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Volume 2.3, 2010

Lactobacillus bulgaricus added to dairy products

in a milk protein matrix.

Probiotics

Probiotics...........................p2

Genus Yersinia..................p5

Marine Isolates

or bioleaching ................p7

Volume 2.3 2010

Lactobacillus bulgaricus added to dairy products

in a milk protein matrix.

Probiotics...........................p2

Genus Yersinia..................p5

Marine Isolates

or bioleaching p7

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Microbiology

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ProbioticsJvo Siegrist, Product Manager Microbiology

ivo.siegrist@sial.com

Probiotics take on a key role in

the unctional ood industry

The name Probiotics is derived romLatin (pro) and Greek (biotic) rootsmeaning or lie. Probiotics are defnedas live bacteria with benefcial eects tothe health o the host organism. Today,primarily lactic acid bacteria (LAB) andbifdobacteria are used as probiotics,however certain yeasts and bacilli arealso known to have positive eects. Inmost cases, probiotics are produceddirectly by ermentation in oods suchas yogurt, or are supplied throughdietary supplements. Although the

term Prebiotics is also used oten insuch cases, the two terms should notbe conused, since prebiotics actuallyreers to non-digestible compounds thatstimulate the growth and/or activityo probiotics in the gut. Prebiotics arerequently used in unctional ood, andtypically consist o oligosaccharides.Prebiotics can be ound in milk(galactooligosaccharides, or GOS) andplants with dietary fbers, however theyare oten produced by ermentation.

At the beginning o the 20th century, probiotics were thought to have a benefcialaect to the host by improving the intestinal microbial balance. At that time, it wasobserved that lactic-acid bacteria inhibit the growth o proteolytic bacteria due to lowpH. Through his studies and observations, the Russian biologist Metchniko noted

that people rom cultures which consumed large amounts o ermented milk, orexample those in Bulgaria and the Russian steppes, generally had a higher lie-span.Thereore, he proposed that lactic-acid bacteria decrease the intestinal pH as a resulto ermentation, thereby suppressing the growth o proteolytic bacteria. Metchnikopropagated the consumption o sour milk ermented with the Lactobacillus bulgaricus[5] as a means o promoting intestinal balance and overall health.

Today, diverse studies report the benefts o probiotics, such as inhibitory eects onpathogens, aid in the management or prevention o chronic intestinal inammatorydiseases or o atopic diseases, and support to the immune system. Potential benefcialapplications abound, and the research around this topic continues to expand.

Recently, scientists in Finland have discovered that cheese can help preserve and enhancethe immune system o the elderly by acting as a carrier or probiotic bacteria. The researchshowed that daily consumption o probiotic cheese helps to bolster immunity in elderly

people. Cheese was confrmed to be an eective carrier or probiotics [1].

Bifdobacteria

Bifdobacterium is one o the most important probiotic bacteria used in the dairyindustry. They are Gram-positive, non-motile, rod-shaped, and oten branched anaerobicbacteria. They were frst isolated rom a breast-ed inant by Henry Tissier rom thePasteur Institute. At that time, Tissier named the organism Bacillus bifdus communis [6].Bifdobacteria have a positive aect on the immune system and help to control intestinalpH . In additional, bifdobacteria produce bacteriocins and bacteriocin-like inhibitorycompounds which inhibit the growth o other bacteria.

Bifdobacteria posesses many glycosylases able to degrade various plant- or milk-derived oligosaccharides. Several such enzymes were identifed on the Bifdobacterium

genome. Also diverse glycosyl hydrolase, ABC transporter and the os gene cluster thatis involved in the processing o health-promoting ructooligosaccharides (prepiotics),called bifdogenic actors, can be ound on the genome [10]. Obviously Bifdobacteriaare able to utilize a broad range o substrates as energy sources, such as plant polymers,glycoproteins and glycoconjugates, as well as having specialized proteins or thecatabolism o oligosaccharides.

Bifdobacteria also have a unique hexose metabolism called the bifd shunt. The keyenzyme, ructose-6-phosphate phosphoketolase is not ound in any other Gram-positiveintestinal bacteria and thereore provides an ideal target or a diagnostic test.

It was ound that live B. lactis bacteria can directly counteract the harmul eects ocoeliac-toxic gliadin and this may prove to be a uture potential treatment o coeliacdisease [9].

In adult intestines, only 3-6% o the ecal ora is composed o Bifdobacteria, whilein breast-ed inants Bifdobacteria can constitute up to 90%. With increasing age, thenumber o Bifdobacteria decreases. It was observed that babies and adults with lowernumbers o Bifdobacteria have a higher risk or diarrhea and allergies. For this reason,Bifdobacteria are added as a probiotic supplement to inant ormulas, drinks, yogurtsand a range o other products.

Because o the wide use o bifdus, Fluka has developed a Bifdobacteria Selective Media(BSM), available as an agar or a broth, as a standard or quality control. This mediumallows or easy and ast quality control o yogurt made with bifdus and can be used tocontrol the count o bifdus bacteria.

Did you knowthat bacteria accountsfor about 7% of humanbody weight?

An adult person carries about 2.5 -7.5 kg o living bacteria in the intestinaltract. There are about ten times asmany bacterial cells as there are tissuecells in the entire body.

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Microbiology

Bifdobacterium grow very well on this medium, while Lactobacillus and Streptococcusstrains are inhibited. Bifdobacterium colonies grow within 24-48 hours (occasionally upto three days because o the highly selective conditions). The Bifdobacterium coloniesare purple-brown and thereore are easy to dierentiate rom other organisms.

In a Swiss governmental evaluation study or the enumeration o bifdobacteria in sourmilk products, the traditional method was compared to Wilkins-Chalgren Agar with100 mg/L mupirocine and BSM Agar. The traditional method produced statisticallysignifcant dierences, while Wilkins-Chalgren Agar and BSM Agar showed similarresults without any signifcant variances. The study concluded, On the BSM Agar, thebifdobacteria orms purple-brown colonies which made the enumeration easy [3].

Lactobacillus Species

Lactobacilli are rod-shaped, Gram-positive, ermentative, acultative anaerobic ormicroaerophilic organotrophs. Normally they orm straight rods, but under certainconditions spiral or coccobacillary orms have been observed. In most cases, they ormchains o varying length. Lactobacilli belong to the lactic acid bacteria and comprisethe major part o this group. As their name implies, they produce lactic acid andderive energy rom the ermentation o lactose, glucose and other sugars to lactate viahomoermentative metabolism. About 85-90% o the sugar utilized in the ermentativeprocess is converted to lactic acid. However, there are some heteroermentativelactobacilli that produce alcohol in addition to lactic acid rom sugars. This acid-producing mechanism inhibits growth o other organisms and avors the growth olactobacilli that thrive in low pH environments. ATP is generated during the process by

non-oxidative substrate-level phosphorylation.Some strains o lactobacilli were shown to produce, like bifdobacteria, a bacteriocin-likesubstance and are able to inhibit a broad range o pathogens [8]. Lactobacilli also produceadhesins (proteins), which perorm a vital role in recognizing specifc host components(extracellular matrix) important or the bacterial adhesion and colonization at host suraces,as well as in bacterial interaction with physiological and immunological processes [8].

In the last several years, several new Lactobacillus species have been introduced asprobiotics, including Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillusjohnsonii. As o this time, probiotics have not been used in the pharmaceutical industrydue to the many open questions that remain to be answered [7].

Figure 1. Yogurt sample cultured on BSM Agar.

Bifdobacteria appears as purple-brown colonies.

Medium or Lactobacilli

Since lactobacilli preer acidic conditions,natural extracts and juices rom tomatoesand oranges, as well as other singlemetabolic acids (e.g. malic acid), are otenused as media ingredients. Casein andyeast extract provide rich amino acidsources, and the maltose is used as a

carbohydrate source or lactobacilli, whichcannot utilize glucose as ermentablesugar. Fructose is the carbohydrate sourceoLactobacillus ructivorans. Polysorbate,sorbitan mono-oleate and other relatedcompounds act as a source o atty acidsand stimulate the lactic acid bacteria.

Today, it is standard practice todierentiate lactobacilli based on theirphenotype using selective media. Classicalphenotypic tests or identifcation olactobacilli are based on physiologicalcharacteristics, like motility, growth

temperature, respiratory type, andgrowth in sodium chloride, as well ason diverse biochemical characteristics,such as ermentation type, metabolismo carbohydrate substrates, production olactic acid isomers, coagulation o milk,and presence o specifc enzymes likearginine dihydrolase. In Bergeys Manual,Lactobacillus is described as a Gram-positive rod, non-spore orming, acidast negative and catalase negative. Thecolony morphology on certain media istaken or the presumptive identifcation.

Organisms (ATCC) Growth Colony Appearance

Biidobacterium longum (15707) +++ red-brown (maroon)

Biidobacterium inantis (15697) +++ red-brown (maroon)

Streptococcus thermophilus (14486) - -

Lactobacillus acidophilus (314) - -

Lactobacillus bulgaricus (11842) - -

Table 1. Bifdobacterium sp. cultural characteristics on BSM Agar

Name Brand Cat. No. Pack Size

BSM Agar Fluka 88517 500 g

BSM Broth Fluka 90273 500 g

BSM Supplement Fluka 83055 5 g

Wilkins Chalgren Anaerobic Agar Fluka W1761 500 g

Mupirocin Sigma M7694 50 mg

100 mg

Lithium Mupirocin Sigma 07188 1 g

100 mg

Table 2. Selective medium and supplements or Bifdobacterium

Figure 2. 3D rendering o isolated Lactobacillus

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Microbiology

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Cat. No. Name Description

Media or Dierentiation

17153 LS Dierential Agar For the maximum growth and dierentiation o lactobacilli and streptococci on the basis oAntibiotic free skim milk powder (Fluka 70166) colonial morphology, T.T.C. reduction and casein reaction.TTC Solution (Fluka 17779)

17158 Litmus Milk For maintenance o lactobacilli and or determining the action o bacteria on milk.

17222 WL Nutrient Agar For the examination o materials encountered in brewing and or industrial ermentationscontaining mixed lora o yeasts and bacteria.

17215 WL Dierential Agar For selective isolation and enumeration o bacteria encountered in breweries andindustrial ermentations.

Nonselective Media

Y3127 Yeast Malt Agar Used or the isolation and cultivation o yeasts, molds and other aciduric microorganisms.

17216 Tomato Juice Agar For the cultivation and enumeration o lactobacilli.

80957 Plate Count Skim Milk Agar For the enumeration o bacteria in milk and dairy products.

17218 Tomato Juice Broth For cultivation o yeasts and other aciduric microorganisms.

T2188 Tryptone Glucose Yeast Extract Agar Recommended or enumeration o bacteria in water, air, milk and dairy products.

17123 Elliker Broth For culturing streptococci and lactobacilli o importance in the dairy industry.

Selective Media (by low pH and selective agents)

R1148 Rogosa SL Agar Used as a selective medium or cultivation o lactobacilli.

85515 Sorbic Acid Agar (Base) For the isolation and dierentiation o lactobacilli rom ood, aeces etc. according to Reuter.

17154 Lactobacillus Bulgaricus Agar (Base) Used with acetate buer or isolation and identiication oLactobacillus bulgaricus.

69966 MRS Broth For the enrichment and isolation o all species o lactobacilli rom all types o material.

41782 MRS Agar, Vegitone This MRS Agar is ree o animal derived material. It is recommended or the isolation andcultivation oLactobacillus species.

69964 MRS Agar For the enrichment, cultivation and isolation o all species oLactobacillus rom all types omaterial according to De Man, Rogosa and Sharpe.

38944 MRS Broth, modiied, Vegitone This MRS Broth contains plant peptone instead o animal peptone. It is recommended or theisolation and cultivation oLactobacillus species.

30912 MRS Agar, original acc. For the enrichment, cultivation and isolation o all species oLactobacillus rom all types oDeMan-Rogosa-Sharpe materials. Recommended by the Schweizerisches Lebensmittelbuch 5th ed., chapter 56A.

75405 Orange-serum Agar For the isolation, cultivation and enumeration o acid-tolerant spoilage microorganisms in ruitjuice and ruit juice concentrates, in particular rom citrus ruit, according to Hays, Troy and Beisel.

17226 Universal Beer Agar For culturing microorganisms o signiicance in the brewing industry.

W2261 WL Nutrient Broth Recommended or the cultivation o bacteria encountered in breweries and industrialermentations.

02538 Raka Ray Agar A medium or selective isolation o lactic acid bacteria rom beer and brewing processes.Lactic Acid Supplement, modified (Fluka 14121)

Cycloheximide solution (Fluka 18079)

83920* Rogosa Agar Selective agar or the isolation and enumeration o lactobacilli belonging to the buccal andintestinal lora, as well as rom ood such as milk and meat.

64198 NBB Agar Base, modiied Selective medium used or the detection o contaminating/spoilage microorganisms in brewery.

50725 NBB Broth Base, modiied Selective medium used or the detection o contaminating/spoilage microorganisms in brewery.

Table 3. Media or lactobacilli detection, dierentiation, isolation, enumeration and cultivation

*Available in Europe only.

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Microbiology

the need or PCR amplifcation. The sandwich hybridization test,called HybriScan, is perormed on a microtiter plate. The range olactobacilli detected by HybriScan tests is listed inTable 4.

More inormation about the test and the technical principlesbehind it are ound on our web site sigma-aldrich.com/hybriscan

Modern Methods to Detect Lactobacilli

As a modern alternative, molecular biology-based methods, likePCR, can be consulted. However, they are oten quite expensive.Under our Fluka Brand, Sigma-Aldrich provides a revolutionarymolecular biology method that is rapid, easy and cost eective.Based on the detection o rRNA, this method completely avoids

Cat. No. Brand Description Specifty Reactions

62533 Fluka HybriScanD Beer Detects all beer spoiling bacteria o the genera Lactobacillus, Pediococcus, 96Pectinatus and Megasphaera

68301 Fluka HybriScanD Drinks Detects yeast o the genera Saccharomyces, Zygosacchromyces, Brettanomyces, 96Torulaspora, Pichia, Candida and bacteria o the genera Lactobacillus,AcetobacteraceaeandAlicyclobacillus

59744 Fluka HybriScanD Lactobac Detects bacteria o the genera Lactobacillus and Pediococcus in ruit juices and 96non-alcoholic beverage.

75724 Fluka HybriScanI Identiication oLactobacillus brevis 48Lactobacillus brevis

80065 Fluka HybriScanI Identiication oLactobacillus buchneri 48

Lactobacillus buchneri

86827 Fluka HybriScanI Identiication oLactobacillus lindneri 48Lactobacillus lindneri

Table 4. HybriScan products (HybriScanD = detection kit; HybriScanI = identifcation kit)

Genus YersiniaJvo Siegrist, Product Manager Microbiology

ivo.siegrist@sial.com

Detection, identifcation, dierentiation and

cultivation oYersinia species

Yersinia is a rod shaped and acultative anaerobic Gram-negativebacteria belonging to the amily o Enterobacteriaceae. It hasa ermentative metabolism, is oxidase-negative, mannitol-positive, glucose-positive and lactose negative. On the Bismuthsulfte Agar (Fluka 95388) Yersinia can be dierentiated romSalmonella because it is not able to produce hydrogen sulphide.It is a psychrophilic organism, surviving and prolierating at lowtemperatures o 0-4 C (e.g., on ood products in a rerigerator).Some Yersinia species are also relatively highly heat resistant.However, they can be quite easily inactivated by oxidizing agentssuch as hydrogen peroxide and potassium permanganate.

Pigs, rodents, rabbits, sheep, cattle, horses, dogs, and cats arethe natural sources oYersinia. At the moment, most humanillness cases caused by Yersinia originate rom Y. enterocolitica.This organism is the cause o yersiniosis, an inectious diseasewith symptoms like ever, abdominal pain, and diarrhea. Otherclinically important species o this genus are Y. pseudotuberculosis(symptoms similar to Y. enterocolitica except in most casesno diarrhea is seen) and Y. pestis (organism responsible orthe bubonic plague). Most inections are acquired throughcontaminated ood, like raw or undercooked pork products,seaood, vegetables, unpasteurized milk or untreated water.However, inections may also occur ater contact with inectedanimals or aeces, or through transmission by eas.

The bacteria received its name rom A. E. J. Yersin, a Swiss micro-biologist, who discovered the Yersinia pestis bacterium in 1894 inHong Kong.

Reerences

1. F. Ibrahim, S. Ruvio, L. Granlund, S. Salminen, M. Viitanen, A.C. Ouwehand,Probiotics and immunosenescence: cheese as a carrier, FEMS Immunology &Medical Microbiology, 59, 1, p53-59 (2010)

2. Rada V. and Koc J.: The use o mupirocin or selective enumeration obifdobacteria in ermented milk products. Milchwissenschat. 55, 6567 (2000)

3. Bundesamt r Gesundheit: Schweizerisches Lebensmittelbuch (SLMB):Kapitel 56, Mikrobiologie Neuausgabe 2000, Stand 2004

4. J. Nowroozi, M. Mirzaii, M. Norouzi, Study o Lactobacillus as ProbioticBacteria, Iranian J. Publ. Health, Vol. 33, No. 2, pp.1 -7 (2004)

5. R.B. Vaughan, The romantic rationalist: A study o Elie Metchniko, MedicalHistory 9: 20115 (1965)

6. H. Tissier, Recherchers sur la ora intestinale normale et pathologique dunourisson, Thesis, University o Paris (1900)

7. G.W. Tannock, Probiotics: time or a dose o realism, Curr. Issues Intest.Microbiol., 4(2), 33-42 (2003)

8. . Ljungh, T. Wadstrm, Lactobacillus Molecular Biology: From Genomics toProbiotics, Caister Academic Press (2009)

9. K. Lindors, etal., Live probiotic Bifdobacterium lactis bacteria inhibit the

toxic eects induced by wheat gliadin in epithelial cell culture, Clin. Exp.Immunol., 152(3), 552-8 (2008)

10. J.F. Kim, et al., Genome sequence o the probiotic bacterium Bifdobacteriumanimalis subsp. lactis AD011, J. Bacteriol., 191(2), 678-9 (2009)

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Cat. No. Supplements

38266 Ferrioxamine E

75258 Yersinia selective Supplement

17778 Ticarcillin Supplement

17777 Potassium chlorate Supplement

Table 3. Selective and growth supplement or Yersinia

Cat. No. Test or Yersinia Diagnostics

80507 Bile Esculin Disks

88597 Catalase Test

56481 Cellobiose Disks

63367 Dextrose (Glucose) Disks

05686 DMACA Indole Disks

49825 DMACA Reagent

67309 Kovacs Reagent or Indoles

78719 Kovacs Reagent Strips

28816 Lactose Disks

94438 Mannitol Disks

08714 Methyl Red Solution

07345 Oxidase Reagent acc.Gaby-Hadley A

07817 Oxidase Reagent acc.Gaby-Hadley B

18502 Oxidase Reagent acc.Gordon-McLeod

40560 Oxidase Strips

70439 Oxidase Test

93999 Rhamnose Disks

92971 Salicin Disks

93998 Sorbitol Disks

94309 Sucrose Disks

Table 4.Test or identifcation and dierentiation

oYersinia

Species Dierentiation Properties

Y. enterocolitica catalase-positive, most strains are ornithine-positive, motile at 22-26 C,non-motile at 37 C, urea-positive, sorbitol- and cellobiose- positive, moststrains are sucrose-positive

Y. pseudotuberculosis motile at 22-26 C, non-motile at 37 C, urea-positive, rhamnose-positive,esculin-positive, ornithine decarboxylase negative, indole negative

Y. pestis produces two antiphagocytic components (antiphagocytic slime), catalase

positive non-motile at 22-26 C and 37 C, esculin-positive, indole negative,ornithine decarboxylase negative

Table 1. Some dierentiation o the important Yersinia species C

Cat. No. Description

Nonselective Broths

53286 Brain Heart Broth

40893 Peptone Water, phosphate-buered, Vegitone

77187 Peptone Water, phosphate-buered

Selective Enrichment Broths

17156 ITC Broth (Base)

69965 Mossel Broth17192 Peptone Sorbitol Bile Broth

Basal Media or Carbohydrates Utilization

A0715 Andrade Peptone Water

28943 Andrade Peptone Water, Vegitone

Medium or Autoagglutination

39484 Methyl Red Voges Proskauer Broth

Nonselective Agars or Dierentiation & Confrmation

27048* Christensens Urea Agar

60787 Kligler Agar

M1428 Motility Indole Ornithine Medium

75315 OF Test Nutrient AgarO5386 Ornithine Decarboxylase Broth

51463 Urea Broth

Selective Agars or Detection and Isolation

95760* Yersinia Selective Agar (CIN Agar)

Selective Agars with Dierential System or Dierentiation, Detection and Isolation

95388 Bismuth sulfte Agar

70135 DCLS Agar

90035 DCLS Agar No. 2

17213 Violet Red Bile Glucose Agar without Lactose

53605 Violet Red Bile Glucose Agar without Lactose, Vegitone

95273 VRB MUG Agar

Table 2. Media or Yersinia

*Available in Europe only.

Reerences

1. K.J. Ryan, C.G. Ray (editors), Sherris Medical Microbiology, 4th ed., McGraw Hill, 36870 (2004)

2. F.M. Collins, Pasteurella, Yersinia, and Francisella. In: Barrons Medical Microbiology (S. Barron et al, eds.), 4th ed., University o Texas Medical Branch (1996)

3. F. Bichai, P. Payment, B. Barbeau, Protection o waterborne pathogens by higher organisms in drinking water: a review, Can. J. Microbiol. 54 (7), 509-524 (2008)

4. Bergeys Manual o Systematic Bacteriology, 1994, 9th ed., Williams and Wilkins, Baltimore

More details about the media or tests can be ound on our website sigma-aldrich.com/microbiology.

Figure 2. Bacteriophage R1-37attached to

Y. enterocolitica YeO3-R1 cell (length o cell = 1,7 m;

Source Mikael Skurnik, University o Turku, Finland)

sigma-aldrich.com/microbiology

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Microbiology

Abhilash, R. Venkat Raman, K.D. Mehta and B.D. Pandey, MEF Division, National Metallurgical

Laboratory (CSIR), Jamshedpur-831007, India

biometnml@gmail.com; www.nmlindia.org

The marine isolates rom sea nodules can be worthy catalysts to explore the possibilities

o aster bioleaching o low index ocean nodules mined rom Indian Ocean. The nativemicrobes were isolated rom Indian Ocean Nodules in Artifcial Sea water NutrientBroth (ASWNB) at pH 7.0. Two new and pure species were isolated ater 12 rounds oculture purifcation on ASWNB Agar. They were termed as 7-BIOSN and -BIOSN. Theirmicrobiological and biochemical eatures are summarized below.

7-BIOSN -BIOSN

Microscopic

Long chains o rod shaped cells Short rod shaped cellsCells entangled in dense network Cells clubbed in pairsGram Positive Gram PositiveNon Acid Fast Staining Non Acid Fast StainingNon-Endospore orming Endospore ormingMesophilic Mesophilic

Biochemical

Indole Test + +

Methyl Red Test + +

Voges Proskauer Test - +

Citrate Utilization Test + +

TSI Agar Test Glucose ermenting Glucose, lactose ermenting

Starch Hydrolysis Test + +

Gelatin Liquefcation Test + -

DNase Test + +

Catalase Test - -

Oxidase Test + +

Salt Tolerance Test Could not grow in Could not grow in>5% NaCl conc. >7% NaCl conc.

Antibiotic Resistant to Penicillin Resistant to PenicillinSusceptibility Test Resistant to Ampicillin, etc. Sensitive to Ampicillin etc.

Generation time 1.3 h 0.7 h

Table 1. Microbiological and Biochemical Features

The pure isolates would be molecularly characterized or phylogenetic homology andsubsequently tested or toxicity on metal ions (Cu, Ni, Co, Fe, Mn) or applications inbioleaching.

The Isolated species rom sea nodules were tested or metal toxicity evaluation on variousconcentrations o synthetic salts o copper, nickel and cobalt. It was ound that both speciesexhibited good resistance towards 1000 ppm Ni (II) concentration, whereas resistancetowards Cu(II) and Co(II) could only be seen at a metal concentration o 100 ppm.

Metal ion Cu Ni Co

Concentration [ppm] -BIOSN 7-BIOSN -BIOSN 7-BIOSN -BIOSN 7-BIOSN

10 R R R R R R

50 R R R R R R

100 R R R R R R

500 S S R R S S

1000 s S R S S S

Table 2. results o sensitivity testing; R = resistance, S = sensitive

These values can be improvised on subsequentsub-culturing or defnite applications inbioleaching o low-index sea nodules and pose apromising alternative technology or the uture.

Native Marine Isolates rom Indian Ocean Nodules

Figures 1 & 2. Two isolates rom Indian Ocean

nodules capable o metal oxidation rom its

sulphides and oxides

(Under Oil Immersion 1000X Magnifcation in PhaseContrast Microscope)

7-BIOSN

-BIOSN

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