Stanley A. Robrish

The use of the o-phthalaldehyde reaction as a sensitive assay for protein and to determine protein in bacterial cells and dental plaque.

Abstract Alkaline hydrolysis of protein followed by reaction with o-phthalaldehyde has permitted the determination of less than 10 ng of protein fluorometrically. When intact proteins were analyzed with o-phthalaldehyde the reaction was less sensitive. This reaction has been applied to analysis of the protein content of dental plaque and bacterial cells.

The analysis of picomole amounts of l(+)- and d(−)-lactic acid in samples of dental plaque using bacterial luciferase

L(+)-Lactic acid (5 pmol) and D(-)-lactic acid (20 pmol) were assayed by coupling the generation of NADH with the use of bacterial luciferase. The binding of NADH to L(+)-lactic dehydrogenase made it necessary to denature the protein so that the assay with bacterial luciferase was effective. The coupled luciferase assay of L(+)-lactic acid was 400 times more sensitive than the fluorometric assay. The luciferase coupled assay was used to analyze the L(+)- and D(-)-lactic acid contents of small samples of dental plaque.

Acid Production from Glucose and Sucrose by Growing Cultures of Caries-Conducive Streptococci

Acid production was determined for several strains of caries-conducive streptococci growing on glucose and sucrose. The yield of acid per mol of sucrose was the same as that from two hexose units. It was determined by macroscopic and microscopic observations of the culture that extracellular polymer was formed from sucrose during growth, but the amount of sucrose diverted to polymer formation was less than that anticipated from visual observations.

Na+ requirement for glutamate-dependent sugar transport byFusobacterium nucleatum ATCC 10953

Resting cells ofFusobacterium nucleatum ATCC 10953, when provided with glutamic acid (Na+ salt) as fermentable energy source, rapidly accumulated [14C]glucose, from the medium. Sugar accumulation was not observed when Na+ glutamate was replaced by ammonium glutamate. However, addition of Na+ (chloride) to the latter system elicited uptake of [14C]glucose by the organism. Of other monovalent cations tested, only Li+ was found to be slightly stimulatory, but K+, Rb+, and Cs+ ions were ineffective. For determination of the role(s) of Na+ in sugar accumulation, the transport of [14C]glucose and [14C]glutamic acid by the cells was studied independently, with lysine as an alternate (and Na+-independent) energy source. In the presence of lysine, cells ofF. nucleatum 10953 accumulated [14C]glucose from a Na+-free medium, but, in contrast, uptake and fermentation of [14C]glutamic acid was Na+-dependent. The glucose transport system is Na+-independent. However, our data indicate dual role(s) for Na+ in the transport and intracellular metabolism of glutamic acid. The Na+-dependent glutamate fermentation pathway provides the necessary energy for active transport of glucose by the resting cell.

A comparison of viable counts and adenine nucleotide analysis to determine the effect of antimicrobial agents on dental plaque

The effects of three antimicrobial agents on smooth surface dental plaque in monkeys were assessed using a plaque index, bacterial viable count, and adenine nucleotide analysis. The effects of the agents were revealed equally well when, viability was assayed by extractable adenosine triphosphate (ATP) or conventional viable cell counts. A persistent effect of one of the agents was revealed by both viable count and extractable ATP. These interpretations were supported by calculations of adenylate energy charge from the adenine nucleotide content of the smooth surface dental plaque samples.

The flavin mononucleotide content of oral bacteria related to the dry weight of dental plaque obtained from monkeys

The analysis of the protein and flavin mononucleotide (FMN) content of dental plaque obtained from monkeys proved to be, an effective method of estimating the dry weight of dental plaque samples. The FMN content of various bacterial cultures was also comparable with the FMN content of dental plaque. FMN could be measured in a suspending medium containing amines or protein; however, both of these saspending media interfered with the estimate of dry weight by protein determination.