UNIVERSITI TEKNOLOGI MARA

BIOCHEMICAL CHARACTERIZATION OF THERMOPHILIC BACTERIA AT HIGH TEMPERATURE

ANNA ANDREW

Project submitted in partial fulfillment of the requirement for the degree of Bachelor of Medical Laboratory Technology (Honours)

Faculty of Heath Sciences

November 2008

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DECLARATION I declare that the work in this thesis was carried out in accordance with the regulations of Universiti Teknologi MARA. It is original and is the result of my own work, unless otherwise indicated or acknowledged as referenced work. This thesis has not been submitted to any other academic institution or non-academic institution for any other degree or qualification.

NOVEMBER 2008

../T^.]. ANNA ANDREW 851109-12-5508 2006838889

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The Final Year Project Report entitled "Biochemical characterization of thermophilic bacteria at high temperature" was submitted by Anna Andrew, in partial fulfillment of the requirement for the Degree of Bachelor (Hons.) in Medical Laboratory Technology in the Faculty of Health Science, and was approved by

Dr. Hjh Zuridah Hj Hassan Supervisor Faculty of Health Sciences Universiti Technologi MARA

En. Zed Zakari Abd Hamid Head of Programme B. (Hons.) Medical Laboratory Technology Universiti Teknologi MARA

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ABSTRACT
BIOCHEMICAL CHARACTERIZATION OF THERMOPHILIC BACTERIA AT HIGH TEMPERATURE

Thermophilic organisms are organisms that have optimal growth temperature between 50C to 80C. These thermophilic organisms can be further subdivided into obligate thermophiles which grow at 55C but not at 37C and facultative thermophiles which grow at both 55C and 37C. This aim of this study is to characterize thermophilic bacteria using conventional microbiological tests from various hot springs in Malaysia, namely Sungai Klah, Labis, Gadek, Pedas and Selayang. Castenholz Tryptone Yeast Extract (TYE) medium (Medium D) was used to isolate these bacteria and eleven conventional tests were used. Five strains of gram negative bacilli and five strains of gram-positive spore-forming bacilli were isolated from these hot springs. Three of the gram negative rods (strains Al, A7 and A8) closely resembling Thermus aquaticus in terms of biochemical reactions and another two were facultative bacteria. Only strains A7 and A8 were found to be protease enzyme producers. All the five gram-positive strains were closely related to the genus Bacillus. Motility, oxidase and catalase tests were among the tests that required modification for use at high temperature. This study provides evidence that the hot springs in Malaysia supports the growth of both Thermus and Bacillus which may have numerous biotechnological applications.

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ABSTRAK
PENCIRIAN BIOKIMIA BAKTERIA TERMOFILIK PADA SUHU TINGGI

Organisma termofilik adalah organisma yang hidup secara optimum pada suhu antara 50 hingga 80C. Organisma termofilik boleh diklasifikasikan sebagai termofilik wajib yang mana boleh hidup pada suhu 55C dan terbantut pada suhu 37"C atau sebagai termofilik fakultatif yang mana boleh hidup pada kedua-dua suhu 55C dan 37C. Tujuan kajian ini adalah untuk mencirikan bakteria termofilik menggunakan ujian mikrobiologi tradisi yang dipencil daripada beberapa kolam air panas seperti Sungai Klah, Labis, Gadek, Pedas dan Selayang. Media Castenholz Tryptone Yis Extrak (Media D) digunakan dalam kajian ini untuk pemencilan bakteria tersebut dan sebelas ujian mikrobiologi digunakan untuk pencirian bakteria termofilik. Lima gram negatif bacilli dan lima gram positif bacilli disertai spora dipencil daripada kolam air panas. Tiga daripada gram negatif rod (Al, A7 dan A8) menyerupai organisma Thermus aquaticus dari segi reaksi biokimia dan dua lagi adalah bakteria fakultatif. Hanya bakteria A7 dan A8 didapati berupaya untuk menghasilkan enzim protease. Semua organisma gram positif dengan spora didapati sangat menyerupai genus Bacillus. Ujian pergerakan bacteria, oxidase dan catalase adalah antara ujian yang memerlukan pengubahsuaian untuk pencirian organisma termofilik. Kajian ini menunjukkan kolam air panas yang terdapat di Malaysia menyokong pertumbuhan bakteria Thermus dan Bacillus yang mungkin mempunyai kepentingan bioteknologi.

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ACKNOWLEDGEMENTS
I'm so grateful to God blessing for enabling me to complete this thesis successfully. I would like to express my gratitude to all those who gave me the possibility to complete this thesis. I am deeply indebted to my supervisor Dr. Hjh Zuridah Hj Hassan whose help, stimulating suggestions and encouragement helped me in all the time of research for and writing of this thesis. I would like to express my heartfelt appreciation to Me. Zed Zakari Abd Hamid, The Head Programme of B. (Hons.) of Medical Laboratory Technology and also to Dr. Roslina Hussain for their sage advice, helpfulness and encouragement. My deep and sincere gratitude were credited to all the lab staffs and all my friends for their advice, suggestion, helpfulness and encouragement. This work has also been partially supported by Fundamental Research Grant Scheme (FRGS) of Ministry of Higher Education, Malaysia (Project code: FRGS/FAS A-1-2006) I cannot end without thanking my family, on whose constant encouragement and love I have relied throughout my time at fulfilling this thesis. Their unflinching courage and conviction will always inspire me, and I hope to continue, in my own small way, the noble mission to which they gave their lives. It is to them that I dedicate this work.

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TABLE OF CONTENTS
Page TITLE PAGE DECLARATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF ABBREVIATIONS CHAPTER 1 INTRODUCTION 1.1 1.2 1.3 2 Problem statement Research objectives Research hypothesis 1 3 3 4 i ii iv v vi vii xi xii xiii xiv

LITERATURE REVIEW 2.1 2.2 2.3 Hot Spring studies Castenholz Tryptone Yeast Extract (TYE) Medium Thermophilic organisms isolated from hot springs 2.3.1 Thermus species 8 5 6

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2.3.2 2.4

Bacillus species

Conventional microbiological tests 2.4.1 2.4.2 2.4.3 2.4.4 2.4.5 2.4.6 2.4.7 2.4.8 2.4.9 Gramstain Motility test Oxidase test Catalase test Indole test Citrate test Spore stain Starch hydrolysis Gelatin hydrolysis 10 11 11 11 12 12 12 13 13 13 14 15 15

2.4.10 Casein hydrolysis 2.4.11 Litmus milk reactions 2.5 Effect of temperature on bacterial growth 2.5.1 Determination of bacterial growth

METHODOLOGY

3.1

Sampling and isolation of bacteria 3.1.1 3.1.2 Materials and equipments Water sampling method 17 17

3.2

Culture media 3.2.1 3.2.2 Materials and equipments Culture media preparation 20 20

3.3

Thermus aquaticus ATCC 25104 3.1.1 3.1.2 Materials and equipments Culture medium for ATCC Thermus aquaticus 25104.. 22 22

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3.4

Conventional microbiological tests 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.4.6 3.4.7 3.4.8 3.4.9 Gramstain Motility test Oxidase test Catalasetest Indoletest Citrate test Sporestain Starch hydrolysis Gelatin hydrolysis 23 24 26 26 27 27 28 28 29 29 30 31 31

3.4.10 Casein hydrolysis 3.4.11 Litmus milk reactions 3.5 Effect of temperature on bacterial growth 3.5.1 4 RESULTS 4.1 4.2 4.3 Sampling and isolation Thermus aquaticus ATCC 25104 Conventional microbiological tests 4.2.1 4.2.2 4.2.3 4.2.4 4.2.6 4.2.7 Gramstain Motility Oxidase test Catalasetest Citrate test Sporestain Data analysis

32 37 37 38 38 38 38 42 42 42

4.2.5 Indoletest

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4.2.8 4.2.9

Starch hydrolysis Gelatin hydrolysis

42 47 47 47

4.2.10 Casein hydrolysis 4.2.11 Litmus milk reactions 4.3 Effect of temperature on bacterial growth 4.3.1 4.3.2 Growth curve of ATCC, Al and A5 at different temperature Comparison of bacterial growth (Al, A5 and ATCC) at different temperature 5 DISCUSSION 5.1 5.2 5.3 6 Sampling and isolation Conventional microbiological tests Effect of temperatures on bacterial growth

51 54

58 60 66 63 69 74 81

CONCLUSION AND RECOMMENDATIONS

REFERENCES APPENDICES BIOGRAPHY

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LIST OF TABLES
Page Hot Springs inoculum sources, temperature, pH and depth of water samples Turbidity of liquid Castehnolz TYE medium, colony characteristics and gram reaction Conventional microbiological tests and results Summary of Casein and Litmus Milk reactions Summary of thermophiles isolated from springs 33 37 62 64 32

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LIST OF FIGURES
Figure 4.1 4.2 4.3 4.4 4.5 4.6 4.7 Growth curve of ATCC Thermus aquaticus 25104 at 55C, 60C, 65Cand70C Growth curve of Al at 55C, 60C, 65C and 70C Growth curve of A5 at 55C, 60C and 65C Growth curve of ATCC Thermus aquaticus 25104, Al andA5at55C Growth curve of ATCC Thermus aquaticus 25104, A1 andA5at60C Growth curve of ATCC Thermus aquaticus 25104, Al andA5at65C Growth curve of ATCC Thermus aquaticus 25104, A1 andA5at70C 57 56 55 54 51 52 53 Page

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LIST OF PLATES
Plate 3.1 3.2 3.3 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 Water sampling equipment Algae mats taken using bottom sampling dredge Hanging drop method Culture plate sealed in plastic bags Colony characteristics GramStain Preheating process before observe for motility Oxidase test Indole test Citrate test Spore stain Starch hydrolysis Gelatin hydrolysis Casein hydrolysis Litmus milk reactions , Page 18 19 25 35 36 39 40 41 43 44 45 46 48 49 50

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LIST OF ABBREVIATIONS
C ml pH TYE % jim g L cm +ve -ve > h spp. Degree Celsius Mililiter Potential of hydrogen Tryptone Yeast Extract Percentage Micrometer Gram Liter Centre meter Positive Negative More than Hour Species

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CHAPTER!

INTRODUCTION

Microorganisms have been classified according to the temperature at which growth rate is most rapid. Based on this, four major groups i.e. psychrophiles, mesophiles, thermophiles and hyperthermophiles have been described. By definition, thermophiles have their optimal growth temperature between 50C and 80C (Brown, 2005). The thermophilic organisms are further subdivided as obligate thermophiles which grew at 55C but not at 37C and facultative thermophiles which grow at both 55C and 37C (Campbell and Williams, 1952; Richmond and Fields, 1966). Thermophilic organisms prefer living at extreme environments which are usually inhospitable for most forms of life and ordinary destroy or inactive the cellular components of most forms of life. Many of the studies have been addressed to the question how do these organisms live at high temperatures (Shen et. al, 1970; Ray et. al, 1971a; Ray et. al, 1971b; Pask-Hughes and Shaw, 1982; Nordstrom, 1993; Llarch et. al, 1997). A microbial cells is surrounded by a membrane lipid which play a key role in the fluidity of membranes and thermophiles adapt to extreme temperature by modification of phospholipids which maintain both the membrane integrity and fluidity (Nordstrom, 1993). On the other hand, Shen et al. (1970) found that thermophilic bacillus contains lipids of higher melting points than mesophiles and this finding was perfectly agreed by Llarch et. al. (1997). For Thermus aquaticus, this organisms increase the proporrtions of monoenoic and heptanoic fatty acids #s the temperature of growth is raised (Ray et. al, 1971a; Ray et. al, 1971b). Thermophiles have high proportions of guanine and cytosine

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in DNA that raise the melting point and add stability to the DNA molecule (Brock and Freeze, 1969; Ramaley and Hixson, 1970; Kristjansson and Alfredsson, 1983). Several intensive studies were performed to isolate thermophilic bacteria from several habitats, aiming the use of these microbes and their enzymes for biotechnological application (Da Costa et. al., 1989). Thermus aquaticus is especially well known for its thermotolerant DNA polymerase (Taq polymerase) which has been patented and used world over in Polymerase Chain Reaction (Sharma, 2005). There has been an increasing interest in the study of enzyme from thermophiles since they are not only thermostable but often resistant to chemical agents and extreme pH values compared to those of mesophiles. One of the enzyme produce by thermophilic organisms is protease, an enzyme that hydrolyzes proteins to short peptides or free amino acids. This enzyme has a wide range of applications, particularly in food, detergent, weave, leather, pharmaceutical and chemical industries. Thermus strains have been recommended as sources of proteases (Matsuzawa et. al, 1983; Kanasawud et. al., 1992) and thermophilic bacillus has also been found to produce this enzyme (Huang et. al, 2006). Besides proteases, other enzymes such as amylase (Carvalho et. al., 2008), lipases (Dominguez et. al., 2007), and cellulases (Abdelnasser and Ahmed, 2007) are useful industries. In Malaysia, not much research was done to study the thermophilic organisms and their application in commercial industry. Sheikh et. al. (2003) had identified extracellular lipase-producing thermophilic bacteria which were useful in food industry. Thermophilic bacterial strain B160 which were isolated from oil reservoir sample has also found to have capability to produce biosurfactant under facultative anaerobic conditions (Fareh et. al, 2007). On the other hand, Wan Azlina (2007) uses thermophilic organisms for recovery of gold and copper from grade ore. After extensive researches on thermophilic organisms, they are found to have high potential in industrial and biotechnological in agriculture, paper, pharmaceutical, water purification, bioremediation, mining and petroleum recovery

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application. There is an opportunity niche to isolate and characterize thermophilic organisms found in Malaysia and this systematic knowledge may be valuable in choosing which of many strains is best for a particular biotechnological purpose. 1.1 Problem statement

Thermophilic bacteria have been known for many years, however the isolation and culture of these organisms is a great challenge since the nutritional requirements of the strains were found to be complex (Campbell and Williams, 1952; Brock and Freeze, 1969; Epstein and Grossowicz, 1969; Degryse et. ah, 1978). Successful cultivation of the high temperatures depends on the composition of the medium and as the incubation temperature is increased, there is an increase in the growth requirements of the particular organism under study. Modification need to be made for some conventional microbiological test used to characterize thermophilic organisms although a number of microbiological tests usually applied to mesophiles were suitable for use at high temperatures. In this study, a mineral base medium known as Castenholz Tryptone Yeast Extract (TYE) will be used to isolate and characterize thermophilic microorganisms from the local hot springs. 1.2 Research objectives

The objectives of this research are: a. To isolate thermophilic bacteria using Castenholz Tryptone Yeast Extract (TYE) medium. b. To characterize thermophilic bacteria using conventional microbiological tests.

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1.3

Research hypothesis

Alternative Hypothesis (Ha): Thermophilic bacteria grow on Castenholz TYE medium in laboratory setting at 60C. Null Hypothesis (Ho): Thermophilic bacteria do not grow on Castenholz TYE medium in laboratory setting at 60C.

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CHAPTER 2

LITERATURE REVIEW

2.1

Hot Spring studies

Thermophilic bacteria can be isolated from both man made and natural thermal environments (Ramaley and Bitzinger, 1974; Kristjansson et. al, 1986). These thermophilic bacteria has been isolated from hot spring (Brock and Freeze, 1969), hot water heaters (Brock and Boylen, 1972), thermally polluted river (Degryse et. al, 1978), soil (Ward and Cockson, 1972), manure pile (Epstein and Grossowicz, 1969), ocean basin cores (Bartholomew and Paik, 1966) as well as from hot composts (Beffa et. al, 1996). Hot spring is one of the favourable natural geothermal areas which are habitat for most of the thermophilic bacteria. Large collections of thermophilic organisms have been isolated from hot springs all over the world which include Yellowstone National Park (Marsh and Larsen, 1952; Brock and Freeze, 1969), Icelandic hot spring (Kristjansson and Alfredsson, 1983; Kristjansson et. al., 1986; Sharp and Williams, 1988), Jordan hot spring (Elnasser et. al, 2007), Egyptian hot spring (Abdelnasser and Ahmed, 2007), and Thai hot spring (Kanasawud et. al, 1992). These thermophilic organisms were found in both neutral and alkaline hot springs. According to Kristjansson and Alfredsson (1983), acidic springs were devoid of thermophilic organism (particularly Thermus species) and presumed that when found in mixed waters, they all originated from the alkaline springs. Malaysia has about 40 known hot springs (Abdul et. al, 1997) and most of them are located at Peninsular Malaysia. Abdul et. al. (1997) noted that the hot springs are

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originated from the deep-lying ground water of the earth crust which moved towards the surface as a result of magmatic heat and pressure. They found that the ground water seeping through the fractures and crevices in the earth's crust is heated by contact with the hot granitic rock which eventually emerges as hot spring. The temperatures of the hot springs around Malaysia range from 36 to 102C and the pH range from 6.5 to 9. 2.2 Castenholz Tryptone Yeast Extract (TYE) Medium

Different culture mediums have been used for the isolation of thermophilic organisms and many studies were done to investigate the nutritional requirements of thermophilic strains. According to previous studies (Epstein and Grossowicz, 1969; Souza and Martins, 2001), successful cultivation at high temperatures depended upon the composition of the medium, and the degrees of complexity usually parallel to the increase of the growth temperature. Since isolation of thermophilic organisms require extreme temperature (55 C and above), liquid enrichment medium is used for initial isolation of thermophilic organism instead of agar media (Morrison and Tanner, 1921). A basal salt medium which is known as Castenholz TYE medium (previously known as Medium D) has been extensively used for the isolation of thermophilic organisms. Since the initial isolation Of Thermus aquaticus by Brock and Freeze (1969) using Castenholz TYE medium from the thermal springs in Yellowstone National Park, many other researches on thermophilic organisms were conducted using the same medium. Brock and Freeze (1969) have performed nutritional studies by using the basal salts medium and found that good growth was obtained in 0.1 and 0.33% tryptone plus yeast extract, but no growth was obtained at 1 % tryptone plus yeast extract. They also noted that the media for Thermus enrichments must be fairly dilute in organic constituents, since the organism is inhibited by tryptone and yeast extract at a concentrations of approximately 1%. No growth was also observed where 0.1% tryptone plus 0.1% yeast extract was made up in deionized water instead of basal salts.

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Degryse et. al. (1978) had devised a mineral base medium for the isolation of Thermus species which was the modification of Medium D (Castenholz medium). They found that Medium D, which has been widely used as mineral base medium for Thermus-like strains was quite inadequate if one wish to optimize growth conditions. However, they proposed that enrichment procedures for Thermus strains using Medium D as a mineral base have to be carried out in the presence of tryptone and yeast extract. Shimura et. al. (1999) had conducted a study where Castenholz medium was used to isolate and characterize thermophilic Bacillus sp. JF8 which were able to degrade polychlorinated biphenyl (PCB) and napthalene. They found that Castenholz D medium supplies low levels of unidentified nutrients which are required for aromatic utilization but also supports low levels of growth. In the study done by Souza and Martins (2001), they found that the thermophile isolated require yeast extract to grow at supraoptimal temperatures, suggesting that at higher temperatures, the enzymes responsible for the synthesis of a particular metabolite present in this compound underwent thermal inactivation and thus the organism required it as an exogenous sources. Besides Thermus aquaticus (Brock and Freeze 1969; Brock and Edwards, 1970; Ray et. al., 1970; Ray et. al., 1971; Brock and Boylen, 1972), thermophilic organisms which were successfully cultivated by Castenholz TYE medium include white Thermus strain (Nordstorm, 1993), Thermus ruber (Sharp and Williams, 1988), Thermus thermophilus Gyl211 (V.T. Marteinsson et. al., 1999), non pigmented Thermus aquaticus X-l (Ramaley and Hixson, 1970) and cellulases producing bacteria such as Anoxybacillus flavithermus, Geobacillus thermodenitrificans and Geobacillus stearothermophilus (Abdelnasser and Ahmed, 2007).

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2.3

Thermophilic organisms isolated from hot springs

Hot springs are well known for its natural geothermal areas most favourable by thermophilic organisms. Many thermophilic bacteria can be found from hot springs but the most common strains isolated from the hot springs belong to the genus Thermus (Brock and Freeze, 1969; Kristjansson and Alfredsson, 1983; Kristjansson et. al., 1986; Sharp and William, 1988; Kanasawud et. ah, 1992) and Bacillus (Marsh and Larsen, 1952; Elnasser et. al., 2007). 2.3.1 Thermus species Members of the genus Thermus are described as thermophilic, gram negative, aerobic, yellow pigmented heterotrophs with an optimum growth temperature of 70C and a pH optimum between 7 and 8 (Brock, 1978). These Thermus species are found to be ubiquitous in nature and can be isolated from both natural and man made thermal sources (Ramaley and Bitzinger, 1974). According to Bergey's manual (ninth edition), the bacteria belong to the genus Thermus is a straight rods, 0.5 - 0.8X5.0 - 10.0u,m and gram stain reveals the bacteria as negative. These bacteria do not posses flagella and therefore is non motile. Most of the strain form yellow (Brock and Freeze, 1969), orange or reddish colonies with pigmentation due to carotenoid pigment. Isolation of white Thermus (Nordstrom, 1993) as well as non pigmented strain (Ramaley and Hixson, 1970; Brock and Boylen, 1972) had also been discovered. Thermus species are aerobic, having a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor. The genus Thermus posses the enzyme cytochrome oxidase and they also produce catalase enzyme. Gelatin is usually hydrolyzed, starch is usually weakly digested and nitrates are reduced to nitrites.

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One of the distinctive features of Thermus is the formation of rotund bodies. Rotund bodies are found to be larger than spherical bodies, which are often seen in older culture (Brock and Edwards, 1970). According to Brock and Edwards (1970), the formations of rotund bodies are result from the association of a number of separate rods or filaments. In these bodies, a number of cells are connected, probably as a result of fusion of their outer cell envelope layer. 2.3.2 Bacillus species Majority of the investigated thermophilic that bacteria belong to the genus Bacillus have been isolated from thermophilic and mesophilic environments (Marsh and Larsen, 1952; Allen, 1953; Richmond and Fields, 1966; Bartholomew and Park, 1966; Strom, 1985; Bertram et. ah, 1992; Elnasser et. al., 2007) and these organisms will take place whenever favorable circumstances occur. According to Bergey's manual (ninth edition), endospore forming gram positive rods belong to the genus Bacillus have a wide diversity of physiological ability with respect to heat, pH and salinity. Bacillus species which grew at both 55 and 37C were classified as facultative thermophiles, e.g. B.licheniformus, B.subtilis, B.coagulans, B.brevis, B.pumilus, and B.macerans (Richmond and Fields, 1966) while those grew at 55C but not 37C were classified as obligate thermophiles, e.g. Bacillus stearothermophilus (Bartholomew and Park, 1966).

According to Allen (1953), the characterization of the thermophilic aerobic sporeforming bacteria to make them readily recognizable is a difficult task. This is supported by Holt et. ah, (2006) where species differentiation is difficult because of their large number and the often incomplete descriptions of a number of newly reported species. In general, organisms belong to the genus Bacillus are rod shaped and straight (Bergey's manual, ninth edition). They are often arrange in pairs or chains, with rounded or squared ends. The bacteria are gram positive and they are motile by peritrichous flagella. The most important feature of the organisms belong to the genus bacillus is the present of

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endospores which are oval, sometimes round or oval. These endospores are heat resistant which make them have biotechnological values in agricultural and industrial practice (Shimura et. al., 1999; Huang et. al, 2006; Carvalho et. al, 2008). However, these thermophilic sporeformers are a nuisance in food industry as they are among the principal cause of spoilage of canned foods and food ingredients (Allen, 1953; Richmond and Fields, 1966). Bacillus are usually catalase positive and they can be either aerobic or facultatively anaerobic. 2.4 Conventional microbiological tests

Conventional microbiological tests are the one that is widely accepted and published as a standard microbiological method or related procedure. Not all microbiological tests applied to mesophiles are suitable for use at high temperatures. Some of the tests require modification for characterization of thermophilic bacteria. 2.4.1 Gram stain Gram staining is the most important method to separate Gram-positive bacteria from Gram-negative bacteria based on the different composition of the cell walls. Grampositive bacteria have a thick peptidoglycan layer whereas Gram-negative bacteria have a thin peptidoglycan layer. In gram staining, the peptidoglycan will trap the crystal violet and form a Crystal Violet-Iodine (CV-I) complex. The decolorization step in Gram staining differentiates Gram-positive bacteria from Gram-negative bacteria by the application of 95% ethyl alcohol. The CV-I complex will be washed up in Gram negative bacteria but Gram positive bacteria will retain the decolorization. A counterstain (usually safranin) will be uptake by the colorless gram negative bacteria (Barrow and Feltham, 1993).

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