APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1992, p. 713-716 Vol. 58, No.

2
0099-2240/92/020713-04$02.00/0
Copyright © 1992, American Society for Microbiology

Detection of Pediococcus spp. in Brewing Yeast

by a Rapid Immunoassay
MICHAEL WHITING,1 MICHAEL CRICHLOW,2 W. M. INGLEDEW 2
AND BARRY ZIOLA1*
ImmunologylVirology Research Group, Department of Microbiology, College of Medicine,' and
Department of Applied Microbiology and Food Science, College of Agriculture,2
University of Saskatchewan, Saskatoon, Saskatchewan, Canada 57N OWO
Received 30 September 1991/Accepted 29 November 1991

A membrane immunofluorescent-antibody test was developed to detect diacetyl-producing Pediococcus

contaminants in brewery pitching yeast (yeast [Saccharomyces cerevisiae] slurry collected for reinoculation).
Centrifugations at 11 and 5,100 x g separate yeast cells from bacteria and concentrate the bacteria,
respectively. Pelieted bacteria resuspended and trapped on a black membrane filter are reacted with
monoclonal antibodies specific for cell surface antigens and then with fluorescein-conjugated indicator
antibodies. Whether pitching yeast is contaminated with pediococci at 0.001% is determined in <4 h. The
sensitivity of the assay is 2 orders of magnitude below the Pediococcus detection limit of direct microscopy.

In brewing, microbial contaminants cause product spoil-
age through formation of metabolic end products which
disturb the delicate balance between unused flavor-active
wort components and metabolic products produced by Sac-
charomyces cerevisiae (8). Lactobacillus and Pediococcus
spp. are the most common spoilage bacteria found in wort,
beer, and pitching yeast (12). Pediococcus spp. are generally
considered to be the most undesirable contaminant (12, 14),
with Pediococcus damnosus being the species responsible
for 90% of all Pediococcus-induced beer spoilage (11). P.
damnosus causes lager beer spoilage by producing diacetyl
at concentrations above the taste threshold of 0.12 mg/liter
(8). As few as 20,000 pediococci per ml in fermenting beer
can produce a diacetyl concentration of 0.36 mg/liter (10).
Pediococci of brewing origin are insensitive to the bacte-
riostatic effect of hop resins and tolerate the range of pH and
alcohol common in worts and beers (12). Pediococci are also
difficult to eradicate from contaminated brewery equipment,
since commonly used sanitizers are not totally effective (17).
Hence, pediococci exist throughout fermentation and stor-
age and they can be found in previously infected areas of the
brewery, in beer, and in pitching yeast.
Because yeast recycling is universally practiced in brew-
eries, pitching yeast can become a recurrent source of
contamination. Detection of spoilage organisms in yeast
prior to pitching is therefore required to ensure that only
yeast of high quality is used (7, 15). Traditional methods of
microbial monitoring include plating on nutrient media,
forcing tests, and direct microscopic examination. Plating
techniques and forcing tests require 5 to 14 days before
sufficient growth occurs (7, 15). Direct microscopic exami-
nation methods are rapid and convenient but can detect only
gross levels of contamination; i.e., approximately 4 x 105
bacteria per ml of a yeast slurry of approximately 4 x 108
yeast cells per ml (3). This means that brewers have been
forced to consider a 0.1% pediococcal contamination level
acceptable in pitching yeast destined for reuse. These slow
or insensitive methods are unsuitable for process control. A
combination of simple methods allowing 0.001% pediococcal
*
Corresponding author.
713
contamination of pitching yeast to be detected within 4 h is
described below.
Yeast. Commercial lager yeast slurries were obtained from
a local brewery and stored at 4°C. Viability was monitored
by methylene blue dye exclusion (1). Bacterial contamina-
tion of the yeast was not detectable prior to addition of
laboratory-cultured pediococci.
Bacteria. Pediococcus strain BSO 77, described by Dolezil
and Kirsop (4), was obtained from the Brewing Research
Foundation (Nutfield, United Kingdom). Bacteria were
propagated in deMan-Rogosa-Sharpe medium. Cultures
were grown at 27°C in a CO2 incubator following two cycles
of evacuation to 100 kPa followed by refilling with CO2
(99.8% minimum purity). The turbidity of broth cultures was
measured with a Klett-Summerson colorimeter with a no. 66
red filter. Stationary-phase cells were used as the inoculum.
Cell walls prepared from bacteria washed with phosphate-
buffered saline (PBS; pH 7.4) were used to immunize mice
employed in production of hybridomas secreting bacterial
surface-reactive monoclonal antibodies (MAbs). Cells were
first sonicated with a 19-mm probe on a Braun-Sonic 1510
sonicator at 20 kHz and approximately 375 W of peak power
(iced samples were given five 2-min ultrasonic bursts with
intervening 2-min cooling periods). This was followed by five
cycles of pressure shearing in a French pressure cell oper-
ated at 69,000 kPa. The press cylinder and piston were
precooled to 4°C, and the product was collected in a tube
immersed in ice. The disrupted cells were centrifuged for 20
min at 14,500 x g to collect the cell wall fraction. Protein
concentration was determined by using the Bio-Rad protein
microassay with bovine serum albumin as the standard.
MAbs. BALB/c mice were immunized intraperitoneally
with 50 ,ug of protein of BSO 77 cell wall antigen emulsified
in Freund's incomplete adjuvant. The emulsion was dis-
persed with an equal volume of 2% (vol/vol) Tween 80 in
PBS just prior to injection (6). At 2 weeks later, the mice
were boosted intravenously with 2 x 106 whole Pediococcus
strain BSO 77 cells in PBS. Mouse spleens were recovered
after 4 days, and MAb-producing hybridomas were estab-
lished as described by Qualtiere et al. (13), except that FO
myeloma cells, alpha calf serum (Hyclone Laboratories),
and polyethylene glycol 1500 (Boehringer Mannheim) were
714 NOTES APPL. ENVIRON. MICROBIOL.

TABLE 1. Recovery of pediococci and yeast cells in the supernatant after centrifugation at 11 x g for 10 min'
Sample Pediococcus strain BSO 77 CFU/mlb Yeast (CFU/ml)b Lossd
vol (ml) Start Finish Recovery' Start Finish (%)
5 2.69 x 103 1.95 x 103 73 4.26 x 107 9.03 x 103 99.98
2.5 3.60 x 103 2.67 x 103 74 4.90 x 107 1.23 x 104 99.97
1 4.01 x 103 3.05 x 103 76 NDe
5 2.13 x 104 1.41 x 104 66 3.79 x 107 4.80 x 103 99.99
2.5 2.23 x 104 1.78 x 104 80 4.61 x 107 1.67 x 104 99.96
1 3.22 x 104 2.91 x 104 90 5.29 x 107 1.47 x 104 99.97
1 3.29 x 104 2.31 x 104 70 ND
5 2.46 x 105 2.03 x 105 83 4.38 x 107 8.27 x 103 99.98
1 3.31 x 105 2.01 x 105 61 ND
1 1.52 x 106 1.04 x 106 68 3.21 x 107 1.19 X 104 99.96
1 3.00 x 106 2.01 x 106 67 4.47 x 107 8.23 x 103 99.98
1 3.72 x 106 2.92 x 106 78 5.94 x 107 2.46 x 104 99.96
1 2.68 x 107 2.08 x 107 78 7.84 x 107 3.98 x 104 99.95
a A 9-ml volume of experimentally contaminated pitching yeast was centrifuged, and the indicated sample was recovered from the top of the supernatant.
b Means of triplicate plates.
The overall average recovery of pediococci was 74.2%.
d The overall average loss of yeast cells was 99.97%.
e ND, not done.

used. Hybridomas secreting a MAb able to bind to the
pediococcal cell surface were selected by a bacterial surface
immunofluorescent-antibody test using a GIBCO BRL
Screenfast apparatus (18). The surface of each well was
precoated with poly-L-lysine (molecular weight, 141,000; 3
,ug/100 pul of PBS; Sigma Chemical Co.) to ensure strong
adsorption of the pediococci. A mixture of 21 MAbs reacting
with the surface of Pediococcus strain BSO 77 was used
(equal volumes of tissue culture fluid from all of the hybrid-
oma cell lines were pooled). The epitope specificities of
these MAbs were not determined.
Contamination. Concentrated yeast slurry containing ap-
proximately 5 x 108 yeast CFU/ml was experimentally
contaminated with various numbers of Pediococcus strain
BSO 77 suspended in sterile 0.1% Bacto Peptone (Difco
Laboratories). The contaminated yeast samples were then
diluted 1:10 with sterile 0.1% Bacto Peptone.
Enumeration. Viable counts of yeast and bacteria were
determined by filtration through 0.45-p.m-pore-size, 47-mm-
diameter GN-6 membrane filters (Gelman Sciences Inc.),
and the filters were incubated on wort agar for enumeration
of yeast cells or on deMan-Rogosa-Sharpe agar with 10 mg of
cycloheximide per ml added (9) for enumeration of pedio-
cocci. Counts were done in triplicate by using dilutions that
gave 20 to 200 colonies per filter. Incubations of 2 to 2.5 days
for yeast and 5 days for the Pediococcus strain used gave
colonies large enough to count.
Separation of bacteria and yeast by centrifugation. A 9-ml
volume of 1:10-diluted experimentally contaminated pitching
yeast was placed in a 12-ml conical glass centrifuge tube.
After vortexing to ensure homogeneity, each sample was
centrifuged in an IEC CL centrifuge (International Equip-
ment Company) at 11 x g (850 rpm) for 10 min. The top 5-ml
portions of the samples were pooled and centrifuged at 5,100
x g (5,000 rpm; IEC CS centrifuge) for 20 min to pellet the
bacteria. The supernatant was decanted, and the bacteria
were suspended in 0.6 ml of sterile PBS.
Membrane immunofluorescent-antibody test (MIFAT). The
MIFAT used was an adaptation of that described by Gares
(5). All manipulations were done at room temperature.
Black, grid-equipped 13-mm-diameter, 0.45-p.m-pore-size
Millipore cellulose acetate-nitrate filters were soaked in PBS
containing 1% bovine serum albumin and 10% calf serum.
The filters were then placed in Swinnex 13-mm-diameter
filter holders (Millipore), mounted via a needle and rubber
stopper on a manifold, and washed under vacuum (up to 85
kPa) with 5 ml of PBS. Control samples (bacteria diluted in
supernatant from 1:10-diluted pitching yeast centrifuged at
5,100 x g) and test samples were added to individual filters.
After the sample fluid was drawn through, each filter was
washed under vacuum with 5 ml of PBS. The vacuum was
released, and 200 ,ul of the bacterial surface-reactive MAb
pool was added to each filter. After 1 h, the filters were
washed under vacuum with 5 ml of PBS containing 10% calf
serum. Fluorescein-conjugated Affinipure goat anti-mouse
immunoglobulin G+M (heavy and light chains) antibodies
(Jackson Immunoresearch Laboratories Inc.), diluted 1:100
in PBS containing 2% bovine serum albumin, were prefil-
tered through a 0.45-pum-pore-size membrane filter, and 200
pul was added to each filter unit. After 1 h, vacuum was
applied and the filters were washed with 5 ml of PBS
containing 10% calf serum. The filters were dried under
vacuum, transferred to a glass slide, and overlaid with
glycerol-PBS (1:1). After a coverslip was placed over the
filter, the presence of pediococci with surface-bound MAb
was determined by fluorescence microscopy at x 1,000 mag-
nification under oil using a Nikon Labophot Biological
Microscope.
In preliminary experiments, 1:10-diluted contaminated
pitching yeast was centrifuged at 3, 11, and 23 x g for 10 min
and the pediococci and yeast cells remaining in the top 1 ml
of the sample were enumerated. Starting with approximately
106 pediococci per ml, bacterial recovery was about 60%
after centrifugation at 23 x g but 10 to 20% higher following
centrifugation at 3 or 11 x g. Yeast removal from samples
initially containing some 5 x 107 yeast cells per ml was
99.80, 99.96, and 99.99% after centrifugation at 3, 11, and 23
x g, respectively. On the basis of these findings, centrifu-
gation at 11 x g was selected for use in further experiments.
After centrifugation at 11 x g for 10 min, bacterial
recovery and yeast removal in the top 1, 2.5, and 5 ml of
supernatant were determined for yeast samples contam-
inated with bacteria at levels ranging from approximately 103
to 107 CFU/ml (Table 1). Regardless of the supernatant
VOL. 58, 1992 NOTES 715

TABLE 2. MIFAT of pediococci in experimentally

contaminated pitching yeast

Starting Recovery Avg count/ Ratioc

Sample
Sample
aFUng
FU/mlCFU/filtera
CFU/filtera Reovr
(%) field (no.
~~~~~~~of
fields)'
Test 3.44 x 104 2.84 x 105 82.6 47.5 (30) 1.7
Controld 2.83 x 104 1.94 x 105 68.6 45.5 (30) 2.3
Test 3.94 x 103 3.09 x 104 78.4 8.0 (50) 2.6
Control 4.40 x 103 3.46 x 104 78.6 9.9 (50) 2.9
Test 5.40 x 102 4.16 x 103 77.0 0.% (50) 2.3
Control 4.16 x 102 2.87 x 103 68.9 1.12 (50) 3.9
a
A 10-ml volume was filtered.
b Field area = 0.015 mm2 at x 1,000 magnification.
c (Average count per field)/(CFU per filter) x 1O-4.
d MIFAT of pediococci in supernatant of 1:10-diluted pitching yeast clari-
fied at 5,100 x g.

volume taken at a given infection level, there was little

difference in the number of bacteria recovered or the number
of yeast cells removed per milliliter. Likewise, regardless of
the numbers of pediococci added, only small differences
were seen in the percentages of bacteria recovered. Overall,
centrifugation at 11 x g gave a supernatant which retained
approximately 75% of the input bacteria but only 0.03% of
the input yeast cells.
Filtration of supernatant from the centrifugation at 11 x g
through a 13-mm-diameter, 0.45-,um-pore-size filter was not
successful. The initial 1 ml filtered, but the flow rate then
decreased rapidly. As particulate matter smaller than bacte-
ria likely contributed to progressive blockage of the filter, we
tested whether pelleting the bacteria at 5,100 x g, with
resuspension in a small volume of PBS, would allow filtra-
tion of 10 ml of supernatant clarified at 11 x g. This approach
was successful, meaning that a MIFAT could be immedi-
ately run to detect the trapped bacteria. As shown in Table
2, pediococci at low levels in pitching yeast and control
samples were readily enumerated by fluorescence micros-
copy. Bacteria trapped on the filter occurred as singles,
dyads, tetrads, and occasionally as multicellular clumps.
Each of these was counted as a single colony to correlate
with enumeration data. The appearance of a dyad against a
background of residual nonfluorescing yeast cells and me-
dium debris is shown in Fig. 1.
We have shown that pediococci and yeast cells can be
effectively separated by 10 min of centrifugation at 11 x g
(Table 1). To our knowledge, only one other investigator has
used low-speed centrifugation to separate bacteria and yeast
cells in samples of contaminated pitching yeast. Shimwell
(16) used this approach so that bacteria could be Gram
stained. However, Shimwell did not detail the centrifugal
force used nor the duration of centrifugation required to
achieve optimal separation of bacteria and yeast cells.
Ideal differential centrifugation would remove all of the
yeast cells and leave all of the bacteria behind. Although the
low-speed centrifugations tested should not pellet pedio-
cocci, we observed that recovery of bacteria from experi-
mentally contaminated pitching yeast samples was always
less than 100% (Table 1). This loss of bacteria is probably
due to simple entrapment of bacteria among the much larger,
rapidly sedimenting yeast cells. Because of this, it is impor-
tant to use a reasonable working dilution of the initial
concentrated pitching yeast slurry. As shown here, a 1:10
dilution giving approximately 4 x 107 yeast cells per ml
resulted in good recovery of bacteria following centrifuga-
tion at 11 x g.
FIG. 1. Pediococci in pitching yeast supernatant detected by a
MIFAT. The sample contained approximately 5 x 107 yeast cells
per ml and 5.40 x 10' pediococci per ml. The cells of the centrally
located Pediococcus dyad each have a diameter of approximately
0.6 ,um.
An important aspect of the present protocol is the second
centrifugation at 5,100 x g to concentrate the pediococci in
the yeast-depleted supernatant. Incorporating this step al-
lowed more of the 11 x g supernatant to be handled per
filter, effectively increasing the sensitivity of the MIFAT. It
is likely that the second centrifugation left in the supernatant
some particulate material smaller than bacteria, which con-
tributed to filter blockage. In any event, the smaller fluid
volume (i.e., 1 versus 10 ml) was rapidly filtered. That the
washing steps effectively removed unbound MAbs and un-
bound fluoresceinated indicator antibody is shown by the
clean background in Fig. 1.
Also seen in Fig. 1 are the unstained ghostlike shapes of
residual yeast cells and particulate matter. Given the com-
paratively smaller diameter of the pediococci, it might be
suspected that some bacteria would be hidden from view.
That this occurs to a limited extent is shown by the ratio
analysis in Table 2. At each level of experimental contami-
nation shown, the ratio for the test sample containing yeast
cells and pediococci was less than the ratio for the control
sample. However, comparison of ratios for the test and
control samples reveals that this effect does not result in a
major loss of MIFAT sensitivity. From Table 2 and other
data (not shown), it was found that between 60 and 80% of
the pediococci trapped on the filters were visible.
At high contamination levels (>105 bacteria per ml),
accurate enumeration of pediococci was not possible be-
cause of decaying fluorescence, making it difficult to distin-
guish bacteria already counted. At lower contamination
levels (<104 bacteria per ml; Table 2), the number of bacteria
present per field could be accurately determined before the
fluorescence faded. While we have shown (Table 2) that
approximately 500 pediococci per ml in the original yeast
sample resulted in one bacterium per field, it must be
emphasized that each field at x 1,000 magnification repre-
sents only approximately 0.02% of the total filter area
involved in trapping bacteria. This means that by scanning
the filter, contamination levels of pediococci down to 10
bacteria per ml can be readily detected.
716 NOTES
Although manual inspection of each filter can be done
within a few minutes, it is likely that enumeration of bacteria
trapped on 13-mm-diameter filters could be done automati-
cally with a scanning fluorescence microscope equipped for
computer-enhanced imaging (2). This should considerably
enhance the sensitivity of the MIFAT method we have
developed. However, whether this is actually necessary in a
brewery operation is questionable, since one pediococcus
per field at x 1,000 magnification (Table 2) means that
pediococcal contamination of pitching yeast at a level of
0.001% has already been detected. This detection level for
pediococci is approximately 2 orders of magnitude lower
than that obtainable by direct microscopic examination and
at least 1 order of magnitude below the lowest pediococcal
contamination level known to cause off flavor in beer (12).
Consequently, even with manual inspection at the end of the
MIFAT, we believe that the method described here could be
routinely used in brewery quality control to avoid reuse of a
pitching yeast slurry that is contaminated with a level of
pediococci which, if viable, could cause beer spoilage.
This research was supported by an operating grant from the
University of Saskatchewan President's NSERC (National Science
and Engineering Research Council) Fund and NSERC operating
grant 6478. M. Whiting was the recipient of a University of
Saskatchewan Graduate Student Scholarship, an Arthur Smyth
Memorial Scholarship, and a Martin Pedersen Postgraduate Schol-
arship. M. Crichlow was the recipient of an NSERC Student
Research Award (University and Industry) and a Challenge '91
Employment Canada grant.
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