DIAGNMICROBIOLINFECTDIS 121

1988;11:121-123

BACTERIOLOGY

Rapid Method for the Isolation of Bacteriophages

from Lysogens

Joseph T. Parisi and Li Meng

A rapid method for the isolation of bacteriophages from lysogens is described. Phages are
induced in broth medium containing mitomycin C, which is then replicated onto agar medium.
Molten medium containing indicator strains is then poured in these plates. Bacterial lysis is
subsequently detected with tetrazolium-containing broth.

Bacteriophage typing for the identification of specific strains of bacteria implicated

in nosocomial infections or other outbreaks still remains a useful tool. Bacteriophages
are also commonly isolated for use in genetics studies. In 1974 we reported on an
improved rapid plate method for the isolation of phages from lysogenic bacteria
(Parisi and Talbot, 1974). Our method was an improvement over that reported by
Siddiqui et al. (1974). Briefly, our method consisted in inoculating 36 cultures of
Staphylococcus epidermidis into trypticase soy broth containing 0.3% yeast extract
(YETS broth) and 400 ~g of CaC12 per ml and incubating these at 37°C for 6-8 hr.
These cultures were then inoculated with a loop onto 36 designated spots on a plate
containing YETS broth and 1.5% agar and 0.1 p,g of mitomycin C (MC; Sigma Chemical
Co., St. Louis, MO) per ml. The YETS broth cultures and YETS-MC agar plates were
then incubated overnight at 37°C. The following day, replicas of the YETS-MC agar
plate were made with sterile velveteen onto 36 YETS agar plates. A velveteen template
could be used to make, at most, eight replica plates before smearing obscured the
spatial relationship of the 36 spots. Replicated plates were then incubated at 37°C
for 6-8 hr. Then, 7 ml of molten YETS broth containing 0.8% agar were inoculated
with 0.1 ml from each of the 36 potential indicator strains growing in the YETS broth
and were poured over the surfaces of the replicated (indicator) plates. After incubation
overnight at 35°C, indicator plates were flooded with 10 ml of YETS broth containing
0.1% 2, 3, 5-triphenyltetrazolim chloride (TTC, Difco Laboratories, Detroit, MI) ac-
cording to the procedure of Pattee (1966) except that the broth was poured off after
incubation for I hr at 37°C. Bacterial lysis due to phage activity is detected as a clear
area against a red background produced by the reduction of TTC by bacterial growth.
In this phage isolation method, each culture served both as an indicator strain and
also as a possible source of phages induced from that strain by the MC.
In our modification of this method, we have eliminated the induction of phages
from lysogens on the YETS-MC agar plate since the titer of phages produced is not
as great as that produced in liquid medium, and hence, phage activity may not be

From the University of Missouri-Columbia, School of Medicine, Columbia, Missouri.

Address reprint requests to: Joseph T. Parisi, Department of Microbiology, University of
Missouri-Columbia, School of Medicine, Columbia, MO 65212.
Received August 5, 1988; revised and accepted October 3, 1988.

© 1988 Elsevier Science Publishing Co., Inc. 0732-8893/88/$3.50

655 Avenue of the Americas, New York, NY 10010
122 J.T. Parisi and L. Meng

FIGURE 1. Phage activity in positions 6, 9, 15, 20, 25, 29, 30, and 31 (beginning at the top,
counting left to right).

as readily seen. We also have e l i m i n a t e d the use of the velveteen replicas in w h i c h

smearing becomes a p r o b l e m as more replicas are m a d e and obscures the detection
of lysis if present. Instead, we e m p l o y a Steers i n o c u l u m replicating device (Steers
et al., 1959) that contains an inoculating head of 32 rods and a stainless steel plate
containing 32 reservoirs. Thirty-two cultures of S. epidermidis, w h i c h will subse-
quently serve both as indicator strains and as sources of phages, are inoculated into
YETS broth and incubated at 37°C for 6 - 8 hr. In the meantime, 0.5 ml of YETS broth
containing 0.1% MC per ml is p i p e t t e d into each of the 32 reservoirs in the steel
plate. These are then i n o c u l a t e d with a loop from each of the 32 incubating indicator
strains. Both the 32 cultures and the 32-holed steel plate, w h i c h is placed in a sterile
petri dish to prevent c o n t a m i n a t i o n and m i n i m i z e evaporation of the YETS-MC broth,
are i n c u b a t e d at 37°C overnight. In the morning, the Steers replicating device w i t h
the 32 inoculating rods d i p p e d into the YETS-MC broth containing the i n d u c e d
phages is used to inoculate 32 p r e v i o u s l y dried (48 hr at 37°C) YETS agar plates
containing 400 ~.g of CaC12 per ml of m e d i u m . The inoculating rods are d i p p e d into
the 32 reservoirs for each i n o c u l a t i o n of a YETS agar plate, thus insuring that a
uniform-sized drop from each reservoir is d e p o s i t e d on each of the 32 YETS agar
plates. If more than 32 indicator strains are available, a d d i t i o n a l YETS agar plates
could also be inoculated with the i n d u c e d phage-YETS-MC broth mixture since the
Notes 123

inoculating rods deliver from 0.003-0.005 ml, depending upon their size, and the
reservoirs may contain 0.5-1.0-ml volumes of medium. The replicated plates are then
incubated at 37°C for 6-8 hr so that the transferred inocula will adhere more firmly
to the YETS agar plates. Since the titer of phages produced in YETS-MC broth is
very high, these replicated plates can be incubated at room temperature overnight
or longer so as to minimize the spreading of phages in the next step. Then, 7 ml of
molten YETS broth containing 0.8 ml agar are inoculated with 0.1 ml from each of
the 32 potential indicator strains and poured over the surface of the replicated (in-
dicator) plates. After incubation overnight at 37°C, indicator plates are flooded with
10 ml of YETS broth containing 0.1% TTC. After incubation for 1 hr at 37°C, the
broth is poured off and the plates are observed for bacterial lysis. Application of the
phages with the inoculating rods results in a more discrete area of lysis than with
velveteen (Fig. 1). Lyric areas can be picked with a needle to serve as a source of
phage for that indicator strain that will, subsequently, be its propagating strain, or
phages can be obtained from the appropriate reservoir in the 32-holed steel plate.
However, this method of induction of prophages is so efficient that sometimes it is
difficult to tell precisely from which well the phages came (see position 25, Fig. 1).
Although we used a Steers inoculum replicating device in this phage isolation
method, one could also use a microtiter plate for the induction of phages in YETS-
MC broth and an 8-12 multichannel pipette for the transfer of induced phages to
indicator plates. One could also use a handheld inoculator similar to the one we
described recently (Parisi and Hamory, 1986) to transfer the induced phages. This
method of isolation of phages from lysogens is rapid (2 days), efficient, sensitive,
and could be used for the isolation of phages from lysogens of other bacterial species.

REFERENCES
Parisi JT, Hamory BH (1986) Simplified method for the isolation, identification, and charac-
terization of Staphylococcus epidermidis in epidemiologic studies. Diag Microbiol Infect
Dis 4:29.
Parisi JT, Talbot Jr HW (1974) Improved rapid plate method for the isolation of bacteriophages
from lysogenic bacteria. Appl Microbiol 28:503.
Pattee PA (1966) Use of tetrazolium for improved resolution of bacteriophage plaques. J Bacteriol
92:787.
Siddiqui A, Hart E, Mandagere U, Goldberg ID (1974) Rapid plate method for the isolation of
lysogenic bacteriophages. Appl Microbiol 27:278.
Steers E, Foltz EL, Graves BS (1959) An inocula replicating apparatus for routine testing of
bacterial susceptibility to antibiotics. Antibiot Chemother 9:307