Journal of the British Interplanetary Society, Vol. 34, pp. 100-102, 1981

HISTOCHEMICAL STUDY OF LIPID-LIKE MATERIAL IN PHOTOCHEMICALLY FORMEDJEEWANU, THE PROTOCELL, WITH FORMALDEHYDE PARTIALLY REPLACED BY OTHER

ORGANIC SOURCES

K. BAHADURChemistry Department, Altahabad Llniversity, Allahabad-211002, U.P-, India

P. K. VARMAZoologl; Department, C.M,P. Degree College,Al:lahabad-211002, U.P., India.

Jeewanu, the protocell having the cell - like morphological appearance and prepared with the interaction ofammonium molybdate, diammonium hydrogen phosphate, biological minerals and formaldehyde were stained with

Sudan black ,B' after fixing them with ilftman'i fixative. These particles showed the presence of lipid like material at

the boundary wall, and the wa11 of the central nuclear like region, by imparting black coloured stain-

Jeewanu, prepared in the mixture in which 60% formaldehyde had been replaced by other organic sources, showed

more synthesis and greater affinity with the stain studied.

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I. INTRODUCTION

THE FORMATION OF CELL membrane is an importantevent in the process of life synthesis, The criticai event

which may best be called as origin of life was the enclosure

of several different self-producing polymers within a semi-permeable membrane [ 1 ] . Jeewanu, the protocell [2]having amino acids [3] , peptides, sugars, enzyme-likematerials, and nucleic acid bases [4] have been prepared

by the action of light on aqueous mixture containingammonium molybdate, diammonium hydrogen phosphate,

biological minerals and formaldehyde'These particles have distinct boundary walls and internal

structures, they can grow from within, multiply by buddingand have metabolic activity [5]. These particles can be

fixed in biological fixatives and stained with gentian violet,eosin and haematoxylin [6]. These microstructures havephbspholipids in their boundary wall [7] . The bright spots

of lipids from particles extracted with.methanol and

chloroform and their presence in the environment medium

too were detected [8]. With all the above background and

researches done on jeewanu regarding the presence ofllpid by paper chromatography, it was desired to confirmthe result by histochemical tests.

Formaldehyde is one of the main consituents of themixture, which formed jeewanu. Bahadur used formalde-hyde because it can be easily formed by the interaction ofshort ultraviolet on aqueous solution of CO2 [9] and

secondly, in the presence of more Ihan 2% formaldehydecontamination was out of the question. An attempt was

made to replace formaldehyde by other organic sources.

It was observed that no jeewanu formation took place on

total replacement of formaldehyde. It was also observed

that only 60% of the total need of formaldehyde can be

replaced, i.e.,40% of forrnaldehyde was absolutelyesiential in the mixture for the formation of jeewanu. Thus,

6O% of ClI2 O was replaced by methanol, ethanol and

lactose as other sources of organic carbon' The jeewanu

thus formed were much bigger in size and revealed clear

internal structures, but the number of the jeewanu was

reduced.

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2. EXPERIMENTAL

(A ) Preparatiort of Jeeutanu

The jeewanu were prepared by using the following solutions:

Solution 'A' - 4 g of ammonium molybdate + 12 g

of diammonium hydrogen phosphate were dissolvedin 1-00 m1 of distilled water.

Solution 'B' - 3 g sodiutn chloride, 0.3 g calcium acetate,

0.5 g magnesium sulphate and 0.5 g potassium sulphatewere dissolved in 100 ml of distilled water.

Lactose Solution It was prepared by dissolving 10 g

of lactose in 12 ml distilled water+ 8 ml formaldehyde'

Whole of solutions 'A' and 'B' were mixed, a whiteprecipitate was obtained, which was digested in leastquantity of concentrated hydrochloric acid at boiling tem-peratures. The mixture was cooled, and the total volumewas made to 300 ml by adding distilied water. The mixturewas sterilised in an autoclave for 30 minutes at l5 1bs.

pressure. This mixture was known as 'control' solution'60 m1 of control solution was taken in four, steri-lised

250 m1 capacity conical flasks. The flasks were cottonplugged and then sterilised in an autoclave for 30 minutesat I 5 lbs pressure. After cooling 12 ml of methanol * 8 m1

of formaldehyde was added in flask No. 1, i2 ml ethanol* 8 ml formaldehyde was added in flask No. 2, 20 m1 oflactose solution was added in flask No. 3, while 20 m1 offormaldehyde was added in flask No. 4 which acted as a

control rnixture. Al1 the flasks were exposed to sunlight forfour days giving four hours exposure each day. After thefourth <1ay the particles were filtered, dried and then fixedin various biological fixatives for cytological studies.

(B) Fixot ittn

For staining with Sudan blac'k 'B' the jeewanu were fixedin Elftman's fixative [10] . The'fixative was prepared by

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dissolving 5 g of mercuric chloride and 2.5 g potassiumdichromate in 100 ml cfistiUea waie]lMercuric chloride i. u.."ugrrunili"utiu., and acts onlipoproteins as an unmasking ug.rri. in-_."r.uri. chloridethe cell was better preserverl than by urly oth.. coagulantfixatives. potassium di.hr";;l. ; l,rronl"lugrrunt fixativeand is important in micro_t".fr"lqr"r'Jfr#y for its fixativeeffect on certain rioios- ceriain',',;il ;;; ;;,e to take upchromium from the.solutro;;i oii.rj"," ol.hromate, andin so doing to lose their. solubiliffij"*ro'rir".ru. This is

:1q1,1".: fixative, wirh the p";t_;i;;;, ,i.,.., " usuat inmrcro-technique, only. phoiptrofipiAs iafie up the metal.Colour test for chromium will th;;;#;;Jveai ttre sites ofphospholipirls in cells i f f 1. 1l1.-;|;Oi"f'.,..rr"r_micro_

scopy of cells fixed by potassium .li.,t.o*il. shows howincomplete this process really is t f f 1 . 1i/lri"rf, potassiumdichromate is almost ur.t.rrin *1".J_,.?riqr.. If mixedwith other fixatives or usecl for p..l_"lro_1ng after fixation,it serves a purpose on account of its action-on unsaturatedlipids [11].The jeewanu were kept in fixative until they becamecompletely fixed. The f*.,1j;;;;;;;;; yellowish incolour and the particles ,."""r"a inJr"_"#rr"ul ,t.u.tu...clearly in fixecl state.

(C) Sraining

Choice and preparation of Stain

The most recommended..selective, and prescribed stain forIipids and phospholipids i, Suaan f,f r;:;:';;s,.ru n u ru.r :e | ;;;i;i;,

" d r',1 iil'X,,i,l ii; l.1L;,lr- "

t1 00 ml of 70% ethanol. fn^. ,oti,iioo *", ,r..i.o gently upto boiling point for about 3 minutes. The mixture wasfittered and coole<1. (Always il;;;;';;o'ri,uuon *u,again filterecl).

Procedure of Staining

(i) After complete fixation the particles were fil ned onalbuminized slides and tept ior;;;;#;r, overnight.(ii) They were washed in running water for lzhour to 1hour ro remo\re the yetlowisi ;;;r';i lixative.

(iii) They were put in 50% alcohol for 15_20 secs.

(iv) Put in stain (Optimum,time duration is 2_5 minutesat roont temperalure, i7oC, but tfr.'p"rtr.f * did notdevelop the colour.even after Z frou.-io"i hour, so theywere left in the stain. The fuli .ofou. aiu.loped afterI d-20 hour:1.

(v) Immersed in 7lVo aicohol for 5_10 secs. for removal ofexcess stain.

(vj) Wasirg.a in water, dried in air and mounted in glycer_ine jeily.

Rt:ults

The particles retained their original shape and size.

The boundary wall of the particles acquired abrilliant black colour.

Some particles showed clear differentiation of thedouble layered boundary, while in .tfr"r. Alfi...rt*tion was not clearly marked.

I he central region also imparted a black colour, which

Study of Lipid_Like Material in Jeewanu

was much darker in shade at the periphery than theinner side of the central,"gion. ' ---."

Conclusions

l_l._-y.,p showed the presence of lipids like material, byrmparting black colour.with Sudan'bi*t :irf,'", the boundarywatt. The boundarv watt appeareJ;;;;;;i. tayered, aso.,l^:n

:^':f -.]; ac g u re d^ t ir.r, .olor.i,

"r. o_. i., ur ilc (.onIrol reeion of lhe particles also got stained andthe black coiour rias ,nr.r-r au*..'i; ,;;;?'irs peripherythan the inner side. This indicates iil"t tf* .*t.ai regionhad its own limitins membrane ,hi;h'rt;"*; the presenceof lipid Jike materia"l.

Experiment to Confirm th-e presence of Lipirts by Non_Speciflc Colouration

The oil soluble colourants are.generally regarded as specificreag€nts f or lipids. Non_sp e cifii .tal"rng-fris' Jeen distin_guished from the true colourati";;idi;lJ"a..otou.utionIl 21. 14ua"rtul that sives a true Sudanophilic reation can bediscoloured and ,e-iloured repeatedly provided it is notex t racr ed by rhe decolourisin g'reageni t'i it'.'"'

ProccJure lA ) Destaining

(i) The particles were stained with Sudan black .B,asdescribed in the earlier .rp..lrn"nl.*" ""'

(it They were immersed in 7O% alcohol for t hr. to1)4 hrs. and observed under a ;;;;;;..Results

The particles lost their black colour andtranspa.renl as the originat. gur in rhese,eo partrcles a lew things were observed:_

(i) Some of the particle.s in their control region showedrhe presence of btack Iipid do;s iF;r. i;-'(ii) In a few, the boundary wall and the limiting mem-brane of the nuclear tit. ..nlrut ,.n"" ,"i.rr.a ,fr"iight black or srevish .olou. giulng*ui'irr#.n.. tt rtthey contain siight amounts ol iipid component(Fie.2).

I:'ig. 1 - Control Jeewanu _ ,showing the prescnce ol lipidJikematerial at the boundarv walt_and

"il ;ii;; ;;;;r"anl ot trrecentral region. (1s00X magniricaitun';;;;,i;; ;'","r*:o)

became almost assufficiently destain_

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K. Bahadur and P. K. Varma

lrig. 2 -- Lactose Jeewanu The boundary wall and the membraneof the central region shou'ing increased intensity ol lipid likematerial (1500X magnification, negative enlarged).

Proc'edure (B ) Restaining

(i) The above destained slides were put again in Sudanbiack 'B'for staining.

(ii) They were dropped in'10% alcohol, quickly andwashed, dried in air, and observed immediately undera microscope.

Result

The particles again took the stain, though not as deep andbrilliant as in the previous experiment, and showed thepresence of lipid like material.

3. DISCUSSION

The above histochemical tests clearly showed fhe presenceof lipids in the abiogenically synthesised jeewanu. Theywere sufficiently present in the boundary wali and take partin its formation along with proteinous element more akinto the modern cell. They were further observed to bepresent in the limiting membrane or the wa1l of the central

(nucleus like) region of the particles.Jeewanu, prepared in the mixture in which 60% formalde-

hyde had been repiaced by methanol, ethanol, and lactosewere better stained and showed more affinity with thestain. The lipid-like material was also synthesised in greateramounts in these particles as compared to the particleswhich had the total part ( 100%) of the formaldehyde as a

source of organic carbon.

ACKNOWLEDGEMENT

The authors are thankful to U.G.C., New Dehii, India forawarding a Teacher Fellowship to the second author.

REFERENCLS

Haldane, J. B. S., New Riol. 16, l2 (1954).Bahadur, K. and Renganayaki, S..JB1.l 23.813 (1970).Bahadur, K., Verma. M. L. and Sin-eh. \'. P-. Zeitschrilt .furAllg. Mikrobioktgie 14, 8l (.191 4).Ranganayaki, S., Raina, V. and Bahadur. K.."rB1S 25,279{t91 2).Bahadur, K., ZbL Bakr. ll7, 585-602 (1964).Bahadur, K. and Gupta, J. L., Zbl. Bokr. 127 ,643 (1912).

Singh, Y. P.,(I974), "Studies in the abiogenesis olall lipids andother compounds of biologrcal interests." D. Phil. Thesis,Chemistry Department, Allahaba4 Universitl'. Allahabad,lnd ia.Narain, K., (1973), "Studies of some self*ustaining molecularassociations," D. Phi1. Thesis, Chemistrl' Department,Allahabad University. Allahabad, India-Garrison, W. M., Morrison, D. C., Hamilton, J. G., Bensen,A. A. and Calvin, M., Science 114,416 (1957).Elftman, H., Stain Tech. 32,29 (1954).Baker, J. R., (1 969), "Cytological Technique, monograph onbiological subjects," Methuen and Co., London.Cassel, W. G. 8., (i959), Histochemical technique," Metheunand Co., London, p. 71.Pearse, E. A. G., (1961). "Histochemistry," J. and A.Churchill, London, p. 307.Lillie, R. D. and Burtner. H. J.,"r. Histochem. and Cytochem1, 8 (1 9s3).

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