TECHNIQUE FOR .SlJPRAU ITAL SUIN3rNG

w:   It is very important to have true vital dyes. In

this country, at present, the National Aniline and Chemical Company,

Inc., New York City, N. Y.,  sells a vital neutral red and a vital Jn-

nus green (Dinzine Green), ~re~,ared under the direction of Doctor H,

J. Conn, Chairman of the Commission on Standardization of Biological

Stains, Geneva, N. Y.   Every sample is tested in my laborr.tory

(Doctor Florence R. Sabin) before it is offered for sale.

       The technique consists of getting ~7. thin, evenly spread

film of two dyes on r?. slide and then m,&ing P prep?.rntion of fresh

blood on this slide.   The two dyes become dissolved in the pl~smn

and stain the co113 differentinlly.   The amount of the dye must be

quite accurately measured.   If there is too much, the cells are

killed nnd de,ad cells in this technique cannot be discriminated. To
get the dry dye on the slide,  the solvent used is absolute alcohol

which then is evaporated off.  The ,?lcohol must be entirely free of

ac id ;  the absolute alcohol, bought commercially, will not do.   It

must be distilled from lime in order to remove the last trrces of

acid.

        I.   Neutral red.   ft is convenient to hs,ve as n stock

solution a saturated solution of neutral red in absolute alcohol.

When procuring the neutral red, one must specify "for vital staining".

A saturzlted solution is 0.25 per cent and is m,ade by dissolving 125


mgms. of the dye in 50 cc. of the p.lcohol.   This solution is stable.


It is fnr too strong for staining cells. To make the dilute solu-
tion, -Ad 1.1 cc. of the sa,turnted solution to 10 cc.  of the abso-

lute alcohol.

        II. Janus green.  The Janus green solution is made by

dissolving 123 mgms. of the dye in 62.5 cc. of alcohol.  This makes

a saturated solution (0.20 per cent).

        III.  Mixed neutral red and J~lus green.   To make the

solution which is nctunlly used on the slides, t&e 3 cc. of the di-

lute neutral red <and to it add 2 drops of the saturated J,anus green

solution.  Have the slides thoroughly clean and free from grease, by

the usual methods.   Flood the slide with the stain nnd drain the ex-

cess immediately back into the bottle.   Then let the slide dry in

w,arm air.   This is conveniently `arranged by working very close to n

lightod Bunsen burner.   There should be no strertks whatever of the

stain on the slide.   The excess wan be removed from the edges by

placing the edges ngninst a piece of cheesecloth.   After the slides

ere once aa&, they keep almost indefinitely.   In anking the fresh

preparations of blood, it is important to seal the edges of the

covers1 ips yith R Vaseline of high melting point.   We study our

preparntions in a warm-box rcgulpted to 37' c. and if one is inter-

ested in motility of cells,  it is necessary to hn.vc the right tem-

porzture.  The supravital differential counts cc?n, however, readi-

ly be made at room temperature p.nd the cells nre much less damaged

by cold thnn by hoat.


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TEE STAINING OF BLOQD CELLS WITH THE UF'BAVIT
$

         1.   Granulocvtes.  The neutrophil ic granules stain

rather faintly nith a salmon red color.  You will notice if any

myolocytes appwar in the circulating blood that the staining of

the gr~anulcs is more intense than that of the mature leucocyte.

The myol ocyt es also contain mitochondria crhich vi11 stain green;

mitochondrin diminish as the cells mature, so the avernge leuco-

cyte shons only a very fe*n nitochondrin.   The grnnulcs of the

basophilic leucocytcs have a very bright red stain, which is the

acid reaction of the dye.  The large, usually ovoid, eosinophil-

ic granules, on the other bend, stain either yellovr or orange,

which is the clolor of neutral red in nlknlino solution.  Besides

the grnnulcs, the leucocytes, esnecinlly the neutroehilic type,

have vacuoles if they have phagocytized material and the fluid in

these vacuoles stains red vith neutral red.

       2. Lvmnhocstos.  The lymphocytes are distinctive on

account of their large content of mitochondria.   They tend to be

in the form of rods.  The lymphocytes occur in three sizes: small,

intermediate, and lexge, but perhaps it is only iqortpnt to sep-

arate the large ones from the other tTo.  The younger the cell ,

the more the mitochondria, and the very young lymphocytes show

also a rather yellont tone in the cytoplasm -rhich is due to the

high content of bnsophilic substance.  This is brought out better

in fixed films.  The lymphocytes also may have n. fey vwzuoles

staining in neutral red.  Both the mitJch,nCria and the VPcUOles


4.

tend to be in the endoplasm rather close to the nucleus.   Thg char-

acteristic pattern of the chromntin of the lymphocytic nuclei shoTs

in the living cell.

                             L

         3.   Monocgtcs.   Monocytes a,re characterized by deli-

wte surface film in contrnst tJ the shnrp 2nd dense borders of the

lymohocytes.  Monocytcs 7x0 Dhr.gocytic cells pnd thus have many vnc-

uolcs, smFl1 or lzge,  which stnin clith the neutral tone of neutral

rd.   These vncuoles are often in the form of 3 rosette, especially

in loner animals and in diseased states in man.   Besides the vacu-

oles that strain in neutral rFd, there are very lnrge numbers of tiny

nitochondrin crhich stain grew.   The chromhtin netnork in the nucle-

us of the living nonocyte is finer than th<Tt in the TIUC~E?US of the

lymphocyte.

         4.   Red blood cells.   You Fill notice in the suprnvitfll

prepornt ions thp t in<my of the red cells shop 8 little reaction to the


neutral red.   This is reticulation but the dilution of the neutral

red used for studying `Thite cells is too great to include all the

reticulocytus.  If the stain is zade cowentrnted enough for bring-

ing out 3.11 the reticulocytes, it -Till kill the white cells.

         5.  Macro-ohnges or clasmntocytes re;lct to the vital neu-

tral red;  their vxuoles vrlry in size and ?rhile each vncuole stains

uniformly, the different v~cuolcs sho*T the full range of shades of

neutral red from yello- to brillinnt scarlet.


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        6.   Awlication to nleural and sscitic fluids, ~.nd tQ

biwsv material.  The method cnn be applied to scrapings from or-

grns and to the cells of pleural, peritoneal, or cerebrospinal flu-

ids,  ~2s nell ns to various exudates.