Light staining microscope: Clinical experience in a Sexual Assault Nurse Examiner (SANE) program Author: Colleen O'Brien, RN, MS, SANE, Madison, Wisconsin

I n December 1996, the YWCA and sexual assault n u r s e examiner (SANE) program of Madison,

Wisconsin, purchased a monocular light s taining mic roscope (Model OS06; Opt ical Services Co., Poway, Calif.). The microscope is used for identi- fication of motile and nonmotile sperm in med ica l - forensic evaluations. The YWCA/SANE Program sees approximately 200 clients per year, both children and adults. As a consequence, we purchased a Model OS06 to give our SANEs a bet ter chance of de tec t ing sperm during evaluations.

The cost of the monocular light s ta ining micro- scope was $1285. Another light s ta ining microscope, the Trinocular (Model OS04), was available at a cost of $3454. The Trinocular has the same objective lens (25X) but the tube length is longer, and as a result it has 33 power (33X). The total sys tem magnifications are 250X and 330X, respectively. We chose the monocular model for financial reasons.

The Optical Services Company also has other equipment available for use with the microscope; 35- mm slides were taken with a 35-mm camera adaptor, which can be used on both the Model OS04 and the OS06. A video sys tem is available for the trinocular model.

The light s ta in ing microscope enhances the image of the sperm by coloring the illuminator light of the microscope with filters. The dark field is illumi- na ted by a yellow filter, and the bright field by a blue filter. The results are yellow-colored sperm agains t a blue background, which makes for much easier iden- tification and el iminates the need for chemical stain- ing of the sperm.

Colleen O'Brien, RN, MS, is Director, YWCA/SANE Program, Meriter Hospital, Madison, Wisconsin. J Emerg Nurs 1998;24:95-7. Copyright © 1998 by the Emergency Nurses Association. 0099-1767/98 $5.00 + 0 18/1186433

Advantages for the SANE We found the light s taining microscope easy to

use because there is only one objective lens. This pro- vides a wider field of vision, which makes it bet ter sui ted to scanning larger fields. We also found that using a z-track or crop dust ing pat tern was most effective in scanning the specimen.

9etc~n9 ~!.p a:,:~d view~;,ing ~the ~pec ime~ is eas i e r a n d i!ess ,:irne c o n s u m i n g t h a n us ing }~o .;finical mic roscope .

Another reason we like the microscope is for the feature the manufacturer calls "constrained focus." For the SANE nurse, this means that a glass slide and coverslip can be set on the s tage of the microscope, and with only minor fine adjustment, the spec imen can be brought into perfect focus. The monoscope does have an inverted-viewing image, which means that when the image moves to the right, the slide is actually moving left. The trinocular scope has an erect image, which means that the spec imen moves in the same direction as the image.

The manufacturer provides a set-up slide with nonmotile sperm, which can be used for set t ing the focal plane. Using a z-track or crop dust ing pattern technique to find the object on the slide, the SANE can then adjust the fine focus for clear visualization. Once a slide has been set up clearly, future slides only need to be set on the s tage and minimal fine-focusing done to obtain a sharp image. We found the set-up slide invaluable for teaching identification of sperm. We recommend that all nurse examiner programs use

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Figure 1 Light staining microscope view of Trichomonas vaginalis.

Figure 2 Yeast cells and budding photographed with 35-ram camera at tached to light staining microscope.

Figure 3 Sperm shown on light staining microscope slide.

a 24 x 60-ram cover g lass to p ro t ec t as m u c h of the s m e a r a rea as poss ib le .

Ano the r a d v a n t a g e to the l ight s t a in ing micro- s cope is t he owner ' s manua l p r o d u c e d by the devel- oper of t he mic roscope , Charles Podvin. It is clear, con- cise, and reasonab ly ea sy to follow. It inc ludes an excel len t p rob lem-so lv ing guide. In contrast , w e have p u r c h a s e d other e q u i p m e n t and found tha t t echn ica l a s s i s t ance , bo th verbal ins t ruc t ions and wr i t t en gu ide -

lines, were imposs ib le to unde r s t and . In one case, w e rewro te the e q u i p m e n t se t -up gu ide l ines to m a k e t h e m clear and u n d e r s t a n d a b l e to the SANE staff. Staff from Opt ica l Serv ices C o m p a n y were knowledgeab l e and readi ly avai lable for p h o n e consul ta t ion.

Overall, in the first 6 months , genera l r eac t ion from the staff to our m i c r o s c o p e has b e e n pos i t ive . They find it m u c h eas ie r to u se t han a cl inical micro- s c o p e w i th mul t ip le objec t ives , aper tu res , and field s tops. Two of the i r r ea sons for f ind ing it eas ie r to use are t he s ingle ob jec t ive lens and the cons t r a ined focus. Se t t i ng up and v i e w i n g the s p e c i m e n is eas ie r a n d less t ime c o n s u m i n g t han u s i n g a cl inical micro- scope . Once the s p e r m or b io logic s p e c i m e n s are visual ized, t hey are eas ie r to ident i fy b e c a u s e of the effect ive color con t r a s t b e t w e e n s p e c i m e n and background .

Thus far, only one d i s a d v a n t a g e has b e e n noted . On severa l occas ions I wou ld have prefer red a h ighe r ob jec t ive lens choice, t ha t is, 40 power. It wou ld not c h a n g e the iden t i f i ca t ion process , b u t i t wou ld have b e e n helpful to magn i fy a par t i cu la r s p e c i m e n , e spe - cially in t e a c h i n g s i tua t ions .

Additional clinical use Al though it w a s our p r imary ob jec t ive to ident i fy

mot i le a n d non-mot i l e s p e r m in t he m e d i c a l - f o r e n s i c e v a l u a t i o n p r o c e s s , w e w e r e a lso i n t e r e s t e d in w h e t h e r w e could s ee o ther b io logic s p e c i m e n s . We a r r a n g e d wi th t he hosp i t a l ' s d e p a r t m e n t of micro- b io logy to have var ious s a m p l e s s en t to t he SANE

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staff. Over t ime, w e v i sua l i zed and p h o t o g r a p h e d Trichomonas vaginalis (Figure 1), y e a s t cells a n d bud -

d ing (Figure 2), and c lue cells. Wi th Kodak 400-ASA K o d a c h r o m e a n d a 35 -mm c a m e r a a d a p t e r on a Canon body, w e were also able to s ecu re s l ides of s p e r m s p e c i m e n s (Figure 3). The Trichomonas spec i - m e n s on the s l ide we re s l ight ly b lur red as a resul t of the con t inuous m o v e m e n t of the flagella. In add i t i on to t h e s e samples , w e were able to v isual ize the p r oce s s of "ferning," an i nd i ca t i on of ovulat ion, from a

cervical m u c u s sample .

O n c e t h e s p e r m or b io log ic s p e c i m e n s are v i s u a l i z e d , t h e y are e a s i e r to identify- b e c a u s e of t h e e f f e c t i v e color c o n t r a s t b e t w e e n s p e c i m e n a n d b a c k g r o u n d .

Visual iz ing t h e s e b io logic s p e c i m e n s unde r the l ight s t a i n i n g m i c r o s c o p e b r o a d e n s t he u s e and i n c r e a s e s the va lue of t he m i c r o s c o p e as a c l inical tool for SANE.

At th is t ime, w e have no t o b s e r v e d an i n c r e a s e in t he f r equency of s p e r m seen . This m a y be due, in

part , to the fact t ha t 62% of our c l ients are children,

and r epor t ing is of ten 72 hours p a s t the t ime of the inc ident . SANE col league, Kathy Peele, RN, C-PNP, in Reno, Nevada , u se s the ]ight s t a in ing m i c r o s c o p e a t t a c h e d to a v ideo camera , v ideo printer, and moni - tor. W h e n s p e r m are iden t i f i ed on the v ideo monitor, the pr in ter has t he capab i l i ty to cap tu re a p ic ture . Law en fo rcemen t a g e n c i e s a p p r e c i a t e th is fea ture b e c a u s e it p rov ides i m m e d i a t e forensic d o c u m e n t a - t ion (personal communica t i on , May 1997).

"in a d d i t i o n to t h e s e s a m p l e s , w e w e r e ab le to v i s u a l i z e t h e p r o c e s s of "ferning," an ind ica t ion of ovu la t ion , f rom a cerv ica l m u c u s s a m p l e .

Summary Our l ight s t a in ing m i c r o s c o p e has d e c r e a s e d the

a m o u n t of s e t -up t ime for staff, has i n c r e a s e d the abil- i ty to see bo th s p e r m and other b io logic s p e c i m e n s , and has b e c o m e an e s sen t i a l tool in bo th our cl inical and forensic prac t ice .

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