Used to confirm the presence of enterococci in water and other sources of sanitary interest
Formula in grams per liter
Casein Peptone ... 5,00 Yeast Extract ...5,00 Dextrose... 5,00 Sodium Azide ...0,40 Methylene Blue... 0,01 Bacteriological Agar ...15,00
Final pH 8,0 ± 0,2 at 25ºC
Preparation
Suspend 30,4 grams of the medium in one litre of distilled water. Heat with frequent agitation and boil until dissolution is complete (approximately one minute).
Dispense in test tubes and sterilize in an autoclave at 121°C (15 lbs. sp.) for 15 minutes. Leave in a slanted position to solidify.
Uses
This medium is used to confirm the presence of enterococci in water and other sources of sanitary interest.
In order to do so aseptically add a volume of Enterococcus Confirmatory Broth, which has the same formulation but lacks the agar, to cover half of the slanted surface. It is preferable that the Confirmatory Broth contain 6,5%
sodium chloride and 65 units of penicillin per 100 ml.
Using growth from the Enterococcus Presumptive Broth, inoculate both the surface as well as the broth in the
Confirmatory Agar/Broth mixture tube. The tubes are incubated at 35-37°C for 12 hours and are examined to detect the presence of small pinpoint colonies. Perform a Gram stain and observe under a microscope looking for large chains of ovoid cells. Immediately perform a catalase test by adding to the tube in study 5 ml. of H2O2. If there is no generation of gases (negative test), this constitutes the confirmation of enterococci in the sample.
Bibliography
Winter and Sandholzer U. S. Det. Interior Fishery, Leaflet 201 Part II, Nov. 1946.
Ewing W.H. 1986. Edwards and Ewing’s identification of enterobacteriaceae 4th edition.
Microbiological Test
Microorganisms Growth
Escherichia coli ATCC 25922 Inhibited
Streptococcus faecalis ATCC 19433 Satisfactory
Streptococcus faecium ATCC 29212 Satisfactory
E.M.B. (EOSIN METHYLENE BLUE) AGAR Cat. 1039
For the isolation and differentiation of coliforms from other enterobacteria of medical and sanitary interest
Formula in grams per liter
Bacteriological Peptone ...10,00 Lactose... 5,00 Sucrose ...5,00 Dipotassium Phosphate... 2,00 Eosin Y ...0,40 Methylene Blue ... 0,065 Bacteriological Agar ...13,50
Final pH 7,2 ± 0,2 at 25ºC
Preparation
Suspend 36 grams of the medium in one litre of distilled water. Mix well. Heat with frequent agitation and boil for one minute. Sterilize in autoclave at 121°C (15 lbs. sp.) for 15 minutes. Cool to 45-50°C. Swirl gently, avoiding the formation of bubbles and pour into Petri dishes.
Uses
It similar to Levine EMB, used for the study of enterobacteria. It is widely used in medical bacteriology, in techniques recommended by the APHA and for the detection and enumeration of coliform microorganisms, which can contaminate foods and drinking water. Due to the lactose and sucrose, the medium can be differential in primary culture: salmonellas and shigellas which are negative can be differentiated from other lactose-negative but sucrose-positive organisms such as Proteus vulgaris, Citrobacter and Aeromonas.
The accompanying microflora which hinders the isolation of medically important organisms are inhibited by the dyes in the formula, especially gram-positives.
It can also be used for the rapid identification of C.
albicans (incubated in CO2) and sometimes to isolate Nocardia.
E. coli Elevated or slightly convex. 2-3 mm. in diameter, with transmitted light blue-black center with a narrow, clear
edge. Blue-green metallic sheen with reflected light. Some strains show no metallic sheen. Small tendency to confluent growth.
E. aerogenes Klebsiella Large colonies, 4-6 mm. in diameter, mucoid with a tendency to run together. Usually no metallic sheen. With transmitted light, gray-brown centers with clear edges.
Salmonella Shigella Slightly elevated, medium size 1-2 mm. in diameter. Transparent, from colourless to amber.
C. albicans Feathery, spider-like colony after 24-48 hours incubation in CO2 at 35-37°C. Never presents a typical colonial appearance.
Coagulase-positive staphylococci Very small punctiform, colourless and inhibited.
Proteus species When there is no swarming, similar to Salmonella and Shigella. Swarming can be minimized by adding a very small amount of alpha-p-nitrophenyl-glycerol.
Bibliography
American Public Health Association. Diagnostic Procedures and Reagents. 2nd Ed. APHA, Inc. New York, 1950.
American Public Health Association. Examination of Dairy Products. 10th Ed. APHA, Inc. New York, 1953.
Society of American Bacteriologists. Manual of Microbiological Methods MacGraw-Hill New York, 1957.
Microbiological Test
Microorganisms Growth Colony colour
Enterobacter aerogenes ATCC 13048 Satisfactory pink
Escherichia coli ATCC 25922 Satisfactory green with metalic shine
Salmonella typhimurium ATCC 14028 Satisfactory colourless
Pseudomonas aeruginosa ATCC 10145 Good colourless
E.S.T.Y. BROTH Cat. 1254
For the cultivation of lactic streptococci
Formula in grams per liter
Tryptone... 5,00 Soy peptone ...5,00 Beef extract... 5,00 Yeast extract...2,50 Ascorbic Acid ... 0,50 Magnesium sulfate ...0,25 Disodium Glicerophosphate... 19,00
Final pH 6,9 ± 0,2 at 25ºC
Preparation
Suspend 37,25 grams of the medium in 900 ml of distilled water. Mix well. Heat to boiling with frequent agitation until complete dissolution. Adjust final volume to 1000 ml.
Sterilize by autoclaving at 121°C (15 lbs sp) for 15 minutes. Allow to cool to 45-50°C an add 50 ml of an sterile solution of 10% lactose.
Uses
Its utilization have been described for bacteriofagues Assays.
It's recommended for initial culture maintenance which produce acids in its metabolism.
This medium has a high buffer capability due to the disodium glycerophosphate which acts as a regulator pH agent, and inhibits the growth of Lactobacillus bulgaricos isolating S. termophilus from yogurt. The Ascorbic acid stimulates the growth of lactic streptococci.
Bibliography
Reiter B., and J.D. Oram. 1962. Nutritional studies on cheese startters. I. Vitamin and aminoacid requirements of single strain starters. J. Dairy Res.
International Dairy Federation 1981 Identification and enumeration of microorganisms in fermented mil KS.
Microbiological Test
Microorganisms Growth
Lactobacillus bulgaricus ATCC 11842 Inhibited
Streptococcus termophilus ATCC 14486 Satisfactory
E.S.T.Y. MEDIUM Cat. 1555
Selective medium for the enumeration of Streptococcus termophilus in yogurt
Formula in grams per liter
Disodium Glicerophosphate ...19,00 Tryptone ... 5,00 Soy peptone ...5,00 Beef extract ... 5,00 Yeast extract ...2,50 Ascorbic Acid ... 0,50 Magnesium Sulphate ...0,25 Bacteriological Agar... 11,00
Final pH 6,9 ± 0,2 at 25ºC
Preparation
Suspend 48,30 grams of the medium in 900 ml of distilled water. Mix well. Heat to boiling with frequent agitation until complete dissolution. Adjust final volume to 1000 ml.
Sterilize by autoclaving at 121° C (15 lbs sp) for 15 minutes. Allow to cool to 45-50ºC and add 50 ml. of an sterile solution of 10% lactose.
Uses
Lactic streptococci produce acid and are difficult to grow, this medium buffers the acid from the lactose fermentation while the ascorbic acid promotes the growth of lactic streptococci. Its a recommended medium for
Streptococcus Termophilus isolation and enumeration in yogurt, due to the glycerophosphate high concentration it inhibits lactobacillus bulgaricus development. It has been recommended by Milky International Federation for this use.
Bibliography
Terzaghi, B.E. and W. E. Sandine. 1975 Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol 29:807-813.
International Dairy Federation 1981. Identification and enumeration of micro-organisms in fermented milks. Joint IDF/ISO/AOAC Group E44.
Microbiological Test
Microorganisms Growth
Lactobacillus bulgaricus ATCC 11842 Negative
Streptococcus termophilus ATCC 14486 Positive