A Method of Obtaining Axenic Rumen Ciliate Protozoa

Abstract

Nutritional studies on rumen ciliate protozoa have long been hampered by the inability of investigators to maintain axenic or gnotobiotic cultures of these organisms. Establishment of such cultures requires the development of a sterilizable culture medium and a reliable method of producing an inoculum of undamaged bacteria-free ciliates. HIND et al.(1) have recently described a medium which appears to satisfy the first requirement. The purpose of this communication is to describe a development of COLEMAN'S (2) methods which satisfies the second requirement.Entodinium biconcavum, Kofoid and MacLennan, 1930, was identified in grass-fed ruminants in New Zealand by CLARKE (3). Clone cultures of this organism were isolated and maintained for 3 years using the methods described by HUNGATE (4) with the following modifications. Cultures of 20 ml each were grown in the anaerobic salt medium described by MAH (5) and supplemented daily with 8 mg each of whole wheat flour and dried milled perennial ryegrass. Half the culture was replaced with fresh mineral salt medium every 2 days. Dilution blanks were prepared from 100 ml of MAH's (5) medium sterilized by autoclaving and cooled under a stream of sterile 02-free C02. Sterile cysteineHCl (0.1 %) and sodium hydrogen carbonate(0.5 %)were added to the cooled medium and 9-ml portions were dispensed in graduated glass conical centrifuge tubes which were flushed with 02-free C02 before the addition and afterwards closed with butyl rubber stoppers.Ciliates were separated from bacteria by repeated washing in sterile diluent. Washed ciliates were usually prepared from cultures diluted 24 hr earlier and refed 2 hr before washing. Ten ml of culture was placed in a stoppered conical glass centrifuge tube, and sedimented at 43 x g for 60 sec in a bench centrifuge fitted with a swing-out head (Baird & Tatlock Ltd., London). The head stopped 385

Glucose control of staphylococcal enterotoxin A synthesis and location is mediated by cyclic AMP

“…Using batch cultures we have been unable to demonstrate any striking effect of medium composition or other culture conditions on SEA pro-duction, apart from the apparent repressive effect of glucose, in common with previous work [8].…”

“…Because outbreaks of food poisoning caused by staphylococcal contamination of food are common [1] and most frequently involve staphylococcal enterotoxin A (SEA)-producing strains [2], there have been several studies of SEA synthesis. These experiments were largely conducted in batch cultures and indicated that toxin accumulation was apparently growth related [5], occurring mainly dur-248 ing the exponential phase of growth [6]• These studies did not identify any particular medium component or other culture condition that specifically controlled toxin synthesis [7], except for a repressive effect of glucose, which has also been reported for synthesis of enterotoxin B (SEB) and enterotoxins C [8]. The present work investigates SEA synthesis and location in chemostat culture using a defined medium•…”

Biosynthesis and Mode of Action of Lantibiotics

“…Resistance to nisin in several Bacillus strains was reported by Jarvis in 1967 and ascribed to the presence of nonproteolytic nisin inactivating enzymes . This followed in the wake of an earlier proposal of an enzyme, nisinase, present in the cell extracts of Streptococcus thermophilus , that was capable of inactivating nisin but not subtilin …”

“…444 Jarvis noted that the treatment of nisin or subtilin with cell-free extracts of Bacillus cereus and Bacillus polymyxa led to loss of their antibiotic activity, whereas antibiotics such as gramicidin S, polymyxin B, and bacitracin were not affected, suggesting the presence of lantibiotic-specific agents. 443 The inacti-vating activity of the B. cereus cell-free extracts was dependent on Ca 2+ , Co 2+ , and Mg 2+ ions and was inhibited by EDTA. Partial purification of the nisininactivating fraction was achieved, but an actual protein was never isolated.…”

“…Resistance to nisin in several Bacillus strains was reported by Jarvis in 1967 and ascribed to the presence of nonproteolytic nisin inactivating enzymes. 443 This followed in the wake of an earlier proposal of an enzyme, nisinase, present in the cell extracts of Streptococcus thermophilus, that was capable of inactivating nisin but not subtilin. 444 Jarvis noted that the treatment of nisin or subtilin with cell-free extracts of Bacillus cereus and Bacillus polymyxa led to loss of their antibiotic activity, whereas antibiotics such as gramicidin S, polymyxin B, and bacitracin were not affected, suggesting the presence of lantibiotic-specific agents.…”