Ultrastructural preservation of biofilms formed by non-typeable Hemophilus influenzae

There is growing evidence to suggest that non-typeable Hemophilus influenzae (NTHi), an important cause of otitis media in children, is able to grow as a biofilm in the middle ear. This observation may help to explain bacterial persistence in chronic infections. We evaluate the usefulness of rapid freezing and freeze substitution as a means of preparing biofilms for ultrastructural examination by comparing the morphology of cryofixed specimens with the morphology produced using more conventional chemical fixation and dehydration methods. Chemical fixation and dehydration methods produced substantial ultrastructural damage to individual NTHi in the biofilm and loss of extracellular matrix, even in the presence of ruthenium red. In comparison, cryofixed and freeze-substituted NTHi biofilms showed significantly improved preservation of bacterial ultrastructure and biofilm organization. The intracellular contents of NTHi prepared using the cryomethods showed little evidence of aggregation, and bacteria within the biofilm were closely packed and surrounded by an abundant extracellular matrix. Although high-pressure freezing of NTHi biofilms followed by freeze substitution was highly effective for preserving ultrastructure when examined by transmission electron microscopy, immersion in liquid propane offered an alternative, “less technical”, freezing method. Immersion in liquid propane followed by freeze substitution and critical point drying was most effective for preserving ultrastructural details in specimens examined by scanning electron microscopy.

Regulation of cellulose-inducible structures of Clostridium cellulovorans

Scanning electron microscopy was used to detect ultrastructural protuberances on the cellulolytic anaerobe Clostridium cellulovorans. Numerous ultrastructural protuberances were observed on cellulose-grown cells, but few were detected on glucose-, fructose-, cellobiose-, or carboxymethylcellulose (CMC)-grown cells. Formation of these protuberances was detected within 2 h of incubation in cellulose medium, but 4 h incubation was required before numerous structures were observed on the cells. When a soluble carbohydrate or CMC was mixed with cellulose-grown cells, the ultrastructural protuberances could no longer be detected. In fact, no protuberances were observed within 5 min following the addition of glucose, cellobiose, or methylglucose to cellulose-grown cells. The presence of these protuberances corresponded with the binding of the Bandeiraea simplicifolia BSI-B4 isolectin to the cell. Cellulose-grown cells had a greater level of observable lectin binding than cellobiose-grown cells, and lectin binding was not detected on glucose- or fructose-grown cells. In addition, lectin binding ability was lost by cellulose-grown cells following the addition of glucose, fructose, or methylglucose to the cellulose medium. A cellulose-affinity protein fraction expressing cellulase activity was also detected in cell extracts of cellobiose- or cellulose-grown cultures. However, this protein fraction was not detected in extracts of glucose-grown cultures, and was rapidly lost (within 5 min) following the addition of glucose to cellulose-grown cultures. The ability of C. cellulovorans to adhere to cellulose was also affected by the energy substrate, but not in the same manner as the protuberance formation or the cellulase-containing protein fraction. Rather, cellobiose-, cellulose-, and CMC-grown cultures adhered to cellulose, but this adherence was not affected by addition of glucose to the medium. This is the first report that soluble carbohydrates caused the rapid loss of some cellulose-inducible systems of C. cellulovorans.Key words: cellulolytic bacteria, bacterial ultrastructure, polycellulosome, scanning electron microscope, lectin binding, cellulosome.