The coconut water egg malachite green medium (CEM) for the Isolation of mycobacterium tuberculosis

Media

Part of The Journal of the Philippine Medical Association

Title: The coconut water egg malachite green medium (CEM) for the Isolation of mycobacterium tuberculosis
Creator: Basaca-Sevilla, V.; Sevilla, Josue S.; Faraon, Potenciana C.; Fernando, Celeste L.; Uvero, Josefina A.
Language: English
Source: The Journal of the Philippine Medical Association Volume 52 (Issue Nos. 9-10) September-October 1976
Year: 1976
Rights
Fulltext: ORIGINAL ARTICLES The Coconut Water Egg Malachite Green Medium (CEM) for the Isolation of Mycobacterium Tuberculosis * • First Prize PMA-Abbott Research Award on Basic Science 1976. V. BASACA-SEVILLA, M.D., M.P.A., ** JOSUE S, SEVILLA, D.D.M., *** POTENCIANA C. FARAON, D.D.M., ** CELESTE L. FERNANDO, A.B. ** and JOSEFINA A. UVERO, B.S. Pharm. ** TUBERCULOSIS in all forms is the sec ond leading cause di deaths in this coun try, fourth in causing disease and the number one disease killer of our child ren. On its early diagnosis depends its cure and control and an assured healthy people. Toward this end, government and private agencies have joined efforts in the detection and treatment of in fectious cases as well as the institution of preventive measures. The most util ized tools of detection are sputum mi croscopy and x-ray. While sputum mi*The Process and Product is covered by Philippine Patent No. 9768 awarded First prize (Pharmaceuticals and Chemical Ca tegory) Invention Contest on the 10th Na tional Philippine Inventor's Week, April 9, 1976. ••Division of Laboratories, Bureau of Re search and Laboratories, Department of Health, Manila. ***Phi lippine Atomic Energy Commission. croscopy lias been the least expensive and most efficient tool in mass case de tection, there is still need for other lab oratory tools in the more scientific stu dy of detected cases. The isolation of Mycobacterium tuberculosis is one of the more reliable aids in the diagnosis of symptomatic cases with negative spu tum for acid fast bacilli, For those cas es under therapy with apparently no cli nical or bacteriological improvement, a bacteriological work-up is necessary to determine sensitivity or resistance to the drug being used, or where a possible change of drug is contemplated. For those where actual identification of the infecting organisms is necessary, a bac teriological work-up is the only tool. Today less than 10 clinical laboratories all over the country can afford to con tinuously and regularly do the routine bacteriological isolation of Mycobacte251 252 Basaca-Sevilla, et aL Sept.rOct., 1976 J. P. M. A. rium tuberculosis. Currently these lab oratories are utilizing a medium princi pally composed of eggs or other protein sources, mineral salts and a dye. With the exception of eggs and the distilled water, the reagents and chemicals that make up the formulas have to be pro cured from abroad entailing difficulties like irregular supply, increased dost, and because too few laboratories are using the special medium, there is very little interest to make the purchase from ab road. The formula of the medium currently in use for the isolation of M. tuberculo sis may be one of the following: 1. Lowenstein-Jensen medium, modified KH2PO4 ........................................................................ 2.4 gm. MgSO4.H2O .............................................................. 0.24 Magnesium citrate .................................................... 0.60 Aspargine .................................................................. 3.6 Glycerol ........................................................................ 12 ml. Malachite green 2% aq. solution ............................ 26 ml. Potato flour ............................. 30 gm. Distilled water .................................... 600 mL Eggs (fresh, whole) .................................................. 1000 ml. 2. Lowenstein-Jensen medium, modified Mineral salt solution KH2PO4 .................................................................. 4.0 gm. MgSO4 .................................................................... 0.4 Magnesium citrate ............... 1.0 Aspargine ................ 6.0 Glycerol ................................................................. 20.0 ml. Distilled water .................................................... 1000.0 ml. Complete formula Mineral salt ........................................................ 300.0 ml. 2% malachite green .......................................... 10.0 ml. 10 beaten eggs, approx. ................ ......... 500.0 gm. 3. American Trudeau Society (ATS medium, modified) Potato peeled and diced .................... 140 gm. Glycerol, reagent grade 2% ............. 335 gm. Egg yolk (fresh) with) 3 white ....... -.... 400 ml. 2% malachite green ................................................ 10 ml. 4. Petragnani medium a. Pasteurized, homogenized whole milk ... 275.0 ml. Potato starch {Fisher) ............................ 20.0 gm. Aspargine (Difco) ............ -................ 1^9 gm. b. Fresh whole eggs- ........... 10 Fresh egg yolk .................................................. 3 Glycerine .... ..................................... 30 ml. c. 2% aq. sol. malachite green 5. Middle-brook-Cohn 7H-10 agar Solution 1 Monopotassium phosphate, ACS .................... 15 gm. Disodium phosphate, ACS .............................. 15 gm. Distilled water .................................................. 250 ml. Mycobacterium Tuberculosis 253 Volume 52 Nos. 9-10 Solution 2 Ammonium sulfite, ACS .................................... 5.0 gm. Monosodium gllutamate .................................... 5.0 gm. Sodium citrate (2 H2O) USP .......................... 4.0 gm. Ferric ammonium citrate .................................. 0.4 gm. Magnesium sulfate (7 H^O) .......................... 5.0 gm. Biotin (in 2 ml. 10% ammonium hydroxide) .......................................................... 5-.0' gm Distilled water ............................... ..................... 250.0 ml. Solution 3 Calcium chloride (2 H^O), ACS .................. 50.0 mg. Zinc sulfate (7 H2O), ACS .............................. 100.0 mg. Copper sulfate (5 H^O), ACS.......................... 100.0 mg. Pyrodoxine HCL .................................................. 100.0 mg. Calcium panthothenate .................................... 100.0 mg. Distilled water ...................................................... 100.0 ml. Solution 4 Reagent grade glycerine Solution 5 Malachite green, 0.01% aq. sol. Solution 6 Albumin-oleate dextrose solution 50 gm. bovine albumin, fraction V in 900 ml. sterile saline Sodium oleate solution 50% aq. solution of dextrose A glance through the above formulas will reveal their varying complexity, the cost it will entail in their preparation, the time involved in the weighing of each constituent especially those where an analytical balance is required, the physical facilities for the preparation and the need for personnel of higher tech nical training. The preparation of for mula nos. 4 and 5 require more skill. Formula no. 3 or ATS medium is a more simple one, but still it makes use of po tatoes which have to be peeled and diced besides eggs. METHODS In answer to the need for maximizing the utilization of isolation and culture of M. tuberculosis in all laboratories, a new medium, the coconut waTer egg mala chite green medium (CEM) has been devised. It has the following formula: Coconut water .................. 300 ml. Whole eggs ........................ 10 (approx. 500 gm.) 2% malachite green Water from young green coconut is sterilized By boiling or autoclaved for 10 min * at 10 lbs. pressure. The eggs are cleansed thoroughly with soap and wa ter; rinsed well and soaked in 70% al cohol, and dried with sterile towel. The eggs are aseptically broken into a sterile conical flask or an Erlenmeyer flask with a pipette or glass rod. The flask is shaken to break up the eggs and the coconut water added with malachite green solution enough to produce a light green color of the mixture. The contents are shaken well to mix, then filtered through sterile gauze. The mixture is Sept .-Oct., 1976 J. P. M. A. 254 Basaca-Sevilla, et al. aseptically distributed into screw-capped test tubes and inspissated at 80° to 85°C one hour in a slanting position. The sputum swab culture method de vised by Nassau was followed in the evaluation of this medium for the isola tion of M. tuberculosis from sputum. The modified Lowenstein-Jensen medium, formula 2, was used as control. One or two sterile swabs are moistened with sterile distilled water and both held in one hand, dipped into the sputum sample and vigorously rotated, mixing well the sample. The swabs are then placed in tubes, two-thirds full with 5% sterile oxalic acid and allowed to stand at room temperature for 35 minutes. Then the swabs are transferred to another tube also two-thirds full with sterile 5% so dium citrate and allowed to stand in the solution for 10 minutes. Two slopes of CEM and of Lowenstein-Jensen me dia are inoculated with each sample. The swabs are firmly rubbed on each slope while the swab is being rotated. RESULTS Laboratory standard strains of Myco bacterium like H37Rv, M. xenopei, M. avium, M. kansasii, a Scotochromogenic strain, 607 strain, BCG strain and M. le prae murium have been cultured and maintained in CEM medium and M. tu berculosis have been successfully isola ted from sputum samples using CEM. Illustrations of these cultures may be seen in Plates 1—4. It has been noted that the growth of the Mycobacterium strains in CEM has been very good. The standard laboratory human Sara nac virulent strain H37Rv has maintained the rough characteristic of its colonies light buff color, somewhat dry in CEM as in the Lowenstein-Jensen medium. A Group II Scotochromogenic strain of the Runyon group of atypical mycobac teria has shown its moist, smooth, con fluent colonies with yellow to orange pigments in both CEM and LowensteinJensen media. Mycobacterium avium, a virulent avian tubercle bacilli, produced smooth glistening colonies with cream or buff non-photochromogenic confluent co lonies in both CEM and Lowenstein-Jen sen media. Another atypical mycobac terium Mycobacterium xenopei in both CEM and Lowenstein—Jensen media produced smooth, pale yellow non-photochromogenic, moist growth. Another lab oratory strain used by the NIST is strain no. 607 of Runyon Group IV which main tains its confluent, finely irregular, light buff colored colonies in CEM as in Lo wenstein-Jensen medium. Mycobacterium kansasii of Runyon Group I showed yel low pigmented somewhat rough colonies when exposed to light in both media. A-20, a strain of the Scotochromogenic group isolated locally from sputum did not lose its yellow orange pigmented colonies when grown in CEM. CEM was found as efficient as Lowens tein^ Jensen medium in the isolation of Mycobacterium from sputum samples; buff colored rough colonies were prod uced, At no instance were negative re sults obtained from CEM when Lowens tein-Jensen medium was positive for iso lation. Using the sputum swab culture tech nique of Nassau, contamination from other bacteria presented no problem in utilizing CEM or Lowenstein-Jensen me dium. CEM was provided by the authors to another independent worker and he was successful in growing both M. leprae mu rium and BCG strain. Volume 52 Nos. 9-10 Mycobacterium Tuberculosis 255 Plate I — Shows the characteristic growth of M. xenopei, M. avium, M. kansasii, H37Rv, H607 and a Scotochromogen strain in CEM and LJ media. Sept.-Oct., 1976 J. P. M. A. 256 Basaca-Sevilla, et al. Plate H — Shows a close-up of H37Rv and a Scotochromogen strain in both CEM and LJ media. Mycobacterium Tuberculosis 257 Volumo 52 Nos. 9-10 — I Plate III — Shows the isolates from a sputum sample (378) on different days after inoculation of CEM and LJ media. 258 Basaca-Sevilla, et al. Sept.-Oct., 1976 J. P. M. A. Plate IV — Shows the isolates from a sputum sample (379) on different days after inoculation of CEM and LJ media. Volume 52 Nos. 9-10 DISCUSSION CEM utilizes coconut water as its main and only source of mineral salts in addi tion to its nutrients like protdin and car bohydrates, to mix with whole eggs as a culture medium for a fastidious group of organism like the mycobacteria. It was definitely shown in this work that used with eggs it can support the growth of mycobacteria. Blauvett1 reported that non-cooked, non-sterilized coconut water from “ripe fresh fruit” added to ordinary nutrient agar and broth nearly doubled the cul ture qualities of the latter in growing Protein .................................................... Carbohydrates ........................... ...... Fats .......................................................... Calcium .................................................... Phosphorous .......................................... Mycobacterium Tuberculosis 259 Staphylococcus aureaus, B. faecalis alcalis alcaligenes, and B. welchii. Paguio et al2 in 1970 utilized coconut water me dium for the isolation of Vibrio cholerae. Agasan3 in 1975 has proven the sensiti vity of coconut water medium in detect ing coliforms in waters and foods and the growth of Salmonella, Shigella and Klebsiella in the same medium. Several workers have analyzed the composition of coconut water from young green coconuts. Peters4 gave the range of concentration of the im portant components of coconut water per 100 ml. as follows: 0.23 — 0,43 gm.% .............. 3.68. — 5.0 gm.% ............. 0.64. — 0.8 gm.% .............. 0.03 gm.% .............. 0.01 — 0.22 gm.% Pradera and his co-workers5 reported the mineral content of 100 ml. of coconut water as follows: Calcium ................. 29.0 — 46.0 mg. Chlorine .................................................... 105.0 — 160.0 mg. Phosphorous .............................................................. 5.5 — 9.0 mg. Potassium .................................................................. 134.0 — 220.0 mg. Child and Nathaniel6 reported the constituents to be: Water .......................................................................... Nitrogen .................................................................... Phosphoric acid ........................................................ Calcium oxide .................................................... Magnesium oxide .................................................... Iron .......................................................................... Total solids ................................................................ Red sugar as invert sugar...................................... Add. sugar as sucrose .......................................... total sugar ....................................................... ash ................................................................... Unidtentified organic solids .................................... Ascorbic acid (Vit. C) ............................................ Nicotinic acid .......................................................... Panthotenic acid ...................................................... Folic add ................................................................ 95.50% 0.50 0.60 0.69 0.59 0.50 mg. in 100 gm. 4.71 gm./lOO ml. ’0.80 gm./ * fi0 ml. 1.28 gm./lOO ml. 2.08 gm./lOO ml. 0.62 gm./100 ml. 2.01 gm./lOO ml 2.20 — 3.70 mgm./lOO ml. 0.640 microgram/100 ml. 0.520 niierogram/100 ml. 0.003 microgram/100 ml. Sept.-Oct., 1976 J.P. M. A. 260 Basaca-Sevilla, et al. Vanderbelt7 found the following Nicotinic acid Panthotenic acid Biotin Riboflavin Folic acid Pradera and his co-workers (5) made a thorough study of the amino acid con tent of the water and found them preGlutamic acid Arginine-1 Leucine Lysine Proline Aspartic acid Tyrosine Alanine Histidine Phenylalanine Serine Cysteine The present work has shown for the first time the full utilization of coconut water to support the growth of a fasti dious group of organisms not just the use of its “growth factor”. Quite a number of workers have Shown this growth factor as capable of stimulating growth of certain bacteria. Ramakrishnan, et al8 in 1958 has shown that coco nut water even at a dilution of 1 in 10,000 when used as a supplement to the ordinary medium for the cultivation of Mycobacterium tuberculosis showed maximum growth in 12 days only in stead of the usual 20 days. We failed to elicit any change in the growth period of the Mycobacterium in CEM compared Materials KH2PO4 MgSO4 Magnesium Citrate Aspargine Glycerol Distilled water Malachite green 10 eggs amount of vitamin B complex as: 0.64 microgram/ml. 0.52 microgram/ml. 0.02 microgram/ml. 0.01 microgram/ml. 0.003 microgram/ml. sent as peptones based on dry protein content of the water to be as follows: 9.76 — 14.50 gm.% 12.75 gm.% 1.95 — 4.18 gm.% 1.95 — 4.57 gm.% 1.21 — 4.12 gm.% 3.60 2.83 — 3.00 2.41 1.95 — 2.05 1.23 0.59 — 0.91 0.97 — 1.17 to Lowenstein-Jensen although in some cases growth has been more luxuriant in CEM especially in isolations from spu tum samples. Tn our particular work the growth factor in coconut water is only an incidental advantage when the water is utilized as a whole. Green coconuts can be procured any where especially in the provinces. The simplicity of the formula and prepara tion does not require much technical skill nor physical laboratory facilities which suit local conditions. A laboratory that is going to start a TB bacteriology service would need to invest in the following if it will utilize Lowenstein-Jensen medium: Cost P 42.00/lb. 56.70/lb. 45.00/lb. 150.00/100 gm. 95.00/480 co. 2.50/liter 50.00/25 gm 4.50 Volume 52 Nos. 9-10 However, if it will utilize CEM, the Green coconut 10 eggs malachite green So that it is very apparent that one has to have about P445.00 to start with the Lowenstein-Jensen medium and only about P56.00 or only 1/8 of the former, for CEM. With CEM, even if malachite green is not available, the medium can still be used. CEM is a medium that can be produced from materials loeally available. Since it is a cheap medium, the pre paration of CEM may be done at a cen tral laboratory and tubes of the medium dispensed to peripheral areas. Actual production of the media can even be done in a small laboratory. The tubes can be inoculated in the peripheral area using the sputum swab culture techni que of Nassau. The inoculated media are then transported to more developei laboratories where it can be incubated, observed and studied. Instead of screw capped tubes, ordinary tubes with rub ber stopper may be used, thus further lowering the cost of each slope. CEM keeps very well at room temperature for at least 3 months and sterile coconut water for about 6 months. The addition of malachite green improves the gross visibility of the colonies. The green co lor of the media brings out very well the colonial characteristics. CEM is an ideal medium for the iso lation of mycobacteria since it supports early and eugonic growth for small ino culum; enables easy recognition of or ganisms; is easily prepared and inex pensive; and keeps the growth of conta minating organisms to a minimum. CEM medium is most useful wherever tuber culosis is a problem; wherever there is Mycobacterium Tuberculosis 261 cost estimate would only be: P 0.30 — 0.70/nut depending on where you procure it 4.50 — if procured in the city 50.00/25 gm. need for a more scientific work-up of detected cases especially for antimicro bial studies; wherever there is a lack or shortage of technical skills and physical laboratory facilities; wherever there is difficulty in the procurement of chemic als and reagents produced or manufac tured in developed countries; wherever there is need to lower the cost of medi cal care delivery and wherever coconut is grown. GEM answers all these needs, which wiM not only be in the Philippines but in many developing countries where tuberculosis is a health problem. SUMMARY For the first time a coconut water egg malachite green medium (CEM), has been devised for the isolation of Myco bacterium tuberculosis. CEM utilizes all the coconut water as its main and only source of mineral salts in addition to its nutrients like protein and carbohydrates to mix with whole eggs as a culture medium for a fastidi ous group of organisms like mycobacte ria. Laboratory standard strains of mycobacteria fike H37Rv, M. xenopei, M. avium, M. Kansasii, a Scotochromo genic strain, No. 607 strain, BCG strain and M. leprae murium have been cul tured and maintained in CEM. Mycobac terium tuberculosis has been successfully isolated from sputum samples using CEM. CEM is simple and inexpensive nor does it require much technical skill and physical laboratory facilities to prepare. It is an ideal medium for the isolation of Mycobacterium since it supports early 262 Basaca-Sevilla, et al. Sept.-Oct., 1976 J. P. M. A. and encourage growth for small inocu lum; enables easy recognition of organ ism and keeps the growth of contaminat ing organisms to a minimum. It utilizes materials locally available. CEM will be most useful not only in the Philip pines but in other developing countries where tuberculosis is a health problem. ACKNOWLEDGEMENT Sincere appreciation is due to Dr. J. SSumpaico, Director of the Bureau of Re search and Laboratories, for his interest in making this study possible; our sin cere thanks to those who in one way or another helped in this study particularly to Mrs. Remedios Fernandez, Mr. Eduar do Fontanos, Mrs. Erlinda Villadelgado, Mr. Bernardo Fotruna and Mr. Sol Dungca; to Mr. Sofronio Tomandao for the photographic illustration in this paper; to Miss Victoria Sanchez for her patience in typing the manuscript of this article; to Dr. Tirso Banzon and Attys. Fermin Galang and Nicolas Tayao for helpful suggestions in the preparation of this ar ticle; to Dr. Eulalia Venzon of the NIST and Miss Esperanza Basaca. of the NSDB for valuable assistance in the li terature research; our special apprecia tion to Dr. Antonio Jacalne of the Ins titute of Public Health, U.P., for his interest and actual trial of the medium and encouragement to go through with the study. REFERENCES 1. Blauvett, LJM.C. Asheville, The Use of Non-Cooked, Non-Sterilized Coconut Milk as an Additional Nu trient Substance on Culture Media, J. Lab. Clin. Med., 24, 4, 420-423 (1938). 2. Paguio, A. and N. Lopez. Coconut Water Medium in the Laboratory Diagnosis of Cholera, J. Phil. Med. Asso., 46:429-439, 1970. 3. Agasan, A.L. Coconut Water as Culture Medium for the Detection of Coliforms in Water and Foods, Thesis (M.S.H.), Institute of Public Health, 1975. 4. Pieters, F.B. The Coconut In the Human Diet, Food and Nutrition Notes and Reviews, Phil. Sci., 83 (4), 1954 5. Pradera, E.S., E. Fernandez and 0. Calderin. Co conut Water — A Clinical and Experimental Stu dy, Am. J. Dis. Child., V, 64 (1942) 977-996. 6. Child, R. and Nathaniel, W.R.N. Utilization of Coconut Water, Tropical Agriculturist (Ceylon) V, 103 (1947) 85-89. 7. Philocoa Compilation on Coconut Water. 8. Ramakrishnan, T., Indira, M. and Sirsi M., J, Indian Institute Science, 4C (15) 1958. 9. Gomez, L.P. Isolation and Characterization of the Growth Factor in Coconut Water, Thesis (B.S. Chem.), issued as Philcoa Technological Research Bulletin No. 7, 1961. 10. Nassau, E. Sputum Swab Culture: Simple Method of Isolating Tubercle Bacilli for Sputum Tuber cle, Lond. (1958) 39, 18. 11. Gradwoht's Clinical Laboratory Method and Diag nosis, Vol, 2, seventh ed., Mosby. 12. Clinical Tuberculosis Essential of Diagnosis and Treatment, Am. College of Clinical Physicians, 1966. 13. Mycobacteria: Isolation, Identification and Sen sitivity Testing, 1968, Butterworths. 14. Philippine Health Statistics, 1973. 15. Jacalne, A. — personal communication.
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