aids in the differentiation of lactose fermenter and non-lactose fermenting colonies.

EMB Agar

EMB agar, first described by Holt-Harris and Teague, contained lactose and sucrose as source of carbohydrates. Levine further modified the medium by adding peptone and phosphate and removed sucrose from the formula and increased the lactose content. This aided in the differentiation of fecal and non-fecal types of the coliforms and also salmonellae and other non-lactose fermenters from the coliforms.

Another commonly used media for selective isolation of Gram-negative rods and differentiation of the member of Enterobacteriaceaeas lactose fermenter and non-lactose fermenter is MacConkey Agar. 

Contents

Principle

EMB agar contains sucrose and lactose, utilized as fermentable carbohydrates substrates, which encourage the growth of some gram-negative bacteria, especially fecal and non-fecal coliforms. Differentiation of enteric bacteria is possible due to the presence of the sugars lactose and sucrose in the EMB agar and the ability of certain bacteria to ferment the lactose in the medium.

  • Lactose-fermenting gram-negative bacteria acidify the medium, which reduces the pH, and the dye produces a dark purple complex usually associated with a green metallic sheen. This metallic green sheen is an indicator of vigorous lactose and/or sucrose fermentation ability typical of fecal coliforms.
  • Organisms that are slow lactose-fermenters, produce less acid, and the colonies appear brown-pink.
  • Non-lactose fermenters, increase the pH of the medium by deamination of proteins and produce colorless or light pink colonies.

Eosin Y and methylene blue are pH indicator dyes that combine to form a dark purple precipitate at low pH; they also serve to inhibit the growth of most Gram-positive organisms. Peptic digest of animal tissue serves as a source of carbon, nitrogen, and other essential growth nutrients. Phosphate buffers the medium.

Composition of EMB Agar

The composition of EMB agar and modified EMB agar (Levine EMB) agar differs slightly. Levine modification contains 10g of lactose (twice as in EMB agar) and contains no sucrose.

IngredientsEMB agar (gm/L)Levine EMB agar (gm/L)
Peptone10 g10g
Lactose5 g10g
Sucrose5g
Dipottasium,PO42g2g
Agar13.5g13.5g
Eosin Y0.4g0.4g
Methylene blue0.065g0.065g

Preparation of EMB agar

  1. Weigh and suspend 35.96 grams of dehydrated media in 1000 ml distilled water.
  2. Mix until the suspension is uniform and 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, and with frequent gentle swirling, pour the media into sterile Petri plates.
    Note: frequent swirling is recommended to restore the blue color o methylene blue and to suspend the flocculent precipitate if any.
  5. Label with initials of the name of the medium, date of preparation, and store the plates upside down (lids below) in the refrigerator until use.

Colony morphology

OrganismColonial appearance on EMB agar
Escherichia coliColonies are 2-3 mm in diameter, and have greenish metallic sheen in reflected light, dark or even black centre in transmitted light
Enterobacter aerogenesColonies are 4-6mm in diameter, raised and mucoid, tending to become confluent.
No metallic sheen, grey-brown centers by transmitted light
Salmonella and Shigella sppTranslucent and colorless colonies

Pseudomonas sppColorless irregular colonies
Proteus sppColorless colonies
Gram positive cocciPartially inhibited or no growth
Coagulase-positive staphylococciColorless, “pin-point” colonies on modified EMB

Quality control of EMB agar

Sterility testing can be performed by incubating 3-5% uninoculated plates from each batch at 37°C for 18-24 hours. Any growth on the media should be regarded as contamination and the whole lot should be discarded.

Performance testing of prepared EMB agar plates can be done by inoculating known strains of bacteria into the medium and observing growth and colonial characteristics.

OrganismGrowth and colony characteristics
E.coli ATCC 25922Good growth, blue-black colonies with a green metallic sheen
Salmonella choleraesuis subsp. Choleraesuis serotype Typhimurium ATCC 14028Luxuriant growth, colorless to amber colonies
Enterococcus faecalis ATCC 29212Inhibition (partial)
Shigella flexneri ATCC 12022Moderate to heavy growth, colorless to amber colonies

Uses of EMB agar

Isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli.

  1. EMB agar is used in water quality tests to distinguish coliforms and fecal coliforms that signal possible pathogenic microorganism contamination in water samples (presence of E.coli in the river/water sample indicates the possibility of fecal contamination of water so does the presence of other pathogenic enterics).
  2. EMB media assists in the visual distinction ofEscherichia coli, other nonpathogenic lactose-fermenting enteric gram-negative rods, and the Salmonella and Shigella genera. Escherichia coli colonies grow with a metallic sheen with a dark center. Aerobacter aerogenes colonies have a brown center, and non-lactose-fermenting gram-negative bacteria appear pink.
  3. EMB agar is also used to differentiate the organisms in the colon-typhoid-dysentery group. For culture of Salmonella and Shigella, selective medium such as MacConkey agar and EMB agar is commonly used.
  4. Levine EMB Agar can be used for the isolation and identification of Candida albicans from clinical specimens. Addition of 0.1g/L of chlortetracycline hydrochloride after autoclaving makes the medium selective by inhibiting the accompanying bacterial flora. The culture medium then is blue in color. Colonies of Candida albicans appear `spidery’ or `feathery’ after 24 to 48 hours of incubation at 35°C in 10% carbon dioxide. Other Candida species produce smooth yeast-like colonies.

References and further readings

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