Calcif. Tissue Int.
31, 45-47 (1980)
Use of Dermestid Beetles for Cleaning Bones
E. H e f t i , U . T r e c h s e l , H. R f i f e n a c h t , a n d H . F l e i s c h
Department of Pathophysiology. University of Berne. Murtenstrasse 35, 3010 Berne. Switzerland
Summary. Various parts of the skeleton of normal
and osteoporotic rats were compared with respect
to t h e i r d r y w e i g h t , a s h w e i g h t , a n d c a l c i u m c o n t e n t
w h e n t h e b o n e s w e r e c l e a n e d b y Dermestes mac,-
latus b e e t l e s o r m a n u a l l y . B o t h t e c h n i q u e s g a v e
similar results. This was also true when whole body
calcium measured by neutron activation and total
skeletal calcium from bones cleaned by the beetles
were compared. Thus dermestid beetles are useful
as a t e c h n i q u e to c l e a n b o n e s , e s p e c i a l l y f o r t h e
p a r t s o f t h e s k e l e t o n w h i c h a r e difficult to d i s s e c t b y
hand.
Key words: Dermestid beetles -- Cleaning bones --
Rats.
Introduction
T h e p r e p a r a t i o n o f s m a l l a n i m a l b o n e s is a t e d i o u s
and time-consuming procedure. Some decades ago,
H a l l a n d R u s s e l l [1] s h o w e d t h a t d e r m e s t i d b e e t l e s
and their larvae clean bones of small mammals
within a reasonable time. Various authors con-
f i r m e d t h e e f f i c i e n c y o f this m e t h o d [2-4]. It w a s n o t
s h o w n , h o w e v e r , if t h e b o n e s c l e a n e d b y t h e b e e t l e s
w e r e s u i t a b l e f o r f u r t h e r q u a n t i t a t i v e a n a l y s i s o f pa-
r a m e t e r s s u c h as d r y w e i g h t , a s h w e i g h t , o r m i n e r a l
content.
I n this p a p e r w e i n v e s t i g a t e d w h e t h e r this t e c h -
nique could be used for this purpose by comparing
the results obtained on various bones with those ob-
t a i n e d a f t e r h a n d - c l e a n i n g . F u r t h e r m o r e , t o t a l skel-
etal calcium measured after cleaning by beetles was
c o m p a r e d to w h o l e b o d y c a l c i u m d e t e r m i n e d b y
neutron activation.
Send offprint requests to E. Hefti at the above address.
Calcified Tissue
International
,~ 1980 by Springer-Verlag
Material and Methods
Dermestid Beetles
The Dermestes maculatus beetles were a gift from Ciba-Geigy
research laboratories and bred in our institute. They were kept at
a temperature between 22~ and 28~ since lower temperatures
decrease their activity [3]. No attempt was made to regulate the
humidity. The animals were kept in steel containers with a size of
30 • 50 • 30 cm. Macrolon plastic cages are not suitable because
the larvae are able to burn holes through the walls of the boxes.
A lid of fine plastic mesh. which permits free circulation of air,
was used to cover the boxes [1]. Each box contained a few thou-
sand beetles since the population should be sufficiently large to
ensure rapid cleaning [3]. The danger of infestation and spread of
the beetles to other rooms is small [2], as these insects feed only
on dead animal parts (cadavers, fur, feathers, leather) and as
room temperature reduces their activity. The beetles were fed
on greaves (remainders of industrial fat production) when no ca-
davers had to be cleaned.
Cleaning of Bones by the Dermestid Beetles
The rats were prepared by removing manually the skin and the
intestine. The animals were then put on a grid in the cages with-
out any additional preparations. It was found that after 24-48 h
the skeleton was free from soft tissues with the ligaments still
holding most bones in position. It is necessary to remove the
skeleton at this moment, since longer exposure results in partial
destruction of parts of the skeleton, the beetles attacking bone
when they are deprived of other food.
Design for Assessing the Accuracy of the Method
In order to establish whether the dry weight, ash weight, or cal-
cium content of the bones processed by the beetles was different
from that of bones processed by hand, we used a group of 22
normal Wistar rats (150-450 g). Furthermore, a group of rats in
which osteoporosis was induced with low Ca diet. pregnancy.
and lactation [5, 6] was used in order to determine if the results
also apply to bones with this abnormality. One leg of the cadav-
ers was removed and cleaned by hand. The remaining portions of
the cadavers were presented to the beetles. Hand-cleaned and
0171-967X/80/0031-0045 $01.00
46
beetle-cleaned tibiae and femora were dried at 85~ for 3 days
and weighed. Afterwards the bones were ashed at a temperature
of 700~ for another 3 days and reweighed. Finally, for calcium
measurements the ash was dissolved in l N HCI.
To establish whether the technique was also applicable lbr the
spine, sections of two or three sacral vertebrae were measured
with in vitro neutron activation [7] before and after cleaning by
the beetles.
Finally, the total body calcium of 5 normal and 5 osteoporotic
rats measured with neutron activation analysis was compared to
the whole skeletal calcium cleaned by derrnestid beetles and
measured after ashing. This is possible since total body calcium
is a good estimate for the' skeletal calcium as 99% of the total
body calcium is contained in the skeleton.
Calcium Determination
Chemical calcium determination was done with a Corning Cal-
cium Analyzer (model 940). Whole body calcium determinations
were performed with neutron activation, the rats being irradiated
with thermal neutron produced by a nuclear reactor, and the cal-
cium y-rays measured with a NaI crystal scintillation counter.
The technique is described in detail elsewhere [8].
Results
F i g u r e 1 s h o w s the c l e a n e d s k e l e t o n a f t e r d r y i n g ,
l i g a m e n t s still h o l d i n g m o s t b o n e s in p o s i t i o n .
F i g u r e 2A s h o w s the d r y weight o f tibiae a n d fe-
m o r a c l e a n e d b y b e e t l e s a n d b y h a n d . T h e s l o p e is
s l i g h t l y a b o v e 1, s h o w i n g that the d r y weight o f the
long b o n e s has a t e n d e n c y to be h i g h e r if c l e a n e d b y
hand. T h i s is e x p l a i n e d b y the fact that a l t h o u g h the
soft t i s s u e s w e r e r e m o v e d a l m o s t c o m p l e t e l y b y the
d e r m e s t i d b e e t l e s , the b o n e s c l e a n e d b y h a n d al-
w a y s s h o w e d r e s i d u a l p a t c h e s o f c o n n e c t i v e tissue
in fine c a v i t i e s , for e x a m p l e , n e x t to t h e t r o c h a n t e r
E. Hefti et al.: Dermestid Beetles for Cleaning Bones
Fig. 1. Final dried skeleton
cleaned by dermestid beetles
m a j o r o f the f e m u r a n d the s u l c u s b e t w e e n the c o n -
d y l e s . F i g u r e s 2B a n d 2C s h o w the a s h w e i g h t a n d
c a l c i u m c o n t e n t o f the s a m e b o n e s . B o t h are a l m o s t
identical if c l e a n e d b y h a n d o r b y d e r m e s t i d b e e t l e s ,
the s l o p e o f the line b e i n g a b o u t 1. This is also true
for o s t e o p o r o t i c r a t s , the ratio o f ash w e i g h t o f nor-
mal v e r s u s b e e t l e - c l e a n e d t i b i a e being 0.999 -+ 0.016
(+_ SD) in t h e s e a n i m a l s .
To i n v e s t i g a t e w h e t h e r p r o l o n g e d e x p o s u r e o f the
b o n e s to the d e r m e s t i d b e e t l e s l e a d s to the d e s t r u c -
tion a n d loss o f b o n e , we c o m p a r e d d r y a n d ash
w e i g h t s o f v a r i o u s b o n e s , c l e a n e d for 1 d a y with
c o r r e s p o n d i n g b o n e s e x p o s e d to the b e e t l e s for up
to 5 d a y s . In long b o n e s the r a t i o b e t w e e n b o n e s
c l e a n e d for 1 d a y a n d t h o s e c l e a n e d for 5 d a y s w a s
1.001 --+ 0.028 for d r y weight a n d 1.013 -+ 0.038 for
a s h weight ( + S D , N = 20). H o w e v e r , this is not the
c a s e f o r m o r e v u l n e r a b l e p a r t s o f the s k e l e t o n , s u c h
as the iliac c r e s t a n d v e r t e b r a e , w h e r e m a c r o s c o p i c
lesions w e r e o b s e r v e d a f t e r l e a v i n g s k e l e t o n s w i t h
the b e e t l e s for s e v e r a l d a y s . T h u s o f s e v e n v e r t e -
b r a e a n a l y z e d b e f o r e a n d a f t e r e x p o s u r e , two w e r e
macroscopically damaged. The two damaged verte-
brae s h o w e d a c a l c i u m ratio o f 1.20 for c a l c i u m be-
f o r e / a f t e r c l e a n i n g . T h e o t h e r five b o n e s w e r e ap-
parently intact. The calcium analysis of these bones
gave a ratio o f 1.004 -+ 0.032 ( m e a n + SD) for cal-
c i u m b e f o r e / c a l c i u m a f t e r c l e a n i n g . It has to be
m e n t i o n e d , h o w e v e r , that s u c h a d e s t r u c t i o n o f
b o n e o c c u r r e d o n l y if the c a r c a s s was e x p o s e d
w h e n the a n i m a l s w e r e d e p r i v e d o f o t h e r f o o d . It
was n e v e r s e e n if the b o n e s w e r e w i t h d r a w n w h e n
m o s t o f the l i g a m e n t s w e r e still p r e s e n t .
F i n a l l y , the t e c h n i q u e is a l s o valid for total s k e l e -
tal c a l c i u m s i n c e the ratio o f t h e t o t a l b o d y c a l c i u m
a n d c a l c i u m o f the s k e l e t o n p r o c e s s e d b y the
b e e t l e s was 1.024 + 0.055 in n o r m a l rats a n d 0.984
E. Hefti et al.: Dermestid Beetles for Cleaning Bones 47

C A L C I UM
600
~ 1000-

O y9 0000 oo9000
C

T
r
C
0
.0

U
"D
C
r
500"

0 J
/
//
j
,Br.

5OO
" 9 Femur
9 Tibia

1;00
0
0
/
/
~

260
,Femur
.Tibia

460 600
50-

0
o
////
//
mur
5'0 16o 1~o 260
ia

0
A Beetle cleaned bones(rag) B B e e t l e cleaned bones (mg) C Beetle cleaned b o n e s (rag)

Fig. 2. Comparison of rat femur and tibia cleaned manually and cleaned by beetles. A Dry weight. 3' = 13.02 + 1.040 x, Y-' = 0.982. N =
43. B Ash weight. 3' = 1.50 + 1.006 x, r2 = 0.992, N = 42. C Calcium content, y = - 1.45 + 1.028 x, r 2 = 0.998, N = 32

_+ 0.034 (mean -+ SD) in osteoporotic rats. The ab-
solute amount of total body calcium was 3725 -+ 209
mg in the normal and 2684 -+ 301 mg (mean -+ SD) in
the osteoporotic rats.
sistance with the illustrations and Mrs. B. Gyger for preparing
the manuscript. This work was supported by the Swiss National
Science Foundation (3.725.76) and by the Ausbildungs- und
F6rderungsfonds der Arbeitsgemeinschaft ftir Osteosynthese
(AO), Switzerland.

Discussion References

1. Hall, E.R., Russell, W.C.: Dermestid beetles as an aid in

Thus dermestid beetles are able to clean bones effi-
ciently within a short time, and the bones obtained
are suitable for further analysis. This is especially
useful for parts of the skeleton that can be dissected
by hand only with great difficulty, such as spine or
skull. So far this method has been used for cleaning
the skeletons of about 200 rats in our laboratory.
No bone substance is destroyed if the skeletons
are carefully o b s e r v e d during the cleaning process
and the bones are r e m o v e d while the ligaments are
still partially intact. Since the speed of cleaning is a
function of population composition, population
size, amount of offered food, temperature, and
light, exposure of the bones to the beetles for a
fixed, predetermined time is not possible. Even if
the bones are attacked by the animals, it is easily
seen by eye, since the process is such that pieces of
conspicuous size b e c o m e detached.
Acknowledgments. We wish to thank Dr. P. Schubiger for per-
forming the neutron activation analyses, Mrs. C. Stieger for as-
cleaning bones, J. Mammal. 14:58-59, 1933
2. Hooper, E.T.: Use of dermestid beetles instead of cooking
pots, J. Mammal. 31:100-102, 1950
3. Russell, W.C.: Biology of the dermestid beetle with refer-
ences to skull cleaning, J. Mammal. 28:284-287, 1947
4. de la Torre, L.: A method for cleaning skulls of specimens
preserved in alcohol, J. Mammal. 32:231-232, 1951
5. Rasmussen, P.: Calcium deficiency, pregnancy and lactation
in rats. Some effects on blood chemistry and the skeleton,
Calcif. Tissue Res. 23:87-94, 1977
6. Rasmussen, P.: Calcium deficiency, pregnancy and lactation
in rats. Microscopic and microradiographic observations on
bones, Calcif. Tissue Res. 23:95-102. 1977
7. Guinn, V.P., Hoste, J.: Elemental analysis of biological mate-
rials by neutron activation analysis (IAEA-SM-227/102). Pro-
ceedings of an International Symposium on Nuclear Activa-
tion Techniques in Life Sciences, International Atomic En-
ergy Agency, Vienna. Austria, 1978
8. Schubiger, P.A., Jakob, K., Ferreira-Marques, F.M., Hefti,
E., Trechsel, U., Miihlbauer, R., Fleisch, H.: Determination
of whole body calcium in vivo in rats by neutron activation
analysis, Int. J. Nucl. Med. Biol. in press.
Received September 25. 1979 / Accepted October 26, 1979