Complete Text (Part 14)

show less proiiiineiitly than with the Wilson stain. ^Malarial parasites are not well stained. The intercellular areas are clear and shoAV no stain. (4) Pitfalls in using Jenner stain. These are almost absent. The chief difficulty is in giving the nuclei a deep enough blue stain. Improper stains show the nucleus to have a faded-out bluish tinge. By changing the water and stain combination and by washing more quick- ly the nuclei can be made to take a bet- ter stain. c. With EhrUch stain. (1) The stain — a complex triacid aqueous- alcohol stain. Do not shake the bottle before using. (2) Preliminary details : (a) Old smears stain better than fresh. Ex. smears 5 to 10 days old are bet- ter than tliose 5 to 10 hours old. It is advisable to have 15 to 25 smears of the same age when an Ehrlich is to be stained. (b) Before staining, smears must be fixed by heat in the following man- ner: 1. Heat a copper bar for 30 to 10 min. in a place free from draughts, so that the flame will not be shift- ing. (X. B. — Place flame under point of bar.) 2. At the end of that time, with a dropper having a small bored point, drop a few small drops of 172 water (3 to 4) on the bar in an etfort to find the spheroidal i)oint. (Spheroidal point equals point on bar where water rolls off in glob- ules.) Let at least 5 min. elaj^se be- tween trials, so that the heat lost by the application of the water will be surely regained. 3. AYhen the spheroidal point has been determined by repeated tests, draw a chalk line across the bar at that place. ( N. B. — Inaccurate determination of the spheroidal point is to blame for many poor Ehrlichs.) 4. Place preparation, smear side up, outside the spheroidal point, with the edge closest to the flame exactly on the line. The method is carried out as follows : Fix a smear 20 sec, another one 30 sec, still another 40 sec, on up at intervals of 10 sec, until 90 to 150 sec (Devise a method for care- fully differentiating the variously heated smears so that in staining no confusion will arise as to the lixa- tion time of any particular smear, for ex. 20 sec. or 90 sec.) 5. In general, the older the smears the shorter the required time of heating. Ex. smear 10 da^^s old may be well fixed at 30 sec. ; smear 1 day old may be well fixed at 150 sec. or more. (3) Actual staining. (a) Place the stain on the smear and let it remain 5 min. At the end of 173 that time pour the stain hack into the t)ottle, and wash off the adher- ing ainonnt of stain in the sink. Dry and blot as directed under the other staining methods. (Ehrlich stains are not easily ruined in the staining and washing, but are generally spoiled by poor fixing.) (4) Criteria for a good Ehrlich. (a) The cells should stand out clearly with no intercellular stain. (b) The K. B. C. should be buff, without the slightest suggestion of any red color, and without being a lemon yellow. (N. B. — Smears Avhich are under- fixed or markedly over-fixed have a reddish color in the K. B. C. Smears which are moderately over-fixed have lemon color in the K. B. C.) (c) The W. B. C. stain as follows : P. M. X. — Nucleus — Blue green or robin's egg blue; no structure visi- ble. Protoplasm — Faint pink. Gran- ules— Lilac, if correctly fixed ; red from short fixation. P. M. E. — Nucleus — Light green color. Protoplasm — Generally none seen. Granules — Dark red or crim- son. P. M. B. — Nucleus — Light green, reticular. Protoplasm — None seen. Granules — No stain, often seen as colorless vacuoles. Lymphocytes. — Nucleus — Light blue green. Protoplasm — ^Light pink 174 or violet, often very faint. Grannies — None seen. Lartie Mononnclears. — Xnclens— r\iint bine or green. Protoplasm — Fink or light violet. Grannies — None seen, (di Platelets are not stained. ( e ) Malarial parasites are not stained. (."")) Creneral tecliniqne nsed in staining with Ehrlich. ( a I From 15 to 25 smears ; select 3 or 4 of the best and lav them aside. (b) Take the less good smears and rnn through the various times for fixing a Irea d v su gges ted . (c) Stain these, and after mounting ex- amine them carefnlly with oil im- . mersion, and note the following points : 1. Under-fixed smears Avill have reddish staining K. B. C, often with stained intercellular spaces. The W. B. C. will have a dirty appear- ance with granules not standing out distinctly. 2. Well fixed smears will have the R. B. C. of a buff color (neither yellow nor red). The W. B. C. will stand out distinctly with neutroph- ilic and eosinophilic granules well stained and standing out sharply against a clear unstained proto- plasm. 8. Over-fixed smears will have the R. B. C. lemon color. The granules in the polymorphonuclear white cells Ayill not be clear cut, and if '175 stained will be seen lying in a stained protoplasm. There is a smudgy look to the polymorpho- nuclear cells. The mononuclears are often so faintly stained as to be missed. 4. If still further over-fixed, the K. B. C. lose their lemon color, and again take on a reddish hue. (d) Having discovered a well-stained smear, note the heating time for it, and then fix any number of good smears (of the same age) for a sim- ilar time. (N. B. — The need of again deter- mining accurately the spheroidal point is evident since by placing these goods smears at a point which is no longer the spheroidal point, they will be ruined.) d. General points with regard to stained blood preparations. (1) Be careful to mount cover slips in a neufral halsam, if any acidity is pres- ent (as frequently occurs) the color will fade at a rate proportional to the degree of the acidity, the nuclear ele- ment, particularly, being attacked. (2) Keep the preparations away from bright sunlight, from acid or alkaline fumes. (3) After using oil, remove it with xylol. (N. B. — Never attempt this if the smear has been freshly mounted.) lY. Differential Counting. The cover slip method must be used for this as follows : . a. With a mechanical stage count across and 176 up and down over the good areas of the spread. Be careful to use such a technique that the same areas will not be counted twice. b. .'iOO W. K. C. must be counted. In order to get the true proportion it is necessary to include in this nund^er brolvcn cells and undertermined cells as well as those which can be classified. c. A W. P. (\ count should always be nuide at the time a ditferential is counted. It is only by this means that relative or abso- lute changes in the number of cells is de- termined. (1. The classiticati(m of cells is as follows: (1) Polymorphonuclear neutrophiles (P. M. X.). The nuclei — irregular, 2 to ^) lobes, often appearing actually polynuclear. The granules — pink or violet (AVilson). pinker (Jenneri, lilac (Ehrlich). (X. B. — With Wilson the granules may not stand out distinctly an<l one sees only the more or less homogenous pink protoplasm.) (2 1 l^)Iymorphonuclear eosinophiles (P. M. E.). Tlie nucleus is irregular, generally bi- lobed and nmy give the appearance of being actually polynuclear. The gran- ules are large as compared with the neutrophilic granules and are all of the 177 same size. They stand out very dis- tinctly and stain a deep pinl-c to crim- son with all stains. These cells tend to be a trifle larger than the neutro- philes and are rather fragile. For the latter reason they can often be found broken witli their granules scattering. Eosinophiles are in general recognized by the large, constant size of their granules rather than by the depth of the granular stain. 5 (o» Polymorphonuclear basophiles (P. M. B.). The nucleus is of the size and shape of the eosinophilic nucleus, but some- times is entirely hidden by the deeply staining basophilic (blue) granules which completely fill the cell. These granules are not stained by the Ehrlich stain. # Wilson Stain Khrlich Stain (1) Lymphocytes. Small and large types with the size of the P. M. N. taken as the arbitrary 178 (^livicUng line; lymphocytes smaller than a P. M. X. are considered small lymphocytes (S. L.). Those as large as or larger than the P. M. N, being considered as large lymphocytes (L. L.). «» I L. Iv. S. L. P. M. N. L. Iv. The nucleus is round and takes a deep blue stain. The protoplasm stains a pale blue (darker than the proto- plasm of the large mononuclear) and gives an impression of thickness. It is sometimes described as waxy (in contradistinction to the thin tissue paper thickness of the protoplasm of the large monos.). The cell is fairly regular in outline and is round in shape. (5) Large mononuclears are large cells with a single nucleus, which is round or kidney shaped. The nuclear stain is lightei' than that of the lymphocyte and the nucleus appears less dense. In the protoplasm, which is stained a pale blue, can be seen the reticular nodosities, which may be so marked as to give a granular appearance. No true granules are ever present, al- though the reddish azurophilic gran- ules are often found. This cell is pro- portionally large, with a relatively 179 large amount of protoplasm. Tt gives the impression of great thinness. (The term transitional is given to those cells when the nucleus is kidney shaped. It probably represents an older type of cell.) ^nS Transitional V, Fresh blood examinations. a. Technique. (1) Place a drop of blood the size of a pin- head on a cover slip (procured as for making smear). (2) Place the cover slip on the convex side of a slide in such a manner that the drop is spread without bubble forma- tion. Let the cover slip remain on the slide. (3) Study first with the low power, then with high, dry and oil immersion. b. Criteria for a good preparation. (1) The spread must be thin, with the cells lying flat and not overlapping. c. Common difficulties in making a good, fresh blood preparation. (1) E. B. C. overlap or lie in rouleaux, due to (a) Unclean glass ware. (b) Too large a blood drop. (2) Uneven spread, due to (a) .Dirty glass ware. (b) Careless spreading of the drop. 180 J. Findings in a fresh blood preparation. (1) R. B. C. (a) Color — Greenish yellow with the intensity depending npon the amount of hemoglobin present. (b) Shape — Ronnd (if lying free T^ith- ont touching other cells), with a biconcavity, the apparent degree of which depends upon the amount of hemoglobin content. Abnormalities in the shape of the cell as a whole constitutes a condition known as jwikiJocytosis. (c) Size — Diameter of 7.5 mikra ; nor- mally there is only the slightest variation in size. With noticeable variation in size the condition is spoken of as anisocytosis. Large cells = macrocytes (ap- proximately above 9 mikra ^. Small cells = microcytes (ap- proximately below 6 mikra). (d) Degenerations — Due to trauma as- sociated with the spreading and to the drying of the preparation, de- pending, of course, primarily upon the inherent fragility of the cells. 1. Total degeneration. The cell shrinks and becomes darker in color ; the margin becomes irregular : varying numbers of prickly points appear, giving the picture of a thorn apple, crena- tion. These points Avhen apiDcaring in pro- file are readily recognized, but when looked straight down upon ma}- give the impression of cellular inclusions. 181 2. Partial degeiieratiou. a. Maragliano bodies — an endoglobular degeneration, generally in the center of the cell, but frequently at the periphery, occurring as a single or multiple degeneration in a cell. The body is round or elliptical and looks like a vacuole. Its size and position in the cell often change. Confusion arises with nucleated R. B. C. and the early stage of malarial parasites. b. Bacilliary degenerations — rod-like hya- line areas in the cells, with a vibratory motion so that the ""area" may move through the whole substance of the cell. Often confused with bacilli. c. Ehrlich^s hemoglobinemic degeneration — cells appearing with a dark center and a light periphery often giving the appearance of a small cell superim- posed upon a larger one. The condi- tion probably is due to areas of con- densed protoplasm with the hemo- globin separated from the stroma. 0 Pessary Form 3. Differentiation of degenerations from actual inclusions in the E. B. C, such as nuclei or malarial parasites, a. Changing the focus upon a degenera- tion results in a variation in the ap- parent size of the degeneration greater than that of the containing E. B. C. 182 b. Changing the focus upon a nucleus or malarial parasite results in no such proportional change in size, the nucleus or parasite merely becomes more or less distinct as the focus is changed. (2) W. B. C. Colorless round bodies, varying from 8 to 17 milvra in diameter. The shape is irregular, and amoeboid motion may occasionally be seen. In the polymor-. phonuclear cell the granules may be easily distinguished, and eosinophiles and basophiles can be differentiated from the neutrophiles by the larger size of the granules of the former. The nuclei appear as hyaline areas ^dthin the cells. The mononuclear cells are recognized with more difiiculty, inas- much as there are no granules. The nucleus appears as a round hyaline area. The protoplasm is clear. Unless the light is well cut down, such cells are easily overlooked. (3) Platelets. They appear as small granular masses lying singly or in groups. Because of their sticky nature, they are never seen floating, but adhere to the glass or to corpuscles. (4) Blood dust. Dancing particles which give the im- pression of cocci. They probably rep- resent extruded granules from poly- morj)honuclear cells. 183 JJ. (^)UTLINE OF TECHNIQUE OF QUANTITA- TIVE BLOOD EXAMINATIONS. I. Blood counting. a. Apparatus and use of solutions. (For de- scription see Emerson, Ed. IV, page 459.) (1) Counting chamber. (a) Never wash with alcohol or ether as the cement is in this manner dis- solved. Clean with a soft cloth and cold or lukewarm water (never hot). (N. B. — The glass platforms on the counting chamber are mounted in balsam as a cement, hence the need of avoiding alcohol, ether, xylol, and heat in any form in clean- ing.) (b) Before using have the counting chamber and cover slip so clean that Newton's rings appear when the cover slip is placed on the slide. Any grease, dust or lint mil pre- ventthe rings from appearing. (2) Blood pipettes. (a) Care of the pipettes. 1. Cleaning, a. Fill the bulb three times with distilled water, shaking each time, emptying and refilling. b. Repeat the above procedure by filling with 95% alcohol three times. c. After using alcohol, fill the bulb three times with ether. In this Avay the pipette will be thor- oughly dried and cleaned. If the .above x-)rocedure has been prop- 184 erlv carried out, the glass bead will not adhere to the sidei of the bulb and there will be no dis- coloration am^Avhere in the j)ip- ette. d. Never let saliva be drawn into the pipettes. Prevent this by either using a force pump for cleaning pipettes or at times re- moving the rubber tubing and blowing out the accumulated saliva. 2. Take all precautions against break- ing the point of the pipette. The slightest nick which enters the bore renders the pipette useless because of the resulting inaccurac}^ in dilu- tions. (b) Use of the pipettes. 1. K. B. C. a. Blood is drawn to point 0.5 with great accuracy. Hayem's Solution (for diluting r. b. c.) Mercuric chloride 0.500 gm. Sodium chloride 1.00 gm. Sodium sulphate 5.00 gm. Distilled water 200.00 cc. b. Diluting fluid (Hay em's solu- tion) is draAvn to point 101 with equal accuracy (resulting dilu- tion in the bulb of the pipette is 1 :200). c. With a finger placed over each end of the pipette, shake the pi- pette for 5 min. with a trans- verse rather than a longitudinal 185 motion. (Shake the bulb to and I'ro across the bnlb — a longitudi- nal motion results in an uneven dilution by slmking cells into ca])illarY tube of pipette.) d. After sufficient shaking expel 2 or o drops from the pipette ( rep- resenting the fluid which re- mained in the bore and was not mixed in the bulb ) and use the third or fourth drop to place in the counting chamber. W. B. C. a. Blood is drawn to point 0.5 wilh Turk's Solution (for diluting w. b. c.) 1% Aqueous gentian violet 1 cc. Glacial acetic acid 1 cc. Distilled water 100 cc. b. Diluting fluid (Turk's solution) is drawn to point 11 with equal accuracy (resulting dilution in the bulb of the pipette is 1 : 20 ) . c. Shake and place drop in count- ing chamber as described above. 3, If a second preparation is to be made from a i^ipette and during the interval the pipette has been laid aside, it is necessary to repeat the 5 min. of shaking, inasmuch as the cells will have settled and no accu- rate count could be made without another thorough shaking, b. Technique of placing the drop in the