Beständigkeit von Färbungen bei Einbettung mit Polyvinyllactopheol

[Alfons Renz]

Hallo, liebe Foristen,

PVLP ist ja ein sehr gutmütiges, wasserverträgliches Einbettungsmittel, speziell auch für niedere Organismen.

Aber wie sieht es mit der Beständigkeit von Färbungen in diesem sauren Medium aus: Hat da Jemand Erfahrung?

Speziell geht es um die Anfärbung von Nematoden-Larven.

Für Hinweise wäre ich sehr dankbar!

Mit besten Grüßen,

Alfons

[B.Neuhaus]

Lieber Alfons,

na ja, als gutmütig würde ich Polyvinyllaktophenol nun wirklich nicht bezeichnen. Folgendes habe ich in der deutsch- und englischsprachigen Literatur dazu gefunden bzw. eigene negative Erfahrungen mit der Selbstzerstörung meiner Präparate von Kinorhynchen gemacht; Kinorhyncha haben ja wie Nematoden eine Kutikula (gut, etwas unterschiedlich aufgebaut), der Vergleich ist aber trotzdem nicht so weit hergeholt, und ich schreibe jetzt von einer Haltbarkeit von ein paar Jahren, nicht von hunderten von Jahren  :

"Polyvinyl lactophenol. Gas bubbles in the medium on the microscope slide may be removed by dropping small droplets of 70% ethanol from a dissecting needle onto the medium; gas bubbles will migrate to the surface of the medium and burst (Koomen & Vaupel Klein 1995, p. 435).
   Polyvinyl lactophenol causes alkaline stains to fade because of the lactic acid in the medium (Salmon 1951a). In addition, granular precipitations occur, but usually slides last longer than four years (Salmon 1951a). Droplet-like structures are supposed to represent a segregation of the chemical components of the medium (Fig. 7F) originating from an unsuitable intermedium during the mounting process (Woelke & Göke 1984, figs 1, 3). These authors also suggest that radially growing crystals or other crystals develop if the medium dries too quickly or at a too high temperature (beyond 40°C: Russel 1961) if the storage temperature of the slides is too low for some time, or if the mounting medium contains chloral hydrate (Woelke & Göke 1984, pp. 212–213, fig. 2). In addition, a coverslip seal is necessary to prevent the evaporation of water and consequently shrinkage of the medium and uptake of humidity from the air; slides may last at least 30 years (Hirschmann & Woelke 1960; Woelke & Göke 1984, p. 211; Hooper 1986b). If the medium sets in an unsealed slide, a specimen may be crushed by the coverslip within two years (Müller 1983), probably because of evaporation of water. Crystals and turbidity are observed in some slides (Bink 1979). The mounting medium turns brown and thickens (polymerization?) after exposure to light for several months (Lipovsky 1953). Huys & Boxshall (1991, p. 451) report too intense maceration of copepods within 10 years, formation of crystals, and drying out. Cuticle and setation of marine mites will be overly macerated within a few years (Bartsch 1988). Hirschmann (1984) stated that slides might last at least 25 years. Phenol in the media seems to have caused shriveling of delicate specimens, so this chemical is omitted in a later formula (comp. Salmon 1951a and 1954). Mounts of Kinorhyncha mounted in polyvinyl lactophenol exhibit numerous cavities and at least four different kinds of crystals, namely irregularly growing crystals, aggregations of spherical crystals, radially growing crystals, and crystals with a rectangular basic structure (Figs 18B–H, 19A–D; Neuhaus 2013, p. 278, fig. 5.5.1.C–D, 5.5.2.A–B). Salmon (1947, 1951a) attributed the development of precipitations in the medium to unbalanced proportions of poly(vinyl alcohol), lactophenol, and water. Lipovsky (1953) stressed that phenol plus lactic acid must not take up more than 50% of the medium by volume to avoid a tacky and soft medium (see also Salmon 1947, 1951a); lack of these components would cause problems with re-mounting a specimen. Danielsson (1985) suggested, on the contrary, that chloral hydrate and phenol were responsible for the loss of volume during drying of the mount and formation of cavities from the periphery of the coverslip and omitted these substances. An incorrect proportion of poly(vinyl alcohol) in the mounting medium is supposed to crack the coverslip over the specimen (Lipovsky 1953). The permeability to oxygen is quite low but increases with the uptake of water (Mills & White 1999, p. 132; Comyn 1986, p. 284). Generally, poly(vinyl alcohols) shrink by about 50% on drying (Heikinheimo 1988), are hygroscopic (especially at relative humidity > 75%) and light stable; they may cross-link over time releasing water but remaining water-soluble (Witte 1976; Mills & White 1999, p. 132; Horie 2011, p. 143). If glycerol, lactic acid, and phenol are included in the medium, the latter seems to become insoluble in water (Brown 1951). However, conflicting evidence is given that re-mounting seems to be possible by soaking for one to several days either in water (Tab. 13: Technique C; Salmon 1954) or in 70% ethanol (Koomen & Vaupel Klein 1995, p. 435) or not possible at all (Ossiannilsson 1958; Singer 1967)."
Quelle: Neuhaus et al. 2017, Seiten 99-101, http://www.mapress.com/j/zt/article/download/zootaxa.4322.1.1/12414

Viele Grüße
Birger

[liftboy]

Hallo erstmal,

meiner einer verwendet Hoyers Gemisch, also Chloralhydrat in Gummi-arabicum, auch nicht gerade harmlos. Da hält sich auch nicht jeder Farbstoff! Außerdem neigt das Chloralhydrat nach längerer Zeit zur Kristallbildung (koimischerweise nicht bei allen Präparaten). Zur Zeit nehm ich Hydromatrix; recht gut aber sauteuer. In meinen Anfagszeiten hab ich auch mit Polyvinyllactophenol experimentiert, bin aber wieder davon abgekommen (steht immer noch hier rum :-} ).

Grüße
Wolfgang