scholarly journals Genetic Transformation as a tool for detection of Neisseria gonorrhoeae

1976 ◽  
Vol 4 (1) ◽  
pp. 71-81
Author(s):  
A Janik ◽  
E Juni ◽  
G A Heym
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Genetic Transformation ◽  
Rapid Method ◽  
Deoxyribonucleic Acid ◽  
Time Interval ◽  
Diagnostic Laboratory ◽  
Primary Isolation ◽  
Transformation Assay ◽  
Mutant Strains ◽  
Pure Cultures

A rapid method for the detection of Neisseria gonorrhoeae, making use of the ability of deoxyribonucleic acid samples from clinically isolated strains of this organism to transform nutritional mutants of a particular strain of N. gonorrhoeae, has been described. In addition to using isolated cultures, transforming deoxyribonucleic acid can be obtained directly from the material that adheres to swabs of the cervix or the urethra. The time interval for transfer of swabs to the diagnostic laboratory is not a significant factor. It is not necessary to use pure cultures on primary isolation plates to obtain definitive results. Nongonorrhoeae neisserias, as well as a large variety of commonly encountered unrelated bacteria, do not react or interfere in the transformation assay when using one of the mutant strains under a standardized set of conditions. The entire assay can be completed in less than 24 h. It has also been shown that type T4 cells of the strain of N. gonorrhoeae employed in the present study are competent for genetic transformation, although type T4 cells are transformed at a significantly lower frequency than are type T2 cells of the same strain.

scholarly journals Identification of Neisseria gonorrhoeae by genetic transformation: a clinical laboratory evaluation

1977 ◽  
Vol 5 (1) ◽  
pp. 108-109
Author(s):  
R E Bawdon ◽  
E Juni ◽  
E M Britt
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Genetic Transformation ◽  
Deoxyribonucleic Acid ◽  
Clinical Laboratory ◽  
Laboratory Evaluation ◽  
Deficient Mutant ◽  
Wild Type

Transformation of a Neisseria gonorrhoeae auxotroph (uracil and arginine deficient) to prototrophy was attempted with wild-type deoxyribonucleic acid from 71 random clinical N. gonorrhoeae cultures. Of these 71 cultures, 97.1% transformed the nutritionally deficient mutant to prototrophy. The procedure was reliable and economical and offered several distinct advantages over other methods used for the confirmation of N. gonorrhoeae.

scholarly journals Genetic transformation assays for identification of strains of Moraxella urethralis

1977 ◽  
Vol 5 (2) ◽  
pp. 227-235
Author(s):  
E Juni
Keyword(s):  
Genetic Transformation ◽  
Deoxyribonucleic Acid ◽  
Clinical Laboratory ◽  
Phenotypic Properties ◽  
Neisseria Species ◽  
Naturally Occurring ◽  
Genetic Lesion ◽  
Mutant Strains ◽  
The One ◽  
Entire Procedure

Studies of 31 strains of Moraxella urethralis have shown that 20 of them are competent for genetic transformation. This finding has led to the development of transformation assays for identification of newly isolated strains of this organism. Crude deoxyribonucleic acid (DNA) samples from all strains of M. urethralis readily transform auxotrophic mutants of competent strains to prototrophy, whereas DNA samples from unrelated bacteria such as Acinetobacter, Moraxella, and Neisseria species uniformly fail to elicit positive transformation of mutant tester strains. One of the competent strains of M. urethralis investigated is a naturally occurring mutant defective in its ability to utilize citrate as a carbon and energy source. DNA samples from 29 of the 30 remaining strains of utilization; the one nonreacting strain is citrate negative and probably possesses the same genetic lesion as the citrate-negative mutant. Three organisms originally identified as strains of M. urethralis, because of their phenotypic properties, are probably incorrectly designated, since DNA samples from these strains failed to transform any of the tester mutant strains used in the present study. The transformation assay for M. urethralis is very simple and can be performed readily in a clinical laboratory. The entire procedure can be carried out in less than 24 h.

Genetic Transformation of Streptococcus sanguis (Challis) with Cryptic Plasmids from Streptococcus ferus

1980 ◽  
Vol 28 (3) ◽  
pp. 692-699 ◽  
Author(s):  
Francis L. Macrina ◽  
Patricia H. Wood ◽  
Kevin R. Jones
Keyword(s):  
Genetic Transformation ◽  
Streptococcus Mutans ◽  
Restriction Enzyme ◽  
Deoxyribonucleic Acid ◽  
Cryptic Plasmid ◽  
Common Ancestry ◽  
Erythromycin Resistance ◽  
Streptococcus Sanguis ◽  
Cryptic Plasmids ◽  
Isogenic Strains

By using the basic methodology initially published by Kretschmer et al. (J. Bacteriol. 124 :225-231, 1975), we have been able to introduce phenotypically cryptic plasmids from Streptococcus ferus (formerly Streptococcus mutans subsp. ferus ) into Streptococcus sanguis by genetic transformation. In this system, the entry of the cryptic plasmids is selected indirectly. This is effected with transforming deoxyribonucleic acid mixtures in which the cryptic plasmid deoxyribonucleic acid is present in an approximate 10-fold molar excess with respect to a plasmid (pVA1) known to confer erythromycin resistance. Under such conditions, 5 to 10% of the pVA1-containing erythromycin-resistant transformants were cotransformed with cryptic plasmid deoxyribonucleic acid. pVA1 may be selectively eliminated by growth of its S. sanguis host strain at 42°C, enabling the construction of isogenic strains with and without S. ferus cryptic plasmids. Comparative physiological studies of such strains have failed to reveal any plasmid-conferred phenotypes in S. sanguis. With this procedure, we have been able to physically separate two small cryptic plasmids (2.4 × 10 6 and 2.8 × 10 6 daltons) of S. ferus. Although these plasmids were found naturally to exist in a single S. ferus host, they were able to replicate independently of one another in S. sanguis. Restriction enzyme fingerprinting indicated that these plasmids did not share a common ancestry.

scholarly journals A Simple, Rapid Method for Demonstrating Bacterial Flagella

2000 ◽  
Vol 66 (8) ◽  
pp. 3632-3636 ◽  
Author(s):  
Hans-Peter Grossart ◽  
Grieg F. Steward ◽  
Josefina Martinez ◽  
Farooq Azam
Keyword(s):  
Rapid Method ◽  
Fluorescent Protein ◽  
Molecular Probes ◽  
Epifluorescence Microscopy ◽  
Image Capture ◽  
Transmission Electron ◽  
Pure Cultures ◽  
The Difference ◽  
General Protein ◽  
A Charge

ABSTRACT We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.

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