C h e m o s p h e r e , Vol.17, No.

3,
P r i n t e d in G r e a t B r i t a i n

STRUUITE

pp 609-615,

1988

(McJNH4PO4.6H20)

CRYSTAL FORMATION

Trypanosoma cruzi
F.J.

Adroher

(I)

(1),

Department

University

A.

of Sciences,

BY

IN CULTURE

Osuna ( 1 ) & J . A .

Lupi~6ez

(2).

Parasitology,

Faculty

of

of

of Granada.

(2) D e p a r t m e n t

0 0 4 5 - 6 5 3 5 / 8 8 $3.00 + .OO
P e r g a m o n J o u r n a l s Ltd.

18071-Granada.

of B i o c h e m i s t r y

University

Spain.

and M o l e c u l a r

of Granada.

Pharmacy,

Biology,

I8071-Sranada.

Faculty

Spain.

ABSTRACT.
This
work
was
to
describe
the
in vitro
formation
of
exocellular
precipitates
of
struvite (MgNH PO. 6H_O) in c u l t u r e s
of
^
,
4
.~"
Z
T r y p a n o s o m a cruzi, grown in m o d i f i e d ~race s m e o m a s u p p l e m e n t e d with IOX
inactivated fetal bovine serum and with NaCl or sorbitol.
This
crystal
precipitation
was
only v i s u a l i z e d in m e d i a after 25 days
of
culture.
Such m e d i a e v e n t u a l l y had a m m o n i u m higher than 0.4 mg/ml and a pH of 7.0
or
more.
Discussed
is the p o s s i b l e influence of osmotic
pressure
on
struvite p r e c i p i t a t i o n .

INTRODUCT I ON
Some m i c r o o r g a n i s m s
bacteria

from

aragonite

(CaC03,

(calcium

phosphate),

ammonium

soil,

reported
"...

organic
the

However,
media

struvite

in which
phosphate

colonies

has
we

(2, 9,

in

during

...'.

only been

aureus

described

struvite

in

crystals

by us

nitrogenous

on nutrient

of them

crystals

was

and s u g g e s t e d

from

it then c o m b i n e s

in a p r e v i o u s

609

as

apatite

matter

crystals which

to form mineral

precipitation

such

(magnesium

organic

that

all

Some

17, 20).

growing

similar

of p r o t o z o a

inform of struvite

and

organisms,

formed

crystals

ammonia

Later,

crystals.

hexagonal),

and

such

the

they are g r o w i n g

other

16,
rich

identified
produce

present

growth,

the c a p a c i t y

II,

material

(18)

(CaC03,

(SrC03) ,

of S t a p h y l o c o c c u s

as struvite;

the m e d i u m

wherein

hexahydrate)

mineral

can produce

calcite

stroncianite

Robinson

generate

or sea w a t e r

microorganisms

matter

identified
of

the

magnesium

within

of

long ago.

that

fresh

can

orthorhombic),

phosphate

Occurrence

in culture

with

occurred
agar

increasing

were
the pH

(2).
crystals

in culture

communication

by T r y p a n o s o m a

cruzi

(I),
in

610

Grace's

medium

the a p p e a r a n c e
in w h i c h

epimastigotes

therefore
the

modified

studied

influence

by O s u n a - C a r r i l l o

of s t r u v i t e
of T.

cruzi

conditions

of NaCl

et al.

(MgNH4PO4.6H20)

under

had

been

which

(14).

crystals
grown

struvite

We

report

in a c u l t u r e

for
is

several

here

medium

days.

formed,

We

including

and sorbitol.

HATERIALS AND METHODS

We

used epimastigotes

Bl&squez,

Instituto

Sanitarias,

Majadahonda,

Nicolle-McNeal
minimal

(NNN)

essential

U.K.)

of T.

Nacional

Madrid,

medium

medium

with

with

20%

supplemented

inactivated

at 56 C

for

the

log-phase

growth

end

of

Grace's

media,

with

cruzi,

strain

Maracay

de M i c r o b i o l o g ] a ,
Spain),

obtained,

overlaid

with

Earle's

salts

(v/v)

30 m i n

a liquid

phase

(MEM;

The

flagellates

inoculated

Eagle's

Middlesex,

serum

(Gibco),

harvested

into m o d i f i e d

of e p i m a s t i g o t e s / m l ,

Dr.

in N o v y -

of

Gibco,

bovine

by

e Inmunologia

at 2 8 C,

fetal

(IFBS).

were

I million

of

(donated

Virologfa

and

at

control

and maintained

at

2 8 C.
The

crystals

(Sartorius)

with

water

until

water

at 0 C

The

were

8-pm diameter

perfectly

struvite

examined

by

were

with
of

They

0.0195)
observed

a Cu a n o d e

then
with

a n d Ni

in

of s t r u v i t e
dried
a

PW

filter

bi-distilled
of

at 37 C.

Philips

1730

filter

involved

on a

in g / l O 0 ml

515

microanalyser,

by a P h i l i p s

the s a m p l e

by retention

were washed

solubility

to an E d a x

diffraction

Preparation

the m e d i a

pores.

(the

connected

X-ray
tube

from

at GO C

crystals

microscope

diffraction

clean

is 0 . 0 2 3 1 ;

electron

source.

isolated

scanning

and

further

diffractometer.

was

used

pulverizing

as

an

purified

X-ray

crystals

in an a g a t e m o r t a r .
To
10%

study

(v/v)

Darmstadt,
cases;
mOsm

Systems

parasites

2 8 C. G r o w t h
Glucose
Bernt

(3).

enzymatic

MO,

flasks

determined

with

the m e d i u m

was

of

whose

with

pH was

Osmette

6.3

plus

(Merck,
in all
550-575

osmometer

U.S.A.).
of o r g a n i s m s / m l

Vineland,

a Neubauer's

suggested

in 2 ml

U.S.A.),

at

Bergmeyer

&

hemocytometer.

in the m e d i u m

kit w a s

NJ,

as described

2-oxoglutarate

170-UV

The

(Gibco)

of NaCl

and approximately

I million

estimated

the p r o c e d u r e

medium

I00 mEq/l

(Merck).

(Bellco,

concentrations

conversion
of

with

Grate's

with

control

Massachusetts,

in

Ammonium

for

inoculated

Leighton

U.S.A.),

prepared

as measured

was

micromodification
Louis,

375 mOsm
media

Inc.,
were

in 17-ml

we

supplemented

or 200 m M of s o r b i t o l

pressure

supplemented

(Precision

of m e d i u m

formation,

as c o n t r o l ,

F.R.G.)

osmotic
for

The

crystal

IFBS,

to

were

measured

L-glutamate

by Sigma

employed.

by

Chemical

from
by

Co.

a
(St.

611

RESULTS
In c u l t u r e s
the culture
crystals
bunsen

flask

were

phosphorus
These

Fig.

IB)

crystals

1.

from

A:

crystals

were

Fig.

In

X-ray

increased
T.

growth

diagram

struvite

(XI00).

ensue,

to 7.0 or over

The
a

microanalyser,

revealed

1.384:1.
yielded

as struvite

the

spectrum

from reference

macle

from

studied

consumes
resulting

(Fig.

3).

(X40).

B:

Electron

Bars = 0.I mm.

::

"

"

25

struvite

(Fig.

15

crystals.

pH

excretion,

to c o n s u m p t i o n

rapidly

with

electron

ammonium
were

excepted.

of

scanning

diffraction

outline

in vitro

(0.4 mg/ml)

risen

Edax

the base

incinerated
by

:)5

cultures,

and

inversely

cruzi

excretion
having

diffraction

parallel

consumption,

controls

identified

of

IA),
not be

at

(4).

~'~

2.

an

crystals

obvious

r a t i o of

to X - r a y

the A.S.T.M.

were

crystals,

to

in a w e i g h t

of struvite

55 t

the

connected

Microphotograph

microphotograph

(Fig.

they could

of

subjected

2. The

15-762

crystals

media

since

Examination

(Fig.

old,

culture

and m a g n e s i u m

in Fig.

to card

in all

inorganic

burner.

microscopy

shown

25 days or more

3);

changes,
ammonium

glucose
excretion

of glucose.

glucose.

High

in struvite

levels

of a m m o n i u m

precipitation,

the pH

612

A~

CD

20

15

"0.2

10

0. 1

10

15

20

DAYS
Fig.

3.

Trypanosoma c r u z i

10~ ( v / v )

IFBG p l u s

millions/ml

((~)!

culture

on G r a t e ' s

100 mEq o f N a C 1 / l i t e r .

(B) pH ( 0 ) ;

(D) Ammonium c o n c e n t r a t i o n ,

medium s u p p l e m e n t e d w i t h

(A) Growth o f p a r a s i t e s ,

(C) G l u c o s e c o n c e n t r a t i o n ,
in mcj/ml

i n mg/mi

in
([7);

(,).

DISCUSSION
We have
(I).

We

correlate

noted- w a s

followed
of pH,

glucose
of

is

after

Interestingly
not

concentration

We

of a m m o n i u m

cruzi

(6).

related

induced

25 or more

even
of

and

of protein

ammonia

of

in

have

excreted

-as

consumption,

reaching

the

3).

The

(5,

7,

metabolism

along with
taking,

5.5,
8),

with

(23).

an

rapidly
due

to

this

is

increase

Formation

higher

than

of
0.4

a pH of 7.0 or more.
crystal

culture.
of

parasite
to

of ammonium,

in the a t m o s p h e r e

higher

excretion

of a m m o n i u m

concentration

for sample

60 days

especially

ammonium

succinic

levels

by T. cruzi

in the m e d i u m m a y

the pH s i n k i n g

(ammonium

opened
after

22),

(Fig.

days of culture)

in flasks

for struvite

think

as a result

thereby

glucose

mainly

of high

12,

amount

period

the m e d i u m
acids,

(I,

precipitation
crystals

catalyzing

The

to

the growth

organic

occur

the struvite
of s t r u v i t e

Enzxmes

from

presumably

of

21).

by the p r o d u c t i o n

struvite

needed

in T.

inversely

production

mg/ml

(19,

at the end of

consumes

reported

the f o r m a t i o n

excretion

media

detected

maximum

did

that

with

sugar-free
been

previously

think

the

precipitation

Perhaps

culture

critical
flask

is

precipitation.

that struvite

precipitation

m a y be p r o m o t e d

by the

elevated

613

osmotic

pressure

of the m e d i u m

ammonium

excretion

had been

in

the G r a t e ' s m e d i u m

or

sorbitol-

normal

observed

-osmotic

w a s struvite

Grace's

used,

medium

since
among

p re s s u r e

crystals

no

several

media

differences
studied.

550-575 mOsm generated

observed.

supplemented

important

with

The

osmotic

in

Only

by

NaCl

pressure

10% IFBS

is a p p r o x i m a t e l y

for

protozoa.

of
375

mOsm.
These
were

c r y s t a l s were
observed

precipitate

of T.

cruzi:

crystals.

formed

in the rosette

the parasite.

This

carried

out

and

an

important

when

factor

working

medium

with

producing
is I,

culture

have

less

of

Ammonium

bacteria,

(17)

were

suggested

in nature,

excretion

by b a c t e r i a

is not

(17);

they

it m a y be

producing
found

strains

that

the

culture

et al.

number
ratio

is increased.

some b a c t e r i a

supporting

sites

although

that

formed without

that

nucleation

non-struvite

in

precipitates

of

in the

Under

modifying

our
the

present.

as

precipitation,

the

of growth.

FerrOn-Vilches

P O ~2- :
+ M ^g

of M g 2+

of

is

calciprecipitans,

struvite

the

by

opened

the s a m p l i n g

of c r y s t a l s

observed

when

crystals

the c u l t u r e s

distribution

Recently,

of

around

substances

the r o s e t t e s

M g 2+ and Ca2+;

is maximal

ions n o r m a l l y

formation

acting

the formation

may

c r y s t a l s may be

because

in P s e u d o m o n a s

is 4 or higher.

struvite

by which

for u n i f o r m

that,

between

freshwater

et al.

struvite

calcite

found

these

of e x c r e t e d

to disperse

if the c o n c e n t r a t i o n

these

Rivadeneyra

isable

hemin

a period

to form r o s e t t e s

struvite,

agitation

Aggregates

in w h i c h

acid after

that

by action

the reason

controlling

struvite

conditions

proportion

al.

(20)

the ratio M g 2 / C a 2+

(I0),

by

vigorous

tended

likewise

to p r e c i p i t a t e

is the r e l a t i o n s h i p

strains

in

could explain

this action

Shinano & Sakai

think

F-81,

becomes

epimastigotes
we

the

medium

the m e d i u m

microhabitat

unable

after

flagellates,

sites

in culture

However,

to s a m p l i n g were

medium

(15)

as c r y s t a l s when

good growth
hemin

aggregation

by Pan

for

m a y have

Morita's

view

abiological

increase

it, since
reported

the number

of c r y s t a l s

(13)

of

precipitation.

the only d e t e r m i n a n t

has been

a role

of

struvite

ammonium

formation

by

Rivadeneyra

produced

is

et

strain

dependent.
Factors

other

precipitation
precipitate
culture

living

stage.

of T.

protozoa

minerals.
be due

struvite

medium

metabolism

than

simple

process.

It

ammonium

m a y be

is t r i g g e r e d

along with
cruzi

high

that

by o v e r p r o d u c t i o n

osmolarity.

remains

protozoa's

to c a p a b i l i t i e s

The

to be studied.

m a y also c o n t r i b u t e

Parasitic

excretion

maintained

trigger

of

T.

of

this

We suggest
of struvite

for struvite

in the evolution

from

the

cruzi

of a m m o n i u m

effect

to d e p o s i t s

abilities

may

the p r o n e n e s s

in

the

in

the

that

free-

and

other

precipitation
its

to

may

free-living

614

Acknowledgements.
We

thank

to Depart amento

Experimental

del

Zaidfn,

diffraction

measurements

Histologfa,

Facultad

the

preparation

are

grateful

manuscript
Thanks
version
This
from

de M e d i c i n a ,

C.

R.

and Dr.

D.

from

de Granada

for a critical
for advice

for

X-ray

Departamento

by electron

Janssen

Estaci6n

de

(Spain),

for

microscopy.
reading

We

of

the

and collaboration.

for r e v i s i n g

the

English

the m a n u s c r i p t .

investigation
Comisi6n

was supported

Asesora

(C.A.I.C.Y.T.)
Ciencia,

Universidad

of material

from

Spain),

Revelles

Benltez-Rodrlguez

Alonso

de A r c i l l a s

(Granada,

F.

Dr. M. A. R i v a d e n e y r a

and Dr.

of

C.8.I.C.
and Dr.

and e x a m i n a t i o n

to

to Dr.

de M i n e r a l o g l a

and

Madrid,

an

de

from

a research

Investigaci~n

INAPE f e l l o w s h i p

Spain)

grant

(No.

1067/82)

T~cnica

Cientffica

(Ministerio

de

Educaci6n

to F.J.A.

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