An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Summary

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Abstract

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

Introduction

The ubiquity of Arthrobacter species in soil environments offers a vast number and diversity of phages capable of being isolated from this species of host bacteria. Bacterial members of the Acintobacteriaceae family are most notable for their catabolic pathways of degrading recalcitrant compounds like atrazine and various other pesticides and herbicides^1,2,3. Though most research has been done using environmental strains of Arthrobacter, clinical isolates of this genus is found in blood, urine, eyes, and many other human sources all displaying phylogenetic heterogeneity⁴.

While there is a rat....

Protocol

1. Preparation of Arthobacter Cells for Phage Isolation

• Culture Arthrobactersp. KY3901 colonies streaked on an Luria Bertaini (LB) agar plate incubated at 30 °C for 2-3 days. Pick a colony and use a sterile loop to add it to 250 ml of LB broth in a baffled culture flask and incubate in a shaking incubator at 225 rpm at 30 °C.
• Allow approximately 24 hr of growth to obtain late exponential/early stationary phase cells for phage infection experiments. Monitor the state of bacterial growth closely to prevent cells from entering the middle to late stationary growth state.
  NOTE: Cell growth should consist of ....

Results

To demonstrate reproducibility of the improved enrichment technique for Arthrobacter phages, 30 different soil samples were used at different times and locations during the spring and summer of 2014. Of these 30 soil samples unique Arthrobacter phages were obtained from 22 of collected soil samples using this enrichment procedure. The standard enrichment procedure yielded unique phages from 3 of the same soil samples. The enrichment samples can have a very high phage titer needing initial dilution to is.......

Discussion

Despite many previous attempts to isolate phages capable of infecting Arthrobacter hosts, we had little success using standard enrichment procedures. The generalized method of bacterial enrichment developed and adapted by van Twest and Kropinski¹⁰ to enrich phages from environmental samples remains the basis for the majority of enrichment procedures. Evidence from previous studies suggests that methods of direct plating have produced detectable plaques on strains of Arthrobacter albeit with v.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB Broth powder	Fisher	BP9722-2	It's best to order these in bulk.
Granulated Agar	Fisher	BP1423-2	It's best to order these in bulk.
0.22 um syringe filters	Fisher	09-719A
0.22 um buchner filters	Fisher	430320	More than 50 ml of liquid can be obtained by carefully swapping the receiving tube.
Eppendorf Tubes	Fisher	05-408-129
5 ml pipets individual	Fisher	13-678-11D
50 ml conical tubes	Fisher	76002844
15 ml conical tubes	Fisher	76002845
10 ml pipets individual	Fisher	13-676-10J
25 ml pipets individual	Fisher	13-676-10K
Whatman qualitative filter paper	Fisher	1001-824