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Jour. Myan. Acad. Arts & Sc. 2005 Vol. HI. No. 4(i) Zoology

Exploration of Yeast and Contaminants in Seed Culture and Fermented Wort from Gyo Gon Alcohol Distillery Ngwe Thein

Abstract Study was made on samples of seed culture and fermented wort from Gyogon alcohol distillery. In a!l samples bacteria contaminants were observed. Samples were cultured on Sabouraud dextrose , Czapek Dox agar, and media and broth. The selected colonies were isolated. Biochemical tests for identification were conducted. The yeast and bacteria contaminants were identified by morphological characteristics and biochemical reactions. The yeast isolated and identified from Gyogon alcohol distillery was Sacchacromyces cerevisiae. The bacteria contaminants isolated and identified were Aeromonas sp. and Pseudomonas sp.

Introduction Alcohol distillation plants are becoming an essential beverages production in the world arid has a ever increasing potential for expansion, particularly in agricultural countries like Myanmar as the raw materials required for alcohol production are abundant and could be easily obtained in this country. The production of wine and beer were the oldest traditional process employing yeasts. Ethyl alcohol is prepared commercially by the action of chiefly yeast on various carbohydrate, crude molasses, potatoes, sugar beets, corn ,tapioca, hydrolyable starch and cellulase (Bryan and Bryan, 1960), Antonie van Leeuwenhock was the first person who described yeast cell by the aid of his famous microscope in 1680 (Peterson and Tressler , 1963; Rose, 1977) It was recognised than as living organism, associated with fermentation (Perterson, 1963). The species commonly used in the manufacture of bread, whisky and alcohol is Saccharomyces cerevisiae (Beeze, 1970). (Fig. 1 A). In 1837. three men Latour, Schwann and Kiitzing proposed that the yeast which appeared during the alcoholic fermentation was a microscopic plant and that the conversion of sugar to ethyl alcohol and carbondioxide

Lecturer, Department of Zoology, University of Yangon 110 Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(i) Zoology characteristic of the alcohol fermentation was [a physiological fermention of the yeast ceil. (Fig.l A;toD) In alcohol distillery fermentation of.carbohydrate has attracted a wide variety of bacterial contamination which will reduce the alcohol yield and effect the quality of the product. The common microbial contamination as claimed by Stainer et a! (1965) are wild yeast, Lactobacillus, Pediococcus, Acetobactors, Acetomonas and Zymomprias.These, organism make the production yield lower. ! Aim : To expore yeast and bacteria contaminants in seed culture and fermented wort from Gyo-goo alcohol distillery. Objectives of this project are 1. To isolate and identify the yeast and contaminant bacteria in seed culture and fermented wort. 2. To control the contamination in ail processing steps of operation in alcohol distillery. 3. To maintain the production in full capacity for maximum profit. 4. To promote higher yield, of alcohol distillery. 5. To obtain best results of pure culture and to maintain the purity of yeasts correctly. 6. To promote the development of the industrial sector, involving private and Government ^Industries. It can be said surely that this will contribute much towards the smooth flow of alcohol production thereby helping, the private sectors and economy of the country. Material and Method Material Collection of samples Two type of samples seed culture (8 hr) and fermented wort (32 hr old) were collected in 100 ml and 500 rnlamount from the selected distillery for the study. The samples were collected and transported to laboratary and cultures were made; and the collection were stored on the refrigerator at 4°C.(Plate A) Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No, 4(i). Zoology 11 1

Glasswares, Equipment and Apparatus 'used;.!; Beakers (all sizes), conical flasks, flat-bottom flasks petridishes, fine pipettes, measuring cylinders, glass slides, test tubes vials with screw caps. Anaerobic, jar, analytical balance, •autoclave, centrifuge hot plate, , microscope, sterilezer, water-bath; camera. (Nikon AF,600, with colourfilm, 200ASA), Olympic camera PM-6j attached with microscope, bunsen burner (gas), were used.

Media, Chemical and Reagent used The chemical media used in the present investigations were products of B.D.H DIFCO OXOID, and FISHER TECH CO. USA. were prepared as described by manufacture's instruction (Cmickshank, 1968)Sterilization by wet heat was carried out by the use of an outoclave under steam pressure.

Methods Direct examination . The sample was taken by stcrlized loop and spread on a clean slide and left to dry. Then the slide was passed over flame. Gram shown morphology of microorganisms.

Culture of yeast and bacteria contaminants. Sterilized melted media of Sabourattfi dextrose agar, Czapek Dox agar and nutrient agar were poured into sterilize petridishes and allowed to harden. With a sterilized loop, the samples of seed and wort were streaked over the surface of the three agar plates which were previously dried in incubators.

Incubation After the incubation the colonies were picked up by sterilized straight wire and transferred to the Sabouraud broth, Czapek broth and peptone water under aseptic condition. It was incubated, aerobically, at 37°C and room temperature. 112 , Jour. Myan. AcatL Arts &Sc. 2005 Vol. III. No; 4(i) Zoology

Yeast identification .. • ;- - The morphological characteristics of yeast isolated from seed and wort were matched with those of particulars of Saccharomyces species as described by Breed,.Murray and Smith (1957), Harrigan and McCance(1966.) :; The isolates were sown in sets of carbohydrate media composing of maltose,.sucrose, giatose, arabinose, sobitol, glyceral and mannitol in phenol red peptone water. Onepercent of.each of carbohydrate (sugars) were used for determining the fermentative reaction. The fermentation was indicated by change of colour from red to yellow (Conn, 1951) was noted positive.

Identification of bacteria ' After recording the growth characters on agar and development of sediment superficial ring or pellicle in broth, gram staining was carried out (Conn, 1951), Motility tests was: done from young broth culture by hanging drop preparation (Seroking and Cullimore, 1969) Growth in air and growth anaerobically were rioted. In Preliminary tests, test, oxidase tests, oxidation fermentation were carried out (Cowen and Steel(1965),Cowen (1974)as follows: (C) Primary tests •

Gatalase activity test Isolates were inoculated on nutrient agar plates, and,incubated at room temperature 29°C and 37°C. Three percent hydrogen peroxide solution was dropped on the colonies on the;solid media. The evolution of gas bubbles observed were noted as catalase positive (Cowan and Steel, 1965).

Oxidase activity test A loopful of the culture was smeared on the surface of a piece of filter paper which was soaked with a few drops of one percent tetramethyl-p- phcnylenediamin in hydrochloric solution. When a dark purple colour developed within 10 seconds, it was noted as oxidase positive (Kovacs, 1956). Oxidation, fermentation test . • ,, The isolates were inoculated by a straight wire in to the O.F medium tubes. One tube was kept in anaerobic jar and another tubes was kept Jour. Myan. Acad. Arts & Sc. 2005 Vol. IIL No. 4(i) Zoology 113

aerobically and both were kept in incubator at;37°C 48 hours. Acid in both tubes were noted fermentation and acid production in open tube .'arid • no change in the other tube in anaerobic jar was noted as oxidation (Hugh and •Leifson, 1953). '

Confirmatory tests of bacteria Secondary tests of bacteria for identificationto. specices levejvof the isolates were done as follows:

Gelatin liquefaction test The isolates were inoculated into the ^sterilized nutrient gelatin tubes and incubated as usual for 2 to 7 days. Liquefaction was checked daily by placing the tubes in a refrigerator for 30 mimites, and liquefaction was noted as positive,(Cruickshank, 1968).

Nitrate reduction test .About two or three drops of nitrate test reagent were dropped iinto 3 days cultured in nitrate broth tubes. Development of a red colour indicated the presence of nitrate and recorded as positive (Conn, 1951).

Indole production test Kovacs reagent of about 0.5 ml were added to 48 hours, culture m peptone water "tubes. Development of a red Colour in the reagent layer was rioted as positive reaction. (Kovacs, 1956).

l red (M.R) test •Two or three drops of reagent were added to two to five days culture in MRVP medium. Presence, of bright red colour indicates the positive reaction (Cmicksnanfc; 1965). ; 114 Jour. My an. Acid. Arts & Sc 2005 Vol. III. No. 4(i) Zoology

Voges Proskauer (V.P) test: One mi of 40 percent potassium hydroxide and 3 ml of 5 percent alpha napthol in absolute etliynol were added to MRVP medium, and cultured as usual. Development of a pink colour in 2 to 5 minutes indicates the positive reaction-(Conn, 1951).

Hydrogen'.sulphide production ; Detected by a slip of filter jpaper soaked in 1 ()%!' lead acetate solution between the tube and the cotton wpol plug. Lead sulphide in the presence of ELS produces bacteria lead to browning or, blackening of the media or filterpaper, (Craickshank, 1968).

Hydrolysis of starch A nutrient agar addition of 0.2 -1% soluble starch, after preparing plates and then over laying 5 ml;,of. starch agar, then streaking the surface, incubate at optimum temperature for 2-14 days. Flooded plate with 5-10 ml of iodine solution, hydrolysed starch appear as a clear zone noted positive

Hydrolysis of lecithin A nutrient agar with 1% NaCl and 10% (v/v) egg yolk emulsion poured dried plate of medium by streaking once across the surface and incubate at optimum temperature for \A days. Lecithinase activity result in formation of opaque zone around7 the region of microbial growth and record die opaque zone, noted positive(Collin and Mlyne (1970).

Fermentation of sugars To peptone water add 0.5>-l~ 1.0% of fermentable substrate such as glucose, sucrose, xylose, fmctose/mannitol, sobitol arabinose, mannose with phenol red 0.01% indicator,. Sterilize by steaming for 30 minutes on 3 successive days then solution by':]Alteration and added to* test tubes, and incubate for up to 7 days. Acid production is shown by a change in colour red to yellow noted positive. Jour, Myan. AcadL Arts & Sc. 290S Vol. III. No. 4(i) Zoology 115

. Results Examined microorganisms Direct microscopic examination of seed culture and fermented wort showed dense growth of contaminated, microorganisms in the samples collected from Gyo gon alcohol distillery. Rounded or oval-shaped yeasts cells were predominent amongst the detected microorganisms. Asexual reproduction, binary and budding processes ipf the yeast cells were also observed. The rod-shaped cells in the microorganisms indicated the presence of bacteria. (Plate. C)

Characteristics of microorganisms after staining;> The stained smear showed that the yeasts were Gram positive with rounded or oval-shape. Yeasts cell were rhucht larger than rod-shaped bacilli that were Gram negative and non-sporing.

Growth of microorganisms The yeasts appeared as small, white particles in broth. On the agar plates, yeast colonies were observed as moist;' white opaque small smooth circular discs. Sometimes, the colonies may be pinkish (Plates B). . Similarly, bacterial colonies were observed on agar plates. The bacterial colonies were white, opaque, small convex circular dies. Greenish colonies were also observed in the isolated samples.

Reproductive nature of yeasts Yeasts reproduce vegetatively by binary fission, (Plate E). budding ind formation of ascospores. Isolated yeast on agar plates underwent the processes of binary fission and budding; Ascospore formation took place jwhen yeasts were cultured on Gypsum block and potato slice after keeping them for a period of 12 to 14 days either at 37°C or at room temperature. Wscospores when stained with malachite green, the rounded spores turned green while the cells appeared as red or pink.' Each ascus consisted of four ispores, (Plate F). 116 Jour. Myan. Acad.Arts & Sc. 2005 Vol. III. No, 4(i) Zoology

Biochemical reactions of yeasts and bacteria Biochemical reactions of isolated yeasts using 10 different carbohydrates, (maltose, sucrose, glucose, fructose, raffmose, arabinose, glycerol, salicin, lactose and mannitol) showed that the yeast isolate fermented maltose, sucrose, glucose, fructoase. Of the remaining carbohydrates raffinose was partially fermented while the rest were not fermented ,(Table 1, a and b). Varying biochemical reactions were recorded Vyith different isolates o( bacteria when tested with 8 kinds of carbohydrates (arabinose, glucose, lactose, maltose, mannitol, salicin, sorbitol and sucrose).Of these carbohydrates tested, only the;-: fermented carbohydrates^ are mentioned together with number, of isolates from the samples of respective studied distilleries. A total of 22 isolates were recorded from Gyogon distillery. Twelve isolates from this distillery ferment arabinose, glucose, -maltose, mannitol, sorbitol, and sucrose, while the other 10 isolates ferment glucose, maltose and sorbitol,(Table 2)

Identification of yeasts and bacteria Based on the findings of cultural characteristics, morphology, primary tests (, spore formation, growth characteristics, motilily, catalase, oxidase and OF tests), and secondary test (Biochemical reactions) isolated yeasts from Gyo Gon distillery was identified as Succharomycv.s'ccrcvisiui'. (Plate D).. - Twelve bacteria-isolates \yare1 identified as Acrornomis species and ten isolates were identifed as • PscuJpnionas species respectively,(Table 2) (Plate G and HV Jour. Myan. Acad. Arts &Sc. 2005 Vol. III. No. 4(i) Zoology

Table 1. Characteristics of yearst isolate from "Gyo gon" alcohol distillery (a) - Cuture Colony morphology Cell morphology test Id t'f d Media/ Temp 30 Colonies Surface Colour Stain Shape . Arrangement Motal Reproduction isolate broth C/34C • Sabouraud Growth+ Small Smooth White Gram+ Ova! Grouping ->neg Vegetative Yeast dextrose circular moist and . round Budding agar pH- opaque convex pink fission 4.5-5.0 Czapek Growth+ Small Smooth White Gram+ Oval Grouping --nsg Vegetative Yeast Dox agar circular moist and round Budding pH5 pink, fission Sabouraud Growth-r White Sediment White ;. Gram+ Oval Grouping --neg Yeast broth particle round Czapek Growth+ White Sediment White Gram+ Oval ; Grouping ' - -neg Yeast. broth particle ..round •., Potato slic Growth+ Small Smooth White Cell, pink Oval Grouping --neg 'Spores Yeast" ' (Sporu circular moist Sporegreen round lation 14 convex days)

Charaters of biochemical reaction (b) Fementation test Identified isolates Isolate Maltose Sucrose Glucose Fructose Raffinose Arabir.ose Glucose Salicin Lactose Mannitil Sobital Nine Partial - Saccharom isolate yces • cerevisiae rrable2. Characters of Bacteria Contaminants Isolated from Fermented Wort ("X" Distillery) Primary Test Secondary Test (Biochemical Reaction

Identified Spacies '.'8 -"8 1 '.§•- 1 •••© '•fi; I % • o Starc h Hydrolysi s Gelati n Liquefactio Sucros e ' • - . No. 3 Reductio n Arabinos e Gro W i n ai r Growt h i n anaerobi c Motalit v GramStai n Temperatur e 37H C Noo f Isolates s -G i i i .6 1 ' Maltos e • "+I GO + >4- Gl + +f + + • .F • + + + Aeiomon + -• •+" as. sp + + - +f F •-". • + +• - + + ± + G2 + + + +

4. + • + - G3 - - + + -rf F + •••+•' +

+ + • + - + + + + + • + G4 - - + +f + F + + ±

•- - + ± + G5 - - + + +f + + + F +

+ -. _. + G6 - - + •+f + + F ++ + + ± + + _-' - + - +f F + + ± + G7 - + + + + + - +• +f F - + + ± G8 - + + + +

- + + • + + F + + + ± + G9 - - + +f ++ +

- + + + - + ± + - + +f F + + G10 - + Jour. My an. Acad. Arts & Sc 2005 Vol.IIL No. 4(i) Zoology 119

' \- Primary Test -^ Secondary Test (Bioc iemical 3leactioh '•.'."""

'Jcr r co , 2 a CO o Identified •s 1 0> Spacies CO "o • i-H " 0) c - (D •1

s CO ^Lactos e , JPigsmen t o J ••%--

- •.. Gir '- 'f :+f; 4- J + F + .+ '--• ± "+- •'+

G12 - .+ + +f •; •"+" '.' -f F - + 4- + .'+•• •± .+ + -. :+ +

•G13 - .. - + ,+f' + :•+ ' + 0 + + - •- ,+' - -- .- + •-'. -• • Pseu

G14 - - + _• + +f + -f , 0 + • + - + + + -

G15 - - ' •+ +f •+• + ';•' + 0 +' ' + , • + - ».+ •- '-

+'.• - G16 •+••; +f •+" :+••'. 0 + •. •+'• "• " •*. - : G17 .-" •;.+ •; •;+• • :+f; Vrl- •' ;'+ 0 + : + + + + . -- - . - ,;

+• G18 - "•.-•"• •+f ;+ O :-+•-•••. -

a:

- •-• + • + - G19 ;+f "y- + 0 + + + + +

,--•; >•- G20- *•* +- + +.f + - + 0 + • + + - + + + 22 -; • • 120 Jour. Myan. Acad. Arts & Sc. 2Q0S Vol. HI. No. 4(i) Zoology

Discussion The yeast samples collected from the alcohol distillery of Gyogon were mature and Wealthy strains of yeast. The samples were found to be full of contaminating bacteria (Plate C). which will retard the production of alcohol. By studying the ceil morphology under microscope it is apparent from the data presenting and the Sacchqromyces cerevisiah, isolated from the seed cultured and fermented wort were able to propergate the vegetable reproduction by one to four ascospore in an ascus,(De Beeze i970), Nickerson and Rose (1970 ), Harrigan and Mcpance (1966), Rose,(l;977). The yeast colony under microscope was found raised at the centre and growing to a large extent and actively growing cells surround the outer part of the colony. . . I ^ . The character of purified'culture were in agreement with those of Sac char omyces cerevisiae as described by Beed et. al (1957), Peiacza and Reed (1965), Harrican and McCarke (1966), and Rose and Harrison (1969). Thus the yeast species currently employed in alcohol distillary of "Gyogon" was identified as Sacch. cerevi$iaej.as they all possessed same characteris of Sacch. cerevisiae, (Plate D).

Bacteria contaminants In both primary and secondary test the organisms are found to be positive to oxidase, catalase, O-F for oxidative, and Liquefy by gelatin. . Negative to M.R.V.P nitrate reduction indole, and hydrogen sulphide:" The isolates are fermentable to glucose, sucrose and fructose. As the contaminant isolated was in agreement with the description by Cowan and Steel (1965), Wilson and Mijes ;|l948), Breed (1957),..it was identified as Pseudomonas sp. and Aeromonas sp. (Plate G and H). The inhabitant and transmission of, Psendomonas, organism was described byHavey (1975) who suggest that contamination may be taken place from heavy dust, and thus cros.s contamination occur at several stages in I the process or utilize water and in the laboratory. Distribution of organism in ;,;'; water, in air and on skin was reported by Stitt el. al (1948), Transport of | organism passively in air, water and soil was also described by Alexander (1971). Since the organism was widely distributed in nature, it contamination in beer brewing causing undesirable result vyas reported by Pfescott and Dunn Jour. Myan- AcMArts&ScPlOOS Vol.'ML Wo. 4(i): Zoology 121

(1959). Starrier et. al (1965) also mention iMt" of ihfefctioh was appear to be accidented and contaminated by mud of soil is.a normal member of the flora of tropical.soil and water. , . •..,„. &<•'... •'"-: . ... , .-,... ', J •'•• - r -u...-. • . •••• ._...'.- .. -.\ • '.>H, ^..-, • ••-., •- ••• ••_ •••-••• . - • The contaminants were found to be Przudomonas sp. mX&Aefomonas sp. These bacteria were an apportunist that piially inhabits in water Especially ihtropies which grow fermently in acidic media assimilates glucose and there for it is undesirablei-iir-tKe^production ofalcbhoi:- : To prornbte efficiency of the, alcohoH.distillery, it is understandable that contaminant have to be guarded in the priniaty steps of seed culture, for further fementation.- process. So attempt should be made to prepare seed culture in the most; purest form, and under aseptic condition. Both Pseudqmouas-sp. and Aeromqrias\sp{aiQ commdnly found in soil and water. Thej/ might contaminants via some' route. In the operation procese of alcohol distipery^ the inoculum is commonly;prepared in a series of step with aseptic conditions and in each steps thefvolurnes was increased by about 30 times as established method of Hawker, Linton, Folkes, and Carlile (1961). Fermentation process in Gyogon ditilleiy is carried out. by,the yeast cell Sacch. cerevisiae in molasses media'containing about 12% sugar with the jiH'adjusted in between 4,0 to 4.5 with the temperature keeping 27-C to 30°C. ,' The seed culture was contaminated with bacteria from the stated seed -culture and fermented wort it is proven that trie contamination originated from the initial step of seed culture. Precaution of seed culture not to be contaminated with from the primary steps of seed culture. Attempts should be made to prepare seed culture in the pure culture form under aseptic 'condition. ' |

. i ; Most bacteria were found to be s aportunist as they underwent fnultiplicatidn any area wherever nutrient; is awailble. Competition thus occurred between the yeasts and bacteria. The results should be wastage of substrate, loss of cash and poor yield of alcoriol production. :••• • T • '*•':' '\ . • y ' T" ' ... . ! -I . . . | ., . •• • I ; ' •' • Suggestions for Future Work 1. Effective preventive measures should be followed in the initial step of inoculum to avoid contamination. ! I' 2. . Confirmation of the contaminated bacteria need to be further recorded. 122 Jour. Myatu Acad, Arts & Sc. 2005 Vol. III. No. 4(i) Zoology

3. Monitoring the contaminants in the process of fermentation should be studied. 4. Standardized methods for the production of good quality alcohol should be sought. Summary 1. The yeast and Most common bacteria contaminant were isolated from the samples and identified as Sacch. cerevisiae and Pseudomona^ls sp. and Aeromonas sp. respective by from alcohol distillery of Gyo gom. I 2. The isolates were inoculated on three different media Czapek Dox agar, Sabourand dextrose, agar and nutrient agar, and were incubated at room temperature 27°C and 37°C, under aerobic and anaerobic conditions. 1 • 3. The pure strain yeast allowed to grow on sporulation media for two weeks, to check the, formation of aseuspores. The isolated contaminants bacteria were incubated at different temperature of 5°C, 29°C, 37°C and 42°C respectively. 4. Jhe isolates of Sacch. cerevisiae and Psendomones sp. and Aeromonas sp. were studied by then morpholvgy characters and biochenical behaviors.-The fermentation test using various type of sugar such as moltuse,: sobital, glycered, salicin and mannitul were performed with these isolates of Sacch. cerevisial and Psendomonas sp. and Aeromonas sp. 5. One and haly litres glucore broth of purified Sacch. cereyisiae of ten days old was handed over to the Gyo-gon alcohol distillery on the 20th April 1979 to employ in fermentation process. It was noted that the fermentation is found to be significantly improved. 6. From the seed culture and fermented wort the bacteria contaminant were found to be various species starting from the initial steps. 7. Suggestions for future work is outlined. Jour. Myan.Acad. Arts & Sc.2005 Vol. III. No. 4(i)^Z6ology 123

Acknowledgements I am greatly indebted to Professor Dr.,U Mya Ob, Deputy Minister, Ministry of Health and'Dr. Soe Yin, Rector, Yangon University; for their encouragement toJ the PhD course. I wish to express my'sincere thank to Pr.ofessbr Dr. Daw Tin Nwe, Head of Zoology Department Yangori University, for her generous advice of this manus cfipt. I next extend my thanks to my colleague Ul;Mg Mg (Retired) Assistant Director, Veterinary Assay Laboratory, forihis advice arid discussion

References Alexander, M. (1971). Microbial Ecology. John Wiley and sons, IncNew York, p-36-40, 67, . 290, and 335-485. K H \ . m Beeze. G. I. De (1970). Yeast in Encyclopedia of Chemical Technology, ed by Kirk Othmer, 2nd ed. Interscience Publishers, New York V.22, p. 507-554. Breed, R.S, E. G. D Murray, and N. R. Smith (1957). Bergevs Manual of Determinative Bacteriology, 7th edition. Battimbre? Trie Williams and Wilkins. Bryan, A.- H arid C. G Bryan^ (1960).Bacteriology Principle arid Practice. 5th ed. New York. Barnes and Nobles: i;;

fii • •, • .jS,. • . Bryan, A. H, C. A. Bryan and C. G.. Bryan (1965) Ba'bteriblogy, Principles and Practice. 6 ed. (Reprinted 65) Barnes and Nobles trie N/Y. Collins. C. H and C. E. D. Taylor (1969) Methods Laboratory Technique series • :2ed. Butterworth's London. P 22-184, 240-254, 141-296. Collins. C. H and P. M. Lyne (1970). Microbial Methods 3 ed University • Park Press, Baltimbre Butterworth's, London. P 22 p, 61-215. Cowan S. T. (1974). Manual for the Identification of Medical Bacteria 2nd ed. rev. Cambridge. 230 pages. ,\ Cowen S, T. and K.J. Steel (1965) Manual for the Identification of Medical Bacteria 2nd ed. New(Cambridge). 217 pages. ; t' - Cruickshank, R. (1968) Mackie and McCartney's Hand Book of Bacteriology. A guide to ! Laboratory Diagnosis and control of Infection. 10th edition. E.S. Livingstone Ltd. Edinburgh, London. 980 pages. Cruickshank, R. (1968). Medical Microbilo^v. 11th ed., The English Language Book Society and E and S Livingston Ltd. Edinburgh, London. 1070 pages. Harrigan, W. F. and M. E. McCance (1966). Laboratory Methods in Microbiology. Academic Press. London. 83-85, 113-150, 201-216. p 30-99, 237.