Journal of the British Interplanetary Society, Vol. 34, pp. 100-102, 1981
HISTOCHEMICAL STUDY OF LIPID-LIKE MATERIAL IN PHOTOCHEMICALLY FORMED JEEWANU, THE PROTOCELL, WITH FORMALDEHYDE PARTIALLY REPLACED BY OTHER ORGANIC SOURCES
K. BAHADUR
Chemistry Department, Altahabad Llniversity, Allahabad-211002, U.P-, India P. K. VARMA
Zoologl; 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 of ammonium molybdate, diammonium hydrogen phosphate, biological minerals and formaldehyde were stained with ,B' after fixing them with ilftman'i fixative. These particles showed the presence of lipid like material at Sudan black the boundary wall, and the wa11 of the central nuclear like region, by imparting black coloured stainJeewanu, prepared in the mixture in which 60% formaldehyde had been replaced by other organic sources, showed more synthesis and greater affinity with the stain studied.
I.
INTRODUCTION
THE FORMATION OF CELL membrane is an important event 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 semipermeable membrane [ 1 ] . Jeewanu, the protocell [2] having amino acids [3] , peptides, sugars, enzyme-like materials, and nucleic acid bases [4] have been prepared by the action of light on aqueous mixture containing ammonium molybdate, diammonium hydrogen phosphate, biological minerals and formaldehyde' These particles have distinct boundary walls and internal structures, they can grow from within, multiply by budding and have metabolic activity [5]. These particles can be fixed in biological fixatives and stained with gentian violet, eosin and haematoxylin [6]. These microstructures have phbspholipids 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 of llpid by paper chromatography, it was desired to confirm the result by histochemical tests. Formaldehyde is one of the main consituents of the mixture, which formed jeewanu. Bahadur used formaldehyde because it can be easily formed by the interaction of short ultraviolet on aqueous solution of CO2 [9] and secondly, in the presence of more Ihan 2% formaldehyde
contamination 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 absolutely esiential 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. 100
2. (A
;
I
I
EXPERIMENTAL
)
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 dissolved
in
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 sulphate were 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 white precipitate was obtained, which was digested in least quantity of concentrated hydrochloric acid at boiling temperatures. The mixture was cooled, and the total volume was made to 300 ml by adding distilied water. The mixture was 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 cotton plugged and then sterilised in an autoclave for 30 minutes at 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 of lactose solution was added in flask No. 3, while 20 m1 of formaldehyde was added in flask No. 4 which acted as a control rnixture. Al1 the flasks were exposed to sunlight for
four days giving four hours exposure each day. After the fourth <1ay the particles were filtered, dried and then fixed in various biological fixatives for cytological studies.
(B)
Fixot ittn
For staining with Sudan blac'k 'B' the jeewanu were fixed in Elftman's fixative [10] . The'fixative was prepared by
I
t
Study of Lipid_Like Material in Jeewanu
dissolving 5 g of mercuric chloride and 2.5 g potassium dichromate in 100 ml cfistiUea waie]l Mercuric chloride i. u.."ugrrunili"utiu., and acts on lipoproteins as an unmasking ug.rri. in-_."r.uri. chloride the cell was better preserverl than by urly oth.. coagulant fixatives. potassium di.hr";;l. ; l,rronl"lugrrunt fixative and is important in micro_t".fr"lqr"r'Jfr#y for its fixative effect on certain rioios- ceriain',',;il ;;; ;;,e to take up chromium from
was much darker in shade at the periphery than the inner side of the central,"gion. ' ---."
Conclusions showed
the presence of lipids like material, l_l._-y.,p by rmparting black colour.with Sudan'bi*t :irf,'", the boundary watt. The boundarv watt appeareJ;;;;;;i.
the.solutro;;i
oii.rj"," ol.hromate, and in so doing to lose their. solubiliffij"*ro'rir".ru. This is fixative, wirh the p";t_;i;;;,
,i.,.., usuat in :1q1,1".: mrcro-technique, only. phoiptrofipiAs " metal. iafie up the Colour test for chromium will th;;;#;;Jveai ttre sites of phospholipirls in cells f i f 1. 1l1.-;|;Oi"f'.,..rr"r_micro_
scopy of cells fixed by potassium
.li.,t.o*il. shows how incomplete this process really is t f f 1 . 1i/lri"rf, potassium dichromate is almost ur.t.rrin *1".J_,.?riqr.. with other fixatives or usecl for p..l_"lro_1ng If mixed after fixation, it serves a purpose on account of its action-on unsaturated lipids [11].
The jeewanu were kept in fixative
completely fixed. The f*.,1j;;;;;;;;;until they became yellowish in colour and the particles ,."""r"a
inJr"_"#rr"ul
clearly in fixecl state.
(C)
,t.u.tu...
;;;i;i;,
Experiment to Confirm Speciflc Colouration
d^ t
ir.r,
.olor.i,
iil'X,,i,l ii;
il;;;;';;o'ri,uuon
again filterecl).
*u,
Procedure of Staining
After complete fixation the particles were fil ned on
albuminized slides and tept
ior;;;;#;r,
overnight.
They were washed in running water for lzhour to hour ro remo\re the yetlowisi ;;;r';i lixative.
1
(iii)
They were put
(iv)
Put in stain (Optimum,time duration is 2_5 minutes at roont temperalure, i7oC, but tfr.'p"rtr.f * did not develop the colour.even after Z frou.-io"i hour, so they were left in the stain. The fuli .ofou. aiu.loped after I d-20 hour:1.
in
50%
tf* .*t.ai region ,hi;h'rt;"*; the presence
th-e presence
of Lipirts by Non_
from the true
Il
colourati";;idi;lJ"a..otou.ution
21. 14ua"rtul that sives a true Sudanophilic reation can be discoloured and ,e-iloured repeatedly provided it is not ex t racr ed by rhe decolourisin g'reageni
(it
)
it'.'"'
Destaining
The particles were stained with Sudan black .B,as described in the earlier .rp..lrn"nl.*" ""'
They were immersed in
7O%
alcohol
1)4 hrs. and observed under a
;;;;;;..
for t hr. to
Results The particles lost their black colour and became almost as transpa.renl as the originat. gur in rhese,sufficiently destain_ eo partrcles a lew things were observed:_
(i)
Some of the particle.s
in their control region
rhe presence of btack Iipid do;s
(ii)
iF;r. i;-'
showed
In a few, the boundary
wall and the limiting membrane of the nuclear tit. ..nlrut ,.n"" ,"i.rr.a ,fr" iight black or srevish .olou. giulng*ui'irr#.n.. tt rt they contain siight amounts ol iipid component
alcohol for 15_20 secs.
(Fie.2).
Immersed in 7lVo aicohol for 5_10 secs. for removal of
excess stain.
(vj)
Wasirg.a
ine jeily.
in water, dried in air and mounted in glycer_
Rt:ults
.+.i
,ffi
The particles retained their original shape and size. The boundary wall of the particles acquired brilliant black colour.
a
Some particles showed clear differentiation of the double layered boundary, while in .tfr"r. Alfi...rt* tion was not clearly marked. I
he central region also imparted a black colour, which
as
The oil soluble colourants are.generally regarded as specific e cifii .tal"rng-fris' Jeen distin_ guished
gently up
to boiling point for about 3 minutes. The mixture was fittered and coole<1. (Always
tayered,
"r.
reagâ&#x201A;Źnts f or lipids. Non_sp
(i)
*", ,r..i.o
(v)
re
had its own limitins membrane of lipid Jike materia"l.
ProccJure lA
The most recommended..selective, and prescribed stain for Iipids and phospholipids i, Suaan f,f r;:;:';; | :e t s,.ru n u ru.r d r',1 l.1L;,lr- " 1 00 ml of 70% " ethanol. fn^. ,oti,iioo
(ii)
ac g u
(.onIrol -.]; reeion of lhe particles o_. i., u ilc :^':f the black coiour rias ,nr.r-r au*..'i; also got stained and ,;;;?'irs periphery than the inner side. This indicates iil"t r
t'i
Sraining
Choice and preparation of Stain
(i)
o.,l^:n
I:'ig. 1 Control Jeewanu _ ,showing the prescnce ol lipidJike material at the boundarv walt_and ;;;;r"anl ot trre central region. (1s00X magniricaitun';;;;,i;;
-
"il ;ii;;
;'","r*:o)
I0l
K. Bahadur and P. K. Varma (nucleus like) region of the particles. Jeewanu, prepared in the mixture in which 60% formaldehyde had been repiaced by methanol, ethanol, and lactose were better stained and showed more affinity with the stain. The lipid-like material was also synthesised in greater amounts in these particles as compared to the particles which 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 for awarding a Teacher Fellowship to the second author. W,;il ':titj:
lrig. 2 -- Lactose Jeewanu The boundary wall and the membrane
of the central region shou'ing increased intensity ol lipid like material (1500X magnification, negative enlarged). REFERENCLS
Proc'edure (B )
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 .fur Allg. Mikrobioktgie 14, 8l (.191 4). Ranganayaki, S., Raina, V. and Bahadur. K.."rB1S 25,279
Restaining
(i)
The above destained slides were put again in Sudan biack 'B'for staining.
(ii)
They were dropped in'10% alcohol, quickly and washed, dried in air, and observed immediately under a microscope.
{t91 2). 5. 6. 7.
Result
13.
Narain, K., (1973), "Studies of some self*ustaining molecular associations," D. Phi1. Thesis, Chemistrl' Department, Allahabad University. Allahabad, IndiaGarrison, 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 on biological subjects," Methuen and Co., London. Cassel, W. G. 8., (i959), Histochemical technique," Metheun and Co., London, p. 71. Pearse, E. A. G., (1961). "Histochemistry," J. and A.
t4
Churchill, London, p. 307. Lillie, R. D. and Burtner. H. J.,"r. Histochem. and Cytochem
9.
DISCUSSION
The above histochemical tests clearly showed fhe presence of lipids in the abiogenically synthesised jeewanu. They were sufficiently present in the boundary wali and take part in its formation along with proteinous element more akin to the modern cell. They were further observed to be present in the limiting membrane or the wa1l of the central
102
Singh, Y. P.,(I974), "Studies in the abiogenesis olall lipids and other compounds of biologrcal interests." D. Phil. Thesis, Chemistry Department, Allahaba4 Universitl'. Allahabad, lnd ia.
The particles again took the stain, though not as deep and brilliant as in the previous experiment, and showed the presence of lipid like material.
3.
Bahadur, K., ZbL Bakr. ll7, 585-602 (1964). Bahadur, K. and Gupta, J. L., Zbl. Bokr. 127 ,643 (1912).
10.
11.
1, 8
(1
9s3).