Microbial limit test

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NICE PPT OF LIMIT TEST

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Slide 1: 

1 MICROBIAL LIMIT TEST FOR PHARMACEUTICALS Presented By: Mr. S.CHELLARAM

INTRODUCTION: 

2 INTRODUCTION Quality control test for Nonsterile pharmaceutical product includes the microbiological testing of raw materials, excipients, active ingredients, bulk, and finished products. Nonsterile samples contain high numbers of microbes and objectionable MOs that change the chemical composition by spoilage, affecting the stability and integrity of the product and package. Microbial bioburden is allowed based upon the product specifications. Quantity and types of MO will determine the safety of that particular pharmaceutical product and efficacy of the manufacturing process.

Slide 3: 

3 Microbial limit test (MLT) is based upon: Chemical composition of product Production process Route of application Intended use of product Delivery system of product USP, EP, and JP had divided MLT into types of test Quantitative test – determines number of bacteria, yeast, and mold present in a given pharmaceutical sample and Qualitative test - determines the presence of specific pathogen indicators, e.g., Salmonella spp., Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Enterobacteriaceae

Slide 4: 

4 Salmonella and E. coli (toxin producers) are gram-negative rods , capable of lactose fermentation , commonly found in fecal sources associated to intestinal disorders P. aeruginosa is a gram-negative, non fermentative rod,typically associated to opportunistic infections S. aureus is a gram positive cocci commonly associated to skin, gastrointestinal, and toxic shock syndrome conditions. USP recommendations and Limits: Plant, animal , and mineral -based formulations – absence of Salmonella Orally administered products - absence of E. coli Topical pharmaceutical formulation - absence of S. aureus, P. aeruginosa Vaginal, rectal, and urethral formulations - absence of yeast and mold

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5 EP recommendations and Limits: Tropical, transdermal, respiratory – NMT 100CFUs/g or ml of bacteria Absence of S.aureus & Pseudomonas Oral & rectal routes -NMT 1000 CFUs / g or ml NMT 100 CFUs / g or ml of Yeast, mold Plant, animal, and mineral-based formulations – NMT 10,000 CFUs/g ml Absence of salmonella species, S.aureus, E.Coli Herbal formulations - NMT 10 5 to 10 7 CFUs /g or ml for bacteria NMT 10 4 to 10 5 CFUs / g or ml for yeast, mold If reconstituted with boiling water - NMT 10 2 CFUs / g or ml If boiling water not added - Absence of E.Coli & salmonella

MICROBIAL LIMIT TEST: 

6 MICROBIAL LIMIT TEST PREPARATORY TESTING is to be done to confirm that the sample themselves do not inhibit multiplication of micro organisms, under the test conditions, that may be present. diluted specimens of the material to be inoculated with separate viable cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella . Innoculum: 1 ml of not less than 10 -3 dilution of a 24-hour broth culture of the microorganism in pH 7.2 Phosphate Buffer , Fluid Soybean–Casein Digest Medium , or Fluid Lactose Medium of the test material.

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7 Observation & Result of the preparatory test: Absence of Microbial growth in the relevant medium invalidates the MLT and necessitates modification of the procedure by (1) an increase in the volume of diluent , the quantity of test material remaining the same, or by (2) addition of suitable inactivating agent in the diluents, or (3) Combination of modifications (1) and (2) so as to permit growth of the inoculum. 0.5 % soy lecithin , and 4% polysorbate 20 are used to neutralize inhibitory substances present in the sample

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8 CULTURE MEDIA Culture media may be prepared as per the formula or dehydrated culture media may be used. Dehydrated media , when reconstituted as directed by the manufacturer or distributor, should have similar ingredients and/or yield media comparable to those obtained from the formulas. Dissolve the soluble solids in the water, using heat, if necessary, to effect complete solution Use Agar containing NMT 15% Moisture content. Use Purified Water . Determine the pH at 25 ± 2. add solutions of HCl or NaOH to adjust the pH. Sterilization of media is by using autoclave at 121 0 C at 15psi for 15 minutes.

Culture media used in Microbial Limit test:: 

9 Culture media used in Microbial Limit test: 1.Soybean–Casein Digest Agar Medium 2.Fluid Soybean–Casein Digest Medium 3.Pseudomonas Agar Medium for Detection of Pyocyanin 4.Xylose–Lysine–Desoxycholate Agar Medium 5.Bismuth Sulfite Agar Medium 6.Triple Sugar–Iron–Agar Medium 7.MacConkey Agar Medium 8.Levine Eosin–Methylene Blue Agar Medium 9.Sabouraud Dextrose Agar Medium 10.Potato Dextrose Agar Medium

Culture media used in Microbial Limit test:: 

10 Culture media used in Microbial Limit test: 11.Fluid Casein Digest–Soy Lecithin–Polysorbate 20 Medium 12.Pseudomonas Agar Medium for Detection of Fluorescin 13. Fluid Lactose Medium 14. Fluid Selenite–Cystine Medium 15. Fluid Tetrathionate Medium 16. Brilliant Green Agar Medium 17. Mannitol–Salt Agar Medium 18. Baird–Parker Agar Medium 19. Vogel–Johnson Agar Medium 20. Cetrimide Agar Medium

Slide 11: 

11 Stock Solution Dissolve 34 g of monobasic potassium phosphate in about 1000 ml of water. Adjust to pH 7.2 ± 0.1 Dispense and sterilize. Store under refrigeration. For use, dilute the Stock Solution with water in the ratio of 1 to 800, and sterilize. Sample size 10-mL or 10-g specimens or as per monograph. For a solid reduce the substance to a moderately fine powder, suspend it in the vehicle specified. For a fluid specimen that consists of a true solution , or a suspension in water or a hydroalcoholic vehicle containing less than 30 percent of alcohol , and for a solid that dissolves readily and practically completely in 90 mL of pH 7.2 Phosphate Buffer or the media specified.

Slide 12: 

12 For ointments, creams, and waxes: prepare a suspension with the aid of a minimal quantity of a suitable, sterile emulsifying agent (such as one of the polysorbates), using a mechanical blender and warming to a temperature not exceeding 45 0 C. For a fluid specimen in aerosol form: chill the container in an alcohol-dry ice mixture for approximately 1 hour, cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible, and transfer the quantity of test material required for the test.

TOTAL AEROBIC COUNT: 

13 TOTAL AEROBIC COUNT Methodology for enumeration of Micro organism is of three types they are: Plate method - soluble & translucent sample Multiple – tube method Membrane method - water samples

Slide 14: 

14 PLATE METHOD Dissolve or suspend 10.0 g of the specimen if it is a solid, or 10 ml , accurately measured, if the specimen is a liquid, in pH 7.2 Phosphate Buffer , Fluid Soybean–Casein Digest Medium , or Fluid Casein Digest–Soy Lecithin-Polysorbate 20 Medium to make 100 mL. For viscous specimens dilute until a suspension is obtained, i.e., 1:50 or 1:100, etc., that can be pipeted. Pipette 1 mL of the final dilution onto each of two sterile petri dishes. add to each dish 15 to 20 mL of Soybean–Casein Digest Agar Medium that previously has been melted and cooled to approximately 45 0 C. Cover the petri dishes, mix the sample and allow the contents to solidify at room temperature. Invert the petri dishes, and incubate for 48 to 72 hours.

Slide 15: 

15 Examine the plates for growth, count the number of colonies , and express the average for the two plates in terms of the number of microorganisms per g or per mL of specimen. If no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the specimen, express the results as “ less than 10 microorganisms per g or per mL of specimen.”

Slide 16: 

16 MULTIPLE-TUBE METHOD 15 test tubes containing 9.0 ml of sterile Fluid Soybean–Casein Digest Medium was taken. Arrange 12 of the tubes in four sets of three tubes each. 1set of three tubes will serve as controls. N A B 100 100 100 10 10 10 1 1 1 Set III Set II Set I Control

Slide 17: 

17 Transfer 1 ml of the sample in to I set of 3 tubes and A. The concentration of the tubes now corresponds to 100 l. Transfer 1 ml of contents in A into set II and tube B so that concentration corresponds 10 l Transfer 1 ml of contents in B into set III tubes such that concentration corresponds 1 l. Negative control should be maintained. After incubation microbial growth is confirmed by the presence of turbidity. Multiple tube method is suitable for the samples which are showing difficulty in measuring CFUs. Most Probable number (MPN) of microorganisms present in the sample is determined from the standard values table listed below

Slide 18: 

18 Observed Combinations of Numbers of Tubes Showing Growth in Each Set MPN per g or per mL No. of mg (or mL) of Specimen per Tube 100 (100 µL) 10 (10 µL) 1 (1 µL) 3 3 3 >1100 3 3 2 1100 3 3 1 500 3 3 0 200 3 2 3 290 3 2 2 210 3 2 1 150 3 2 0 90 3 1 3 160 3 1 2 120 3 1 1 70 3 1 0 40 3 0 3 95 3 0 2 60 3 0 1 40 3 0 0 23

MEMBRANE METHOD: 

19 MEMBRANE METHOD Useful to eunumerate Microbial count in the samples of Purified water & WFI. 100 ml of Purified water or WFI was filtered through sterile membrane filter of pore size 0.44  or 0.22. The membrane was then rinsed with aliquots of sterile peptone water. The membrane was then aseptically transferred into sterile petri plate containing Soyabean casein digest agar. The petri plates were incubated for 48 -72 hrs. After incubation number of CFUs was reported SPECIFICATIONS: Purified water : NMT 100 CFUs / 100 ml WFT : NMT 10 CFUs / 100 ml

TEST FOR DETECTION OF STAPHYLOCOCCUS AUREUS: 

20 TEST FOR DETECTION OF STAPHYLOCOCCUS AUREUS To the specimen add Fluid Soybean–Casein Digest Medium to make 100 ml, mix, and incubate. Examine the medium for growth, and if growth is present, Transfer loopful on to the surface of Vogel–Johnson Agar Medium or Baird–Parker Agar Medium , or Mannitol–Salt Agar Medium each plated on petri dishes. Cover and incubate. Selective Medium Characteristic Colonial Morphology Gram Stain Vogel-Johnson Agar Medium Black Surrounded by yellow zone Positive cocci (in clusters) Mannital-Salt Agar Medium Yellow colonies with yellow zones Positive cocci (in clusters) Baird-Parker Agar Medium Black, shiny, surrounded by clear zones 2 to 5 mm Positive cocci (in clusters)

Staphylococcus species growth in Vogel Johnson agar: 

21 Staphylococcus species growth in Vogel Johnson agar Pancreatic Digest of Casein 10.0 g Yeast Extract 5.0 g Mannitol 10.0 g Dibasic Potassium Phosphate 5.0 g Lithium Chloride 5.0 g Glycine 10.0 g Agar 16.0 g Phenol Red 25.0 mg Water 1000 mL S. aureus reduces potassium tellurite to the metal tellurium (Black colonies) Fermentation of mannitol is indicated by the yellow zones around the black colonies and changes the red color of the medium to yellow . Tryptone provides nitrogen, vitamins, minerals and amino acids Yeast extract is a source of vitamins, particularly of the B-group. Mannitol is the fermentable carbohydrate providing carbon and energy Potassium tellurite, Lithium chloride and Glycine inhibit other MO. Phenol red is the pH indicator . Dipotassium phosphate is a buffer . Agar is the solidifying agent. 3.5% Potassium Tellurite is added as enrichment ingredient

Slide 22: 

22 Staphylococcus species growth in Mannitol Salt Agar Pancreatic Digest of Casein 5.0 g Peptic Digest of Animal Tissue 5.0 g Beef Extract 1.0 g D-Mannitol 10.0 g Sodium Chloride 75.0 g Agar 15.0 g Phenol Red 0.025 g Water 1000 mL Enzymatic Digest of Casein, Animal Tissue,Beef Extract – N 2 , vitamins,& C D-Mannitol - carbohydrate source. High concentrations of NaCl inhibits bacteria other than staphylococci. Phenol Red is the pH indicator. Agar is the solidifying agent. Typical pathogenic staphylococci grow in the presence of a high salt concn.& ferment mannitol produce acid products, turning the Phenol Red pH indicator from red to yellow. Non-pathogenic staphylococci do not ferment mannitol and form red colonies.

Staphylococcus species growth in Baird Parker Agar: 

23 Staphylococcus species growth in Baird Parker Agar Pancreatic Digest of Casein 10.0 g Beef Extract 5.0 g Yeast Extract 1.0 g Lithium Chloride 5.0 g Agar 20.0 g Glycine 12.0 g Sodium Pyruvate 10.0 g Water 950 mL Enzymatic Digest of Casein, Beef Extract - carbon and nitrogen sources Yeast Extract supplies B-complex vitamins Glycine and Sodium Pyruvate stimulate growth of staphylococci. Lithium Chloride and 1% Potassium Tellurite Solution , suppresses the growth of other Micro organisms. Staphylococci that contain lecithinase break down the Egg Yolk and cause clear zones around the colonies. An opaque zone of precipitation may form due to lipase activity. Reduction of Potassium Tellurite is a characteristic of coagulase positive staphylococci, & causes blackening of colonies. Agar is the solidifying agent

TEST FOR DETECTION OF STAPHYLOCOCCUS AUREUS: 

24 Coagulase Test (for Staphylococcus aureus ) transfer representative suspect colonies from the agar surfaces of the Vogel–Johnson Agar Medium or Baird–Parker Agar Medium , or Mannitol–Salt Agar Medium to individual tubes, each containing 0.5 mL of mammalian, preferably rabbit or horse, plasma with or without suitable additives. Incubate in a water bath at 37 o C, examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. If no coagulation in any degree is observed, the specimen meets the requirements of the test for absence of Staphylococcus aureus . TEST FOR DETECTION OF STAPHYLOCOCCUS AUREUS

TEST FOR DETECTION OF  PSEUDOMONAS AERUGINOSA: 

25 TEST FOR DETECTION OF PSEUDOMONAS AERUGINOSA Oxidase and Pigment Tests (for Pseudomonas aeruginosa ) To the specimen add Fluid Soybean–Casein Digest Medium to make 100 mL, mix, and incubate. Examine the medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of Cetrimide Agar Medium . Cover & incubate. streak suspect colonies from Cetrimide Agar Medium on the Pseudomonas Agar Medium for Detection of Fluorescin and Pseudomonas Agar Medium for Detection of Pyocyanin. Cover and invert the inoculated media, and incubate at 35 ± 2 o C for not less than three days. Examine the streaked surfaces under UV light. Fluorescein - yellow-green or yellow-brown fluorescent pigment produced by Pseudomonas Pyocyanin - blue water-soluble pigment produced by P seudomonas aeruginosa.

TEST FOR DETECTION OF  PSEUDOMONAS AERUGINOSA: 

26 TEST FOR DETECTION OF PSEUDOMONAS AERUGINOSA Selective Medium Characteristic Colonial Morphology Fluorescence UV Light Oxidase Gram Stain Cetrimide Agar Medium Generally greenish Greenish Positive Negative rods Pseudomonas Agar Medium for Detection of Fluorescin Generally colorless to yellowish Yellowish Positive Negative rods Pseudomonas Agar Medium for Detection of Pyocyanin Generally greenish Blue Positive Negative rods

Pseudomonas species growth in Cetrimide agar: 

27 Pseudomonas species growth in Cetrimide agar Pancreatic Digest of Gelatin 20.0 g Magnesium Chloride 1.4 g Potassium Sulfate 10.0 g Agar 13.6 g Cetyl Trimethylammonium Bromide (Cetrimide) 0.3 g Glycerin 10.0 mL Water 1000 mL Enzymatic Digest of Gelatin - Nitrogen, vitamins, and carbon source. MgCl & KCl enhance the production of pyocyanin and fluorescein . Cetrimide (cetyltrimethylammonium bromide) is the selective agent acts as a cationic detergent causing nitrogen and phosphorous to be released from bacterial cells other than Pseudomonas aeruginosa . Agar is the solidifying agent. Glycerol is supplemented as a source of carbon Water soluble , yellow-green or yellow-brown fluorescent pigment pyoverdin (fluorescein) combines with the blue water-soluble pigment pyocyanin to produce characteristic green colour .

Pseudomonas Agar medium for detection of Fluorescein: 

28 Pseudomonas Agar medium for detection of Fluorescein Pancreatic Digest of Casein 10.0 g Peptic Digest of Animal Tissue 10.0 g Anhydrous Dibasic Potassium Phosphate 1.5 g Magnesium Sulfate (MgSO 4 ·7H 2 O) 1.5 g Glycerin 10.0 mL Agar 15.0 g Water 1000 mL The medium enhances the formation of fluorescein by Pseudomonas and inhibits the pyocyanin formation . The fluorescein pigment diffuses from the colonies of Pseudomonas into the agar and shows yellow fluorescent colouration . Casein enzymic hydrolysate and proteose peptone provide the essential nitrogenous nutrients, carbon, sulphur and trace elements for the growth of Pseudomonas . Dipotassium phosphate buffers the medium while Magnesium sulphate provides cations for the activation of fluorescein production . Salt concentration exceeding 2% affects pigment production. UV illumination may be bactericidal, so make sure that there is good growth before placing culture under UV light.

Pseudomonas Agar medium for detection of Pyocyanin: 

29 Pseudomonas Agar medium for detection of Pyocyanin Pancreatic Digest of Gelatin 20.0 g Anhydrous Magnesium Chloride 1.4 g Anhydrous Potassium Sulfate 10.0 g Agar 15.0 g Glycerin 10.0 mL Water 1000 mL This medium enhances the formation of pyocyanin but inhibits the formation of fluorescein pigment. The pyocyanin pigment diffuses from the colonies of Pseudomonas into the agar and shows blue colouration. Potassium sulphate and magnesium chloride, which enhances the pyocyanin production and suppresses the fluorescein production. Gelatine peptone - nitrogen, carbon, sulphur and trace elements and is low in phosphorous to minimize the inhibitory action on pyocyanin production. Glycerol serves as a C-source . Potassium sulfate and magnesium chloride is necessary for the activation of pyocyanin production

TEST FOR DETECTION OF  PSEUDOMONAS AERUGINOSA: 

30 TEST FOR DETECTION OF PSEUDOMONAS AERUGINOSA Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with N,N -dimethyl- p -phenylenediamine dihydrochloride : if there is no development of a pink color , changing to purple , the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa .

TEST FOR SALMONELLA SPECIES: 

31 TEST FOR SALMONELLA SPECIES To the specimen, contained in a suitable vessel, add a volume of Fluid Lactose Medium to make 100 mL, and incubate. Examine the medium for growth, If growth is present,Pipette 1 ml portions into vessels containing, respectively, 10 mL of Fluid Selenite–Cystine Medium and Fluid Tetrathionate Medium, mix, and incubate for 12 to 24 hours. (Retain the remainder of the Fluid Lactose Medium.) Portions from both the selenite-cystine and tetrathionate media taken on the surface of Brilliant Green Agar Medium, Xylose–Lysine–Desoxycholate Agar Medium , and Bismuth Sulfite Agar Medium contained in petri dishes. Cover & incubate

TEST FOR DETECTION OF  SALMONELLA SPECIES: 

32 Selective Medium Characteristic Colonial Morphology Brilliant Green Agar Medium Small, transparent, colorless or pink to white opaque (frequently surrounded by pink to red zone) Xylose-Lysine- Desoxycholate Agar Medium Red, with or without black centers Bismuth Sulfite Agar Medium Black or green TEST FOR DETECTION OF SALMONELLA SPECIES

Salmonella species growth in Brilliant Green agar: 

33 Salmonella species growth in Brilliant Green agar Yeast Extract 3.0 g Peptic Digest of Animal Tissue 5.0 g Pancreatic Digest of Casein 5.0 g Lactose 10.0 g Sodium Chloride 5.0 g Sucrose 10.0 g Phenol Red 80 mg Agar 20.0 g Brilliant Green 12.5 mg Water 1000 mL Enzymatic Digest of Casein & of Animal Tissue - Nitrogen, Amino Acid, & carbon. Yeast Extract supplies vitamins required for organism growth. Sodium Phosphate is the buffering agent. Lactose and Sucrose are the carbohydrates in the medium. Brilliant Green inhibits Gram-positive bacteria and most Gram-negative bacilli other than Salmonella spp. Phenol Red is the pH indicator and turns the medium yellow with the formation of acid when lactose and/or sucrose is fermented. Agar is the solidifying agent.

Salmonella growth in Xylose-Lysine- Desoxycholate Agar: 

34 Xylose 3.5 g L-Lysine 5.0 g Lactose 7.5 g Sucrose 7.5 g Sodium Chloride 5.0 g Yeast Extract 3.0 g Phenol Red 80 mg Agar 13.5 g Sodium Desoxycholate 2.5 g Sodium Thiosulfate 6.8 g Ferric Ammonium Citrate 800 mg Water 1000 mL Salmonella growth in Xylose-Lysine- Desoxycholate Agar Yeast extract as a source of nutrients and vitamins. Sodium desoxycholate as the selective agent and is inhibits gram-positive MO. Xylose is fermented by practically all enterics except shigellae. H 2 S indicator system (sodium thiosulfate & ferric ammonium citrate) is included for the visualization of the hydrogen sulfide produced , resulting in the formation of colonies with black centers.

Salmonella species growth in Bismuth sulphite agar: 

35 Salmonella species growth in Bismuth sulphite agar Beef Extract 5.0 g Pancreatic Digest of Casein 5.0 g Peptic Digest of Animal Tissue 5.0 g Dextrose 5.0 g Sodium Phosphate 4.0 g Ferrous Sulfate 300 mg Bismuth Sulfite Indicator 8.0 g Agar 20.0 g Brilliant Green 25 mg Water 1000 mL Enzymatic Digest of Casein & Animal Tissue, Beef Extract provide N, C &vitamin source Dextrose is the carbohydrate present in Bismuth Sulfite Agar. Disodium Phosphate is the buffering agent. Bismuth Sulfite Indicator and Brilliant Green inhibit Gram-positive bacteria and coliforms, allowing Salmonella spp. to grow. Ferrous Sulfate is used for H2S production. When H2S is present, the iron in the formula is precipitated, and positive cultures produce the characteristic brown to black color with metallic sheen. Agar is the solidifying agent.

TEST FOR SALMONELLA SPECIES : 

36 If colonies of Gram-negative rods matching the description are found, then proceed with further identification by transferring representative suspect colonies individually, by means of an inoculating wire, to a butt-slant tube of Triple Sugar–Iron–Agar Medium by first streaking the surface of the slant and then stabbing the wire well beneath the surface. Incubate. If examination discloses no evidence of tubes having alkaline (red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from hydrogen sulfide production), the specimen meets the requirements of the test for the absence of the genus Salmonella .* TEST FOR SALMONELLA SPECIES

Test for Escherichia coli: 

37 Test for Escherichia coli By means of an inoculating loop, streak a portion from the remaining Fluid Lactose Medium on the surface of MacConkey Agar Medium . Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description the specimen meets the requirements of the test for absence of Escherichia coli . Gram Stain Characteristic Colonial Morphology Negative rods (cocco-bacilli) Brick-red; may have surrounding zone of precipitated bile

Growth of Escherichia coli in Mac Conkey medium: 

38 Pancreatic Digest of Gelatin 17.0 g Pancreatic Digest of Casein 1.5 g Peptic Digest of Animal Tissue 1.5 g Lactose 10.0 g Bile Salts Mixture 1.5 g Sodium Chloride 5.0 g Agar 13.5 g Neutral Red 30 mg Crystal Violet 1.0 mg Water 1000 mL Growth of Escherichia coli in Mac Conkey medium MacConkey agar contains lactose, bile salts, neutral red and crystal violet .  It is a selective media because Gram-positive organisms are inhibited by the bile salts and the crystal violet. When bacteria ferment lactose and produce enough acid products to reduce the pH below 6.8, the neutral red turns from colourless to red .  Thus, MacConkey is a differential media on which lactose fermenting colonies appear red (or pink). Nonlactose fermenters are colourless.

Test for Escherichia coli: 

39 If colonies matching the description are found, proceed with further identification by transferring the suspect colonies individually, by means of an inoculating loop, to the surface of Levine Eosin–Methylene Blue Agar Medium , plated on petri dishes. Cover and invert the plates, and incubate. Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light , the specimen meets the requirements of the test for the absence of Escherichia coli . Test for Escherichia coli

E.Coli growth in Levin Eosin Methylene Blue agar: 

40 E.Coli growth in Levin Eosin Methylene Blue agar Pancreatic Digest of Gelatin 10.0 g Dibasic Potassium Phosphate 2.0 g Agar 15.0 g Lactose 10.0 g Eosin Y 400 mg Methylene Blue 65 mg Water 1000 mL Enzymatic Digest of Gelatin is the nitrogen source in EMB Agar, Levine. Lactose is the carbohydrate source Dipotassium Phosphate is the buffering agent. Eosin Y and Methylene Blue are the indicators. Methylene Blue is also a selective agent. During strong acidic conditions, the dyes impart a metallic sheen to certain lactose fermenters, such as E. coli .

Total Combined Molds and Yeasts Count: 

41 Total Combined Molds and Yeasts Count Proceed as for the Plate Method under Total Aerobic Microbial Count , except for using the same amount of Sabouraud Dextrose Agar Medium or Potato Dextrose Agar Medium, instead of Soybean Casein Digest Medium, and except for incubating the inverted petri dishes for 5 to 7 days at 20 o C to 25 o C.

REFERENCES: 

42 REFERENCES www.pharmacopoeia.cn www.neogen.com (Info and photographs regarding culture media) United States Pharmacopoeia European Pharmacopoeia International Pharmacopoeia Japan Pharmacopoeia