Dl;)i r<!

nn any
~_,' -Ill.

J)l'ecipitatae ~d·-;h v.,c o ny<1rolyscd, the ref -

ore , ~t .-.
d~ - --- · ••&~

exp eri me nta l tub ows. the ,Pr ese n~ of trypsin . . Iii. the contro1
it does n.o t contain
trypsin and th c t~ere8 no extract, therefo.re1 the additio·n of a,cetic
aci d gives the e ca . . . ·1 not hydrolysed and
c1p1tate.
TI MA TI ON OF HA EM OG LO BI N
BY liAEMOMETER .
EX PE RIM EN T No. 7
O~Ject :. To est iina te tl,e amount of haem<,Jglobin inrat{I) frog•~
er verteb e animal.
hlood, (11) luunan hlo ofi and (iii ) blo od of any oth

J.

··•.

•••~n-·
tobe ·

.. . .-
I •

standard
tubes

• I

A
A- 20 cmm micropipette. ~H ald ane ·s haemometer.
 592 , PRACTICAL ZOOLOGY_· CHORDATES

.lleflalt~ents·:-: (i) }fa~mometer (Haldane's haeJJtoglobinometer);

(ii): dc,cibomil' .(N/l~) MCI ('1 ·2 cc. .of:conc.,HCJ dissolved in 100
cc
of:distillod water) ; (/ll)'distil~d water-; (if) 'living:· frog· or any other
f li\'idg vertebrate.
. . Appa~tus : ~e ·h.aemometet .co~sisi~ ._o f tw_~ . sealed lateral
~mparison tubes containing a ·su~perision of a~id .haematin. These
, i'tt held 10.· a.black frame against a· Wl\ite· ground glass. Besides, fita
gi,iduated test-tube of the same diameter is"aJso 'provlded whkh can
itf tbe haeinomet~i' in between the ,two _side; tubes for comparison. A
inicropipette · of 2~ :ciitm is alsc{ ·':pfov1,fac1. The _. o~_her things_
1

I prov ided are ·a ··small glal·$·rod·_ stik're't/ a: >tm~II . bottle. 'brus

....
h and
. ;
a.
'1 "·> ·,_.,..,.., ,\1., .........r;- ..

·dro p~·- , ab'd ··aJso a· ~mall ·· bottle to· ·coritai~ ·!_ .11,, ~ deqtn'9riii~f·: acid
, '- : .1 ' .. . .... ·. •
!
solution. , . ... ,.. _ . . : ,. . · ·;- ,· . - · ·. , ~- · .; .,.,.
. Priadple : .,.·_1Th:e ~~tho..d'~<i estiinat~\'"-' ihb haemog)obii;i content
of blood is bas~ bn t~~ ptincjple :Q(,_'m~king '~ri-·acid · ·haematin- solu-
ti~n ~f blood un_~er expe~inientation in\ the grad uated tube and then
~~~-~ ~t-ti.~"-~,e~ ~~~e~ comp~H~_o~• t~l;,es::' :cp~t.ai1:1ing "··the stan-
danfacid haematin.' ,...... , "'· .. ' ,,1
. . ~ • e·: ·The g~aduate~ ~ube .is ·first ~leaned- with distill ed
. water ·anc1 then with -~ethy.Jjted·' spi'rit ,or '90% alcohol/ . It is thoro
u-
l<gbl)' dried ·tip before'$l ij ?ll;~~d.:,. Nbw with the, ~help ·or a dropp
er
. ~ -·. N/10 Ha,· !~l~ti~n~:ri~"·.;filled _'Ill the:gra;~u~~~-ttib_e up to 2 gms
mark. Th~-- ~icfbpi~~e is,.no~\fiH~d--up. ~y s_~cid,ig :fresh : blood of
' . • - ~ ' ••

·the vertebrate · undet,.\p.pttimentat1ont:.: up .tQ tftcfmark 'of 20 cmm.

. .~..... . , .',,: , . . . ..... . ~!
•' •· ' \ • .. .. . . ' •

.- 'The smal l ~o~ t;'.~ '.-tiJpi j~~~ic.frins\ ~~?t~e outersid~~-of ·_ mforopipe tte
', ·should .be:;~ipecl:Qtf'~by::sJerjji~d .cotton~· The blood of niicropipet
te
is iiow ad~ -~~ :· 1
~-~ ~:-~-~~~r:?~-uti~n·1n
·_t~~ _gr~d¥~~e~ The
r
:_ w~.
pipette should1--.-~ -- 1ntr.,Q~~4j~l ·car~fu.Hy -~l}to _the tµbe ,and 1ts lowe
O

mouth.sho~ki ~~~di~r tip t'~:~e botto~ .into,H~J -~9l~tjq,.- When

blood has;;b . . e,d:~
. een ~~n,
is,rin•s~d -twice,: o\f :~
.~~!, ~tte . .
. . ,. e;;pj ~
' - •_,-~y
' .' r- . ~is-
., -,, . ~ ~

tilled wat~t.~..- -~~ ~:J i"~ifpt:e~~nt~n~s,9(.micro.pipette shou ld be .ex-

pel_led into.•the sr.au~,~~tt~:·,·.
Thc{ acid . :hae~a.fin :~s-olution is now
thoroughly stfrr ~ with :t4e llelp of a gl~s .- ro,_d. , arid .theti allowed to
stand at -least for 10 min ~·i · Afterwards ·the .acid haematin solution
is iradually diluted by adding•distilled water iil a: dropwise manner.
·
With the additio11 -of each ·'drop of distilled wate~ -the· solut~on should
be. stirred and its coloq,r matched with that of the stand~d sealed
tubes. . This shou ld be continued t~ll the c~Jour of the acid ·heamatin
solution just· fades .·away as ·compared to th~t of the sta_ndard com-
.parison .tubes. The re~ g before the colour Just fades 1s taken as
the correct and final reading. ·
 ~ _
· .
593 ..
~ ··EXPERIMHN~AL PHY.SIOLOGY
I . ., . • •
:...
1
~.
••
. • :l

O~se r.v ation s : The rea~in_g s_are t~bul~ ted in the ~~l-~l .tng ~
; '
~ mann er . . . : . ,,~..- *~~ . .
r
Frog's blood Hum,pn blood- .,
'!

' ,, . ' . ·. .f . .,t- '/ l

/
,. I •
i "l
I 7.8 .12.45 t j

II 7.10 12.60

III 7.9. 12.50

Final - 7.9 • .
12.50
.
'-r l

... Results: The blood offro ~j;nta ins 7.9 nigm of?_ae~ogl ob~q in 1_~0 ml l \

_ l
ofblo od. ~ - _. · . . _.
, The blood o f ~ ~ontains 2.50 mgm of haem~glob1_n 1n I oo _ ml of .
~

· !

blood . · . ....
· · ,• ,
i
i

Precautions :_Th~ f<;>ll~wing precautions are to -~~ take~ ;.~ .. ,·:.

i
I'\ -_·
~

. 1. In case huma~ blood is being tested,"give ~ _light p_rj_c,lttq :t~e-fin~er

tip of the perso n provid ing ~lood. ·_ · ·_, : ,_., · . ''\ / ... _ ~ 0
·• --~---:· - • , .

· 2. The finger and the prick_(needle)'°sliould~~~ldi~infe;ct ep. fir.st with

or
methy lated spirit or alcohol (90%· atiso_lµtej~ . _i, .· --:~ -~)~:~~--:-'- ::- .• C . ~-- •

3. Avoid unclea~ed tubes' aitd i,tp~ttes, ~tc~:- . _, -~~-- ~-~·:)..(? J- ·: :-r_ .•. . . . - -· \
1

4. Avoid incorrect·filling·up ofrriicr.op1pette~·-- · _' :, '~ ·. ·· ·

5. A void inclusion -of blood stjcking:c.it· the out~r-sutfac e··of- ~lie-'mo:uth ·
of the pipett e · · : . ,. · ··- :~-~- .: _:\ :..,, --.;•..~i=. .. · ..·.. I . ---~>- · · · · --~- · · -A~~ -~

~~- oJ
·h~....
• •
•.• ~.
~-'
. •
. \... _,..

. ..
' •

6. Readi ngs shoul a IJe takei;talway s))f,t~_¢:bpp~ r meris cuf · :_ ··_. ·.

•

f ~ '"'t; "t • • --..

7. Exper iment shoul~l ~-e perf~ ~e<t< t~ickl i ~ !t~out ~~st~'of tim'e so
- • ~... .... 'i "' J '
'•

that fresh blood is not ·allow'-• ed~ to 69agu laf~, p~fore :!t[a_nsfer. 'to ,(iecir torma l,
~--. ,
'¥'•
•--.::_.l . •• -,~•..._,~ ••
- . ~~-
' ' \-.•\. ~.,_ '•~":.' \ •
- .
HCl
. ~-\ '\ -~ -- . ._':'.. _1._: <.'~!t\..- - .. _, .:,·. '· ..
~n"'·n ., nnn rnoU iTQr 1 ~~