Table 1 Typical applications of non-resinous embedding media used for light microscopical histology.

EMBEDDING MEDIUM	TYPICAL APPLICATION
AQUEOUS MEDIA
Agar	embedding small specimens and tissue fragments for orientation and double embedding
Gelatine:sodium carboxy-methyl cellulose	whole organ sections; whole animal sections for autoradiography
Polyvinyl alcohol	water soluble medium suitable for lipid and enzyme studies
WATER MISCIBLE MEDIA
Polyethylene glycols	water soluble wax used in lipid and enzyme studies
Polyethylene glycol monostearate	water soluble wax used for histochemical and botanical investigations
WATER TOLERANT MEDIA
Diethylene glycol	0.5-1 µm thin wax sections for high distearate resolution light microscopy
Ester wax	supporting difficult materials such as dense tissues and chitinised specimens; agar-double embedding of arthropods; also formulated for use in tropics
Polyester wax	supporting heat labile tissues; used for combined light and scanning electron microscopy studies; immunohistochemistry
HYDROPHOBIC MEDIA
Nitrocellulose (celloidin;low viscosity nitrocelulose)	supporting hard, dense specimens such as bone; sectioning heat-labile tissues; neuroanatomical studies; paraffin wax-double embedding of hard, dense tissues; chitinised specimens
Paraffin wax	routine sections, 2-50 µm, of a wide variety of tissues; serial sections

Table 2 Viscosities of dehydrants, transition solvents, and embedding waxes. a - estimated; b - values from Gordon.¹⁴⁷ Other values from various sources.^59,148,149

SOLVENT	TEMPERATURE °C	VISCOSITY CENTIPOISE
acetone	20	0.3
tetrahydrofuran	20	0.5
n-butyl acetate	20	0.5
methanol	20	0.6
chloroform	20	0.6
toluene	20	0.7
xylene	20	0.7
1,1,1-trichloroethane	20	0.8
perchloroethylene	20	0.9
amyl acetate	20	0.9
ethanol	20	1.2
dioxane	20	1.2
kerosene (paraffin)	20	1.2
2-ethoxyethanol	20	1.7
trichloroethylene	20	2.1
methyl benzoate	20	2.1
isopropanol	20	2.2
methyl salicylate	20	2.7a
normal butanol	20	2.9
tertiary butanol	30	3.5
d+limonene	20	20.0a
terpineol	20	24.0a
cedarwood oil	variable	variable
paraffin wax mp 52°C	55	9b
Ester wax 1960	50	18b
Polyester wax 70/30	40	33b
Ester wax 1947	50	50b
Polyethylene glycol 1500	50	100b

Table 3 Evaporation rate, exposure standards, relative ventilation requirements and fire hazards of processing solvents.
a - at 22°C from Reed & Scala⁸³
b - estimated
c - recalculated from Flick¹⁴⁸
d - ppm, from Worksafe Australia Standards¹⁵⁰
e - gram/minute/cm2x100, recalculated from Reed & Scala⁸³ as (evaporation rate x 24)/(molecular weight x TWA).

SOLVENT	EVAPORATION RATEa	EXPOSURE STANDARD (TWA)d	RELATIVE VENTILATION REQUIREMENTe	FIRE HAZARD
1,1,1-trichloroethane	25.8	125	37.3	D
2-ethoxyethanol	0.4	5	21.3	F
acetone	12.2	500	10.1	F
amyl acetate	1	100	1.9	F
chloroform	6.7b	10	135	D
d+limonene	<1		PO
dioxane	0.9	25	9.3	F
ethanol	2.9	1000	1.5	F
isopropanol	2.6	400	2.6	F
methanol	4.9	200	18.4	F
methyl benzoate	<1		PO
methyl salicylate	<1		PO
n-butanol	0.6	50	3.5	F
n-butyl acetate	1.2	150	1.2	F
perchloroethylene	4	50	11.6	D
t-butanol		100		F
tetrahydrofuran	9.6c	200	16	F
toluene	2.8	100	7.3	F
trichloroethylene	5.9	50	21.6	D
xylene	0.9c	80	2.6	F

When substituting one solvent for another, solvent properties, laboratory ventilation, working practices and conditions must all be considered and evaluated. For example, from the relative ventilation values provided here it can be seen that under identical conditions, 1,1,1 trichloroethane (1,1,1 TCE) will require about 14 times more ventilation than is required for xylene in order to remain below the permissible atmospheric level. Thus substitution of 1,1,1 TCE for xylene in a poorly ventilated laboratory might seriously exacerbate the problem of solvent vapour levels in the work place.

Table 4 Processing schedules for a tissue-transfer processor. Day schedule for urgent specimens, tissues 2 mm, fixed in Carnoy's fluid. Overnight schedule for routine processing. Tissue blocks 2-3 mm, single load. For a double load, immersion times should be equal. Weekend processing: tissues are held in fixative, or preferably 70% ethanol until Sunday.

STEP	DURATION
	DAY	OVERNIGHT
Fixative		120 minutes
Fixative		120 minutes
70% ethanol		60 minutes
90% ethanol		60 minutes
absolute ethanol	30 minutes	60 minutes
absolute ethanol	30 minutes	60 minutes
absolute ethanol	30 minutes	60 minutes
toluene or substitute	30 minutes	60 minutes
toluene or substitute	30 minutes	60 minutes
paraffin wax	30 minutes	90 minutes
paraffin wax	30 minutes	90 minutes
paraffin wax	30 minutes	90 minutes
paraffin wax (under vacuum)	30 minutes	30 minutes
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TOTAL TIME	4.5 hours	16 hours

Table 5 Processing schedules for a fluid-transfer processor.
Rapid (30 minutes) day schedule for endoscopic or needle biopsy. Overnight schedule for routine lightly fixed specimens. P/V pressure vacuum option.

STEP	DURATION
	DAY					OVERNIGHT
	Time	Temp (°C)	P/V	Time	Temp (°C)	P/V
Fixative				3.0 hours	35
Fixative				1.5 hours	35
70% ethanol	15 minutes		on	1.0 hours	40
90% ethanol	15 minutes	40	on	1.0 hours	40
absolute ethanol	15 minutes	40	on	0.5 hours	45	on
absolute ethanol	15 minutes	40	on	0.5 hours	45
absolute ethanol	15 minutes	40	on	0.5 hours	45
absolute ethanol	15 minutes	40	on	1.5 hours	45	on
toluene or subst	15 minutes	40	on	0.5 hours	50
toluene or subst	15 minutes	40	on	1.5 hours	50	on
paraffin wax				0.5 hours	60	on
paraffin wax	15 minutes	60	on	0.5 hours	60	on
paraffin wax	15 minutes	60	on	1.5 hours	60	on
paraffin wax	15 minutes	60	on	1.5 hours	60	on
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TOTAL TIME (exclusive of fluid transfer time)	2.75 hours			15.5 hours

Table 6 Recovery of tissues accidentally returned to fixative or dehydrant following wax infiltration. Discard all contaminated reagents.

STEP	DURATION
	Specimen Returned To
	Fixative	Dehydrant
70% ethanol	rinse
95% ethanol	rinse	rinse
absolute ethanol, 2 changes	rinse	rinse
toluene or substitute	rinse	rinse
paraffin wax, 3 changes, each under vacuum if possible	30-60 minutes	30-60 minutes
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Table 7 Chemical dehydration method for manual or machine processing of fixed tissues.
To prepare the reagent, mix 100 ml of dimethoxy propane (DMP) or diethoxy propane (DEP) with 50 µl (approximately 1 drop) of concentrated hydrochloric acid.
The reagent is stored at 4°C in a spark-proofed refrigerator.
Fixed tissues are rinsed in distilled water to avoid precipitation by the reagent.

STEP	MANUAL	MACHINE50
acidified DMP, 2 changes, each	5-15 minutes	5-15 minutes
toluene at 40-50°C	10 minutes
methyl salicylate		15 minutes
paraffin wax, 3 changes, each	20 minutes	45 minutes
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TOTAL TIME	80-100 minutes	3 hours

Note:
(a) Tissues may be transferred directly to paraffin wax and infiltrated under vacuum after the second change of DMP. The acetone is boiled off¹⁰.
(b) For fatty tissues, double the dehydration times.

Table 8 Manual processing: one and two day schedules for well fixed tissues processed using a magnetic stirrer.

STEP	DURATION
	Tissue Thickness
	1-2 mm	3-4 mm
70% ethanol	20 minutes	1.5 hours
90% ethanol	20 minutes	1.5 hours
absolute ethanol	20 minutes	1.5 hours
absolute ethanol	20 minutes	1.5 hours
absolute ethanol	20 minutes	1.5 hours
chloroform or substitute	20 minutes
chloroform or substitute	20 minutes
methyl salicylate		overnight
paraffin wax	20 minutes	1.0 hours
paraffin wax	20 minutes	2.0 hours
paraffin wax		1.0 hours
paraffin wax under vacuum	20 minutes	0.5 hours
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TOTAL TIME	2 hours	1.5 days

Table 9 Manual processing of large, thick, hard, well fixed tissue blocks using terpene transition solvents. Other terpenes such as terpineol or limonene-based solvents can be substituted for the cedarwood oil.

STAGE	DURATION
70% ethanol	3 hours
90% ethanol	6-24 hours
95% ethanol, 2 changes, over	6-24 hours
absolute ethanol-cedarwood oil 1:1	3- 6 hours
cedarwood oil, 2 changes over	6-24 hours
toluene or chloroform or substitute, 2 changes, each	0.5-1 hours
paraffin wax, 4 changes, each	1-1.5 hours
TOTAL TIME	29 hours-5 days

Table 10 Manual processing schedule using n-butanol (after Stiles³⁸). Absolute alcohols are used to prepare the solutions. Tissues can move directly from step 6 to step 8; better results are obtained if butanol is allowed to evaporate in step 7. If t-butanol is used, 100% solutions must be kept above the freezing point of 26°C. Total time: 6.5 - 7.25 days

STEP	SOLUTION	VOLUME in ml	DURATION
1	n-butanol	10
	ethanol	40	2 hours
	water	50
2	n-butanol	20
	ethanol	50	2 hours
	water	30
3	n-butanol	40
	ethanol	50	4 hours
	water	10
4	n-butanol	55
	ethanol	40	6 hours
	water	45
5	n-butanol	75	2 changes over 6-12 hours
	ethanol	25
6	n-butanol		2 changes, each 3-6 hours
7	butanol-paraffin wax 1:2		12-24 hours
8	paraffin wax		2-3 changes over 4-5 days
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Table 11 Manual processing using dioxane as the dehydrant. Tissues must be regularly agitated during the infiltration in all baths incorporating paraffin wax.

STEP	DURATION
30% dioxane, aqueous	2 hours
60% dioxane, aqueous	2 hours
100% dioxane	4 hours
60% dioxane in paraffin wax	1 hour - overnight
30% dioxane in paraffin wax	1 hour
100% paraffin wax	2 hours
100% paraffin wax	1 hour
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TOTAL TIME	13-22 hours

Table 12(1) Schedules for microwave-stimulated processing. Method of Kok, Visser & Boon¹⁰⁰

STEP	Temp (°C)	TISSUE BLOCK THICKNESS
		<1 mm	1-2 mm	2-5 mm
100% ethanol	67	5 minutes	15 minutes	60 minutes*
100% isopropanol	74	3 minutes	15 minutes	45 minutes
PARAMAT wax	67	2 minutes	15 minutes	30 minutes
	82	5 minutes	20 minutes	60 minutes
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Total time		15 minutes	60 minutes	195 minutes

* Reduce to 10-15 minutes if tissues do not contain fat. Bath volumes 200 ml, power level 450 watt.

Table 12(2) Method of Wong.¹⁰¹ Tissue blocks 13 mm thick.

STEP	BATH	VOLUME	DURATION
100% ethanol	one bath	240 ml	5 minutes
xylene	one bath	240 ml	4 minutes
PARAPLAST wax	one bath	400 ml	5 minutes
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Tissues fixed in 10% neutral buffered formalin, stored in 70% ethanol. Total processing time 14 minutes at 125 watt (500 watt oven, 25% power), temperature 68°C.

Table 13 Schedule for ultrasonic-stimulated tissue processing. Method of Gagnon & Katyk⁴⁰

STEP	RAPID METHOD	ROUTINE METHOD
	Tissues 1-2 mm	Tissues 5 mm
Fixation	20 minutes	60 minutes
100% ethanol	1. 5 minutes	1. 15 minutes
	2. 10 minutes	2. 30 minutes
	3. 25 minutes	3. 90 minutes
methyl benzoate*	5 minutes	30 minutes
toluene or substitute	1. 5 minutes	1. 15 minutes
	2. 5 minutes	2. 15 minutes
paraffin wax	1. 10 minutes	1. 30 minutes
	2. 10 minutes	2. 30 minutes
	3. 10 minutes	3. 30 minutes
TOTAL TIME	1 hour 45 minutes	5 hours 30 minutes

* Or methyl salicylate, clears from 95% ethanol.
A 1:1 toluene-paraffin wax bath can be interposed between the last clearing and first wax steps.

Table 14 Polyethylene glycol-LVN method⁶⁷ suitable for manual processing, or an automatic processor with agitation slowed to half speed.

STEP	DURATION
PEG 200, 2 changes, each	4 hours
PEG 200 plus 5% LVN	2 hours
PEG 600 plus 5% LVN	2 hours
PEG 1000 plus 5% LVN	2 hours
PEG 1500 plus 5% LVN, 2 changes, each	5 hours
Embed in PEG 1500 plus 5% LVN
Blocks are stored in heat-sealed polythene bags.

Table 15 Polyester wax method.¹²²

STEP	DURATION
30%, 50%, 70%, 85% ethanol, one change, each	30-60 minutes
absolute ethanol, 3 changes, each	15 minutes
absolute ethanol: polyester wax 1:1*	30 minutes
polyester wax,* 3 changes, each	30-60 minutes
polyester wax*	1-12 hours
Embed in pure wax, freshly melted and filtered, using paper boat or Peel-a-way plastic moulds. Allow blocks to cool at room temperature for 24 hours.

*agitate periodically

Table 16 Celloidin-alcohol-diethyl ether: paraffin wax double infiltration method. Blocks 3-4 mm thick. Processing must be undertaken within a fume cupboard because of the extreme flammability of diethyl ether.

STEP	DURATION
70%, 90% and 95% ethanol, one change, each	1 hour
absolute ethanol, 3 changes, each	1 hour
absolute ethanol: diethyl ether 1:1	4 hours
1%-2% celloidin in ethanol:ether	4-24 hours
chloroform, or subst. 2 changes, each	2 hours
paraffin wax, 2-3 changes, each	1-2 hours
paraffin wax under vacuum	0.5 hours
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TOTAL TIME	11.5-37.5 hours

Table 17 LVN-methyl benzoate:paraffin wax double infiltration method.¹⁴³

STEP	DURATION
70%,90%, and 95% ethanol, each	1-2 hours
0.25% LVN in methyl benzoate	2-6 hours
0.5% LVN in methyl benzoate	2-6 hours
1.0% LVN in methyl benzoate	2-6 hours
1.5% LVN in methyl benzoate	2-6 hours
toluene or substitute	1 hour
paraffin wax, 2 changes, each	1-3 hours
paraffin wax under vacuum	1-3 hours
TOTAL TIME	10-37 hours

Table 18 Method for hard tissues³² containing compact smooth muscle or collagen.

STEP	DURATION
neutral buffered formalin	2 hours
60% ethanol	1 hour
80% ethanol	1 hour
95% ethanol	1 hour
5% phenol in absolute ethanol	1 hour
5% phenol in absolute ethanol	1 hour
5% phenol in absolute ethanol	1 hour
5% phenol in absolute ethanol	1 hour
chloroform or substitute	1.5 hours
chloroform or substitute	1 hour
chloroform or substitute	1.25 hours
Tissue Prep.610	1.75 hours vacuum
Tissue Prep.610	2.5 hours vacuum
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TOTAL TIME	16 hours

Table 19 Methods for yolk-laden gonads,¹⁴⁶ smooth and skeletal muscle and whole specimens of large shellfish and lower vertebrates.
A - gonads, 3-4 mm thick.
B - body muscle and ripe gonads, 3-4 mm thick.
C - muscle, cartilage and decalcified bone, 3-4 mm thick.
D - whole animals, 5-8 mm thick.
Isopropanol, phenol and methyl salicylate do not exacerbate tissue hardening.

STEP	DURATION (hours)
	Tissues
	A	B	C	D
70% ethanol	2	2	2	2
90% ethanol	1	1	1	1
absolute ethanol, 3 changes,each	1
5% phenol in isopropanol		1	1	1.5
5% phenol in isopropanol		1	1	1.5
0.5% LVN in methyl salicylate			3	6-12
1% LVN in methyl salicylate				6-12
toluene, chloroform or substitute	1	1	0.5	2
toluene, chloroform or substitute	1	1	0.5	2
paraffin wax	1.5	1.5	1.5	2-4
paraffin wax	1.5	1.5	1.5	2-4
paraffin wax	1.5	1.5	1.5	2-4
paraffin wax	0.5	0.5	0.5	1
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TOTAL TIMES (hours)	11	12	14	27-47