(full paper is archived in the Miller Library)
Title: Enumeration of cultivatable and non-cultivatable bacteria in egg case sheats of Loligo opalescens
Student Author(s): Tyburczy, Joe
Faculty Advisor(s): Epel, David
Location: Final Papers Biology 175H
Date: June 1999
Symbiotic bacteria inhabit the accessory nidamental gland of the squid, Loligo opalescens, and are transmitted vertically to its egg cases. Previous work has resulted in the cultivation, isolation, and characterization of two dominant bacterial strains (genera Shewanella and Roseobacter) from homogenates of egg case sheath and accessory nidamental gland.
Do these cultivated isolates accurately represent the diversity of the sheath bacterial community? Are there bacteria present in the sheaths that are not cultivatable and therefore have not been described or characterized? What are the numbers of bacteria in the sheaths and what percent of these can be cultured?
In order to obtain release of the bacteria from the sheath for quantitative enumeration, a means of disrupting the tough egg sheath was developed using sodium periodate and glass beads. Total bacterial counts ranged between 10(8) and 10(9) bacterial per sheath which is slightly higher than the 10(6) to 10(8) estimated by Biggs and Epel (1991) with electron microscopy. A live/dead staining assay with DAPI and propidium iodide indicated about 68% of bacteria in sheath homogenate were alive. Serial dilutions of the native sheath homogenate were plated on various culture media and colony forming units were counted. Using comparison of direct bacterial counts from the homogenate, the viability stain, and the number of colony forming units on plates, the percent of bacteria cultivatable was found to be between 30 and 50%.
Because a significant portion of the bacteria were not cultivatable, further investigation of egg sheath bacteria may be necessary to adequetely describe its diversity and composition.