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Arch Microbiol (1981) 130:19-22 
Archives of 
Hicrobinlngy 
�9 Springer-Verlag 1981 
Bacterial Mesosomes: Method Dependent Artifacts 
Hans Rudolf Ebersold t,z, Jean-Louis Cordier ~, and Peter Ltithy ~ 
Mikrobiologisches Institut der ETH, CH-8092 Ziirich, Schweiz 
2 lnstitut ffir Zellbiologie, Elektronenmikroskopie I der ETH, CH-8092 Ziirich, Schweiz 
Abstract. The occurrence of mesosomes was investigated 
during septum formation of vegetative and sporulating cells 
of Bacillus cereus. It has been demonstrated that bacterial 
mesosomes which are considered by numerous micro- 
biologists as an integrated constituent of Gram positive 
bacteria, are in reality artifacts arising during the preparation 
for electron microscopy. The conventional fixation methods 
allowed enough time for the cytoplasmic membrane to react 
to the changed conditions and to form the typical pocket-like 
membrane invaginations. With cryofixation followed by 
freeze-substitution it was shown in ultrathin sections that 
mesosomes do not occur. The extremely rapid freezing and 
the substitution of the ice by an organic solvent containing the 
fixative prevented the formation of membraneous artifacts. 
Key words: Bacillus cereus - Mesosomes - Chemical 
fixation - Cryofixation - Freeze-substitution - Freeze- 
fracturing 
Nanninga (1971) found that size and number of mesosomal 
structures varied with the preparation technique. Later on, 
other authors were able to confirm these observations and 
arrived at the conclusion that the conventional idea about 
mesosomes had to be revised (Fooke-Achterrath et al. 1974; 
Higgins et al. 1976; Silva et al. 1976). In this contribution 
evidence is presented, using Bacillus cereus and three other 
aerobic spore formers as additional control, that the classical 
mesosomes represent artifacts due to the preparation 
technique. 
Materials and Methods 
Organism and Culture Conditions. Bacillus cereus (ATCC 10702) was 
cultured in a semisynthetic medium (Yousten and Rogoff 1969). 
Incubation was carried out in 500 ml Erlenmeyer flasks containing 100 ml 
of medium on a rotary shaker at 303 ~ K. Cells were harvested 5 h from 
inoculation, i. e. in the log-phase, and after 8 h when they had entere d the 
early stage of sporulation. 
In bacterial textbooks mesosomes are cited as regularly 
occurring compounds of Gram positive bacteria. It has been 
suggested that they might be involved in the synthesis of cell 
membranes, in the endospore formation, in replication and 
segregation of DNA and in various other processes including 
energy production (Salton 1971). But nobody has been able to 
assign a definite function to them. 
The first report about mesosomal structures was made by 
Chapman and Hillier (1953). The term mesosome was 
introduced by Fitz-James (1960) who observed these com- 
ponents investigating the role of the cytoplasmic membrane 
during growth and spore formation of Bacillus megaterium, 
Bacillus cereus and Bacillus thuringiensis var. alesti. 
Mesosomes can easily be demonstrated by electron micro- 
scopy following chemical fixation of the bacterial cells. They 
appear along the cytoplasmic membrane and at sites of 
septum formation and they can be described as pocket-like 
invaginations containing so-called vesicles, lamellae and 
tubules. For the demonstration of mesosomes, the cellular 
structures were immobilized by chemicals such as glutar- 
aldehyde and/or osmium tetroxyde (OsO4) as well as by 
freezing with cryoprotectants. The fixed preparations were 
then processed either as thin sections or freeze-fractures. 
In the more recent literature doubts have been expressed 
by several authors about the classic mesosome theory. 
Offprint requests to: P. Liithy 
Non-standard abbreviations." OsO 4 = osmium tetroxide; UO2Ac = ura- 
nylacetate; PHB = poly-fi-hydroxy-butyric acid; M = mesosome; CW 
= cell wall; CM = cytoplasmic membrane; PF = plasmatic fracture of 
the cytoplasmic membrane 
Chemical Fixation. The washed bacterial cells were fixed in glutar- 
aldehyde (3 %) and OsO~ (1%) in the presence of ruthenium red (0. l 5 %), 
an inorganic stain used as a contrasting agent for carbohydrates. The 
samples were then dehydrated in 2,2-dimethoxypropane (Muller and 
Jacks 1975) and embedded in Araldite/Epon (Luft 1961). 
Cryofixation Combb~ed with Freeze-Substitution. For cryofixation a gold 
grid dipped in the bacterial suspension was placed between two low mass 
copper platelets and the assembly was frozen in the propane jet (Mtiller et 
al. 1980a). Methanol containing OsO+ (1%), uranyl acetate (UO2Ac) 
(0.5 %) and glutaraldehyde (3 %) was used as substituent. The frozen 
specimens were incubated in the substituent at 183 ~ 210 ~ and 343 ~ K for 
8 h at each temperature step. After a final incubation at 273 ~ K for 1 h, the 
samples were exposed to anhydrous acetone and subsequently embedded 
in Araldite/Epon. The thin sections were contrasted with UO2Ac and 
lead citrate (Reynolds 1963). The technical details of the fi'eeze- 
substitution have been described by Miiller et al. (1980b). 
Freeze-Fracturing. Freeze-fractures were prepared from cryofixed speci- 
mens in a Balzers BAF 300 at a pressure of 10-s Pa. Contamination was 
prevented by starting the evaporation of platinum/carbon prior to 
fracturing (25 nm PtC/20nm carbon). The replica were cleansed with 
H2SO4 (20%) for I h and HCIO (14%) for 1 h. 
Microscopy. A Philips EM 301 at 100 kv was used. The micrographs were 
taken on Agfa Scientia 23 D 56 cut films and developed in Gevatone G 5c 
for 3.5 rain at 293 ~ K. 
Results 
The Occurrence of Mesosomes in Chemically Fixed Cells. The 
typical mesosomal structures were present in chemically fixed 
cells. Figure 1 a represents a cell in the dividing stage with 
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Figs. 1--3 
mesosomal vesicles located at the base of the developing 
septum. The hypertonic conditions (3 ~o glutaraldehyde in 
0.1 M sodium cacodylate buffer) used in our experiment led to 
a reduction in the size of the invaginations, followed by a so- 
called "flattening out" of the mesosomal content as already 
described by Ryter (1969). Figure lb shows a cell during 
formation of the spore septum with a large mesosomal 
complex. It has to be added that the spore septum consists of 
two cytoplasmic membranes without visible presence of cell 
wall material. 
Absence of Mesosomes in Cryofixed and Freeze-Substituted 
Cells. Bacterial cells processed by cryofixation and freeze- 
substitution never contained mesosomes. The non-existence 
of mesosomes is demonstrated again duringthe dividing stage 
of a vegetative ceil (Fig. 2a) and during septum formation in 
the early sporulation phase (Fig. 2 b). Striking morphological 
differences between conventional chemical fixation and 
cryofixation/freeze-substitution technique could be observed. 
With the latter method, cytoplasmic membrane and cell wall 
remained intimately associated while interspaces were in- 
duced by chemical fixation. This proves that in the septum 
development of vegetative cells the formation of the cytoplas- 
mic membrane and the cell wall are closely linked processes. 
Absence of Mesosomes in Freeze-Fractured Specimens. The 
freeze-fractures confirmed the results obtained with 
cryofixation/freeze-substitution. Mesosomes could never be 
detected. Figure 3a is a freeze-fracture of a dividing cell while 
Fig. 3 b shows a sporulating cell during septum formation. 
Discussion 
The preservation of the native state represents one of the 
major problems in the investigation of structure/function 
relationship in cell biology. The traditional methods of 
electron microscopy such as chemical fixation or freezing in 
the presence of cryoprotectants are known to induce struc- 
tural alterations. It is important to realize that fixativesdo not 
lead to an immediate immobilization of membranes. This is 
also true for the bacterial cell where the formation of 
mesosomes is especially enhanced if the fixation occurs 
slowly, allowing enough time for the rearrangement of the 
cytoplasmic membrane, This phenomenon is pronounced in 
the case ofOsO 4 which is known to be a slow fixative (Silva et al, 
1976), depending in addition on the concentration and the tem- 
perature at which the treatment is carried out (Fooke-Achter- 
rath et al. 1974; Gosh and Nanninga 1976). Higgins eta l . 
(1976) showed that cells fixed with glutaraldehyde at different 
temperatures and processed by freeze-fracturing varied in the 
number of mesosomes. Not only chemical fixatives but the 
21 
mere addition of glycerol (20 ~o) as a cryoprotectant was able 
to generate mesosomes (Higgins et al. 1974). Higgins et al. 
(1976) as well as Fooke-Achterrath et al. (1974) were able to 
reduce the number of mesosomes drastically if the fixation 
was performed on chilled specimens or in the absence of 
cryoprotectants. 
In contrast to chemical fixation, cryofixation (without 
cryoprotectants) leads to an immediate immobilization of the 
intracellular structures. The extremely high cooling rate 
prevents the formation of ice crystals exceeding 15nm. 
Crystals of this size are not able to induce structural 
alterations that are detectable in the electron microscope. The 
further processing by substitution of the ice by an organic 
solvent with concomitant fixation at low temperatures should 
prevent artifacts also during this step. 
The results obtained with B. cereus show that the classic 
mesosomal structures (Ryter 1969) are not present if the 
bacterial cells are fixed in a way which preserves the native 
structures and which reduces the chance of artifact formation 
to a minimum. This could be achieved with the cryofixation/ 
freeze-substitution technique where, by the way, fixatives and 
their concentrations used remained the same as in the 
chemical fixation procedures. Therefore it can be concluded 
that mesosomes are artifacts generated during exposure to the 
fixatives. 
The results were confirmed with Bacillus subtilis, Bacillus 
megaterium and Bacillus thuringiensis, permitting a general- 
ization for the genus Bacillus and very likely also for the other 
Gram positive bacteria. 
Acknowledgement. We thank Dr. M. Miiller, Institute of Cell Biology, 
Electron Microscopy I, Swiss Federal Institute of Technology, for his 
valuable advices. 
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Fig. 1 a. Chemically fixed Bacillus cereus cell during cell division, in the stage of septum formation. The typical mesosomal vesicles (M) are located 
between cytoplasmic membrane (CM) and cell wall (CW). The bar equals 400 nm in all six figures 
Fig. lb. Chemically fixed cell in the early sporulation phase during formation of the forespore septum (FS). The characteristic mesosomes have formed 
along the forespore membrane 
Fig. 2a. Cryofixed and freeze-substituted vegetative cell during septum formation. No mesosomes have been generated. Cytoplasmic membrane and cell 
wall are in close contact 
Fig. 2b. Cryofixed and freeze-substituted sporulating cell. No mesosomes can be seen along the forespore membrane. PHB, poly-fi-hydroxy-butyric acid 
granule 
Fig. 3 a. The freeze-fractured preparation of a dividing cell confirms the absence of mesosomes and the integrity of cytoplasmic membrane and cell wall 
Fig. 3b. No mesosomes have been generated in a freeze-fracture showing the septum of a sporulating cell. PF, part of the plasmatic fracture of the 
cytoplasmic membrane 
22 
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Received March 27, 1981