Microbiological Analyses of
Dry and Slurry Yeasts for Brewing
Marisa Manzano 1,2, Cristina Giusto 1, Ingrid Bartolomeoli 1,
Stefano Buiatti 1 and Giuseppe Comi 1
ABSTRACT
J. Inst. Brew. 111(2), 203–208, 2005
Classical microbiological methods in association with molecular
methods (DNA amplification, Temperature Gradient Gel Electro-
phoresis (TGGE) and Denaturing Gradient Gel Electrophoresis
(DGGE) were used. These methods, developed to rapidly ana-
lyze microbial communities on the basis of sequence-specific
separation of DNA amplicons, allowed the detection of DNA
differences in the amplicons tested and the identification of the
strains analyzed by the comparison of unknown sequences with
sequences of known species. TGGE allowed the comparison of
the different Saccharomyces cerevisiae strains used in brewing
while DGGE allowed the identification of lactic acid bacteria
(LAB) in beer. These methods are a reliable tool for fast com-
parison of strains of Saccharomyces cerevisiae collected from
different craft breweries where they were used as starters to
check the presence of possible yeast contaminants in the brew-
ing process and for rapid LAB identification.
Key words: Beer, lactic acid bacteria, PCR-DGGE, PCR-TGGE,
Saccharomyces cerevisiae.
INTRODUCTION
At present beer is the most common beverage in the
world after tea, soft drinks and milk 1. Beer production has
become more reproducible and product quality more con-
sistent in the last years and the use of yeast starters has
been essential in beer production since 1883 4. Saccharo-
myces cerevisiae is the prevalent yeast species involved in
brewing and it influences the quality and the quantity of
the secondary products formed during the alcoholic fer-
mentation. Different yeast strains will produce different
levels of esters and higher alcohols under similar condi-
tions and these compounds will directly influence beer
flavour. Some yeasts appear to be more vigorous in com-
peting for nutrients than others and therefore leave less
highly assimilable nitrogen and fermentable residue in
beer. Such beer may be less susceptible to microbiological
contamination 11. Beer is a poor and rather hostile environ-
ment for most microorganisms: its ethanol concentration
is usually around 4–5% v/v and its pH ranges from 3.8 to
1 Department of Food Science, via Marangoni 97, 33100 Udine, Italy.
2 Corresponding author. E-mail: marisa.manzano@uniud.it
Publication no. G-2005-0831-285
© 2005 The Institute of Brewing & Distilling
4.7. Only a few bacteria are able to grow under such in-
hospitable conditions and are able to spoil the beer 13. To
prevent microbial contamination during the production
process, the microbiological purity of brewing yeast start-
ers is a necessary condition to maintain high product qual-
ity. Many strains are available on the market and their
characterization is necessary for quality control in dry
yeast production 7. The contamination of production strains
with wild yeasts (non-Saccharomyces or Saccharomyces)
and bacteria may contribute negatively or positively to
beer properties and characteristics. Monitoring of micro-
bial contaminants during the brewing process is important
to obtain reproducible and high-quality beers 16. The plate
count method for enumerating microbiological contamina-
tion has remained unchanged for over a century, but it
requires several days before the microorganisms are iden-
tified after plating 17. Consequently, the products are often
already released for sale before the microbiological re-
sults become available. Polymerase Chain Reaction (PCR)
assays have emerged for the specific detection of micro-
bial contaminant in beer and primers for the most com-
mon spoilage organisms, especially for lactic acid bacteria
have been designed 6. The PCR technique is a fast and
more specific method to detect bacteria in beer compared
to classical microbiological methods which are less spe-
cific and more time consuming. Also real time PCR was
used to improve sensitivity in bacteria detection from beer
and to date this technique still needs to be applied in rou-
tine brewery practice 15.
Restriction fragment length polymorphism (RFLP) pat-
terns of PCR-amplified DNA fragments and Temperature
Gradient Gel Electrophoresis (TGGE) of PCR products
were successfully used for the identification of yeasts iso-
lated from wine 9 and PCR-DGGE was described as useful
in identifying bacteria during sausage fermentation 3. The
aim of this work was to identify and quantify the presence
of wild yeasts and lactic acid bacteria contamination in
dry and slurry yeasts used in beer production, and to in-
vestigate intraspecific differentiation of Saccharomyces
cerevisiae strains by PCR-TGGE.
MATERIALS AND METHODS
Samples of dry and slurry yeasts used in three different
micro-breweries (named A, B and C) in the Friuli Venezia
Giulia region (Italy) were analyzed to evaluate the con-
tamination due to wild yeasts and lactic acid bacteria dur-
ing the brewing process. Two dry yeast samples, b1 (lager
VOL. 111, NO. 2, 2005 203
yeast) and b2 (same brand, different yeast strain, ale
yeast) obtained from the brewery coded B, were used for
one fermentation each, and analyzed before and after in-
oculation in the wort. Two samples of slurry yeasts (a and
c) obtained as “pitching yeast” (lager yeasts) harvested
from the bottom of a fermenter no more than 12 h prior to
sampling, and utilized respectively in the breweries coded
A and C were analyzed before and after their utilization in
the fermentation process. Yeast a was used for the follow-
ing eight fermentations (aF1, aF2, aF3, aF4, aF5, aF6, aF7
and aF8). Yeast c was used for the following three fermen-
tations (cF1, cF2, and cF3). Samples from the pitching
yeast and samples after the fermentations were subjected
to morphological, biochemical and molecular analyses.
Morphological assessment and viability
determination
One g of each of the samples a and b were transferred
to 9 mL of physiological solution (kept at 30°C for 1 h
prior to analysis) to obtain a 10–1 preliminary dilution. To
revitalize yeasts, the dilution was vortexed 4 times for 30 s
every 5 min. Dilutions (up to 10–8 ) were prepared. One
drop of the 10–3 dilution was put onto a Bürker camera
and observed using a light microscope Axiophot (Zeiss,
Milan, Italy) for morphological analyses. To assess the
dry yeast viability, a 1 mL vial of the 10–3 dilution was
thoroughly mixed with a 1 mL vial of Methylene Blue
staining solution by shaking for 30 sec and again after 10
min. The mixture was observed with a Bürker camera
using a light microscope (40×)12. Based on the results, the
viability of strains was calculated (the percentage of liv-
ing cells compared to their total number). Results were
reported as number of live cells /g dry weight. To stan-
dardize the analysis for the slurry yeasts, it was necessary
to dry an aliquot of yeast and determine the dry weight
using a thickener balance (Mettler PM 400, Switzerland).
Evaluation of the number of Saccharomyces
cerevisiae strains and wild yeasts
To evaluate the presence of S. cerevisiae strains, 0.1
mL of each dilution starting from 10–5 and higher was
surface-plated on WL agar medium (Oxoid, Milan, Italy).
Plates were incubated at 28°C for 5 d. After incubation the
number of colonies appearing 5 mm in diameter and from
cream-coloured to light green 2 was counted and, based on
the data, the total number of CFU /g was calculated 18. Cell
morphology was analyzed using a light microscope.
To detect and evaluate presence of non-Saccharomyces
wild yeast strains, lysine medium (Oxoid, Milan, Italy)
supplemented with 10% lactic acid (Oxoid, Milan, Italy)
was applied following the instructions of the manufac-
turer. Plates were incubated at 25°C and after 5 days the
number of colonies forming units (CFU) was determined.
Detection and evaluation of the number
of lactic acid bacteria (LAB)
To detect and evaluate the presence of LAB, 1 mL of
dilutions from 10–1 to 10–4 were transferred to duplicate
plates of MRS agar (Oxoid, Milan, Italy) supplemented
with the antifungal/antimould agent Delvocid ® Instant
(DSM Food Specialties, The Netherlands). Plates were in-
204 JOURNAL OF THE INSTITUTE OF BREWING
cubated at 30°C for 3 d under microaerophilic conditions.
After incubation the number of colonies was counted and
based on the data the total number of CFU/g was calcu-
lated.
Choosing yeast colonies for molecular analyses
Yeast colonies grown on both media, WL agar and Ly-
sine medium, were carefully screened and those which
were visually morphologically different were selected for
further analysis. They were purified onto YPD agar me-
dium (Oxoid, Milan, Italy) (72 h at 28°C) and pure col-
onies were stored in YPD broth (Oxoid, Milan, Italy)
supplemented with 30% glycerol (Sigma, Milan, Italy) at
–80°C until use. Bacteria were grown overnight at 30°C
on MRS agar (Oxoid). Three bacterial colonies of differ-
ent morphology were selected from the plate, observed
under the microscope and subjected to Gram staining and
catalase activity assay. Gram positive and catalase nega-
tive colonies were purified onto MRS agar plates and pure
colonies were transferred into MRS broth supplemented
with 30% glycerol and stored at –80°C until use.
Galactose fermentation
To differentiate S. cerevisiae strains the fermentation of
galactose test was performed. Substrate (7 mL) for fer-
mentation tests (peptone 5%, yeast extract 0.7%) was sup-
plemented with 2% galactose (Sigma, Milan, Italy) and
added to a tube containing a Durham tube. An inoculum
of 0.1 mL with each strain isolated for the molecular
analyses was prepared and tubes were incubated at 28°C
for 15 d. After this time the presence of gas bubbles in the
Durham tube indicated that the fermentation of galactose
occurred.
DNA extraction from yeast and bacteria
Yeast and bacteria control strains used in this study to
optimize the TGGE and DGGE protocols are reported in
Table I.
Table I. Reference strains used.
Yeasts Lactic acid bacteria
Saccharomyces cerevisiae Lactobacillus sakei DSM° 6333
ATCC* 36024 Lact. casei DSM° 20011
S. cerevisiae UCDVE $ 812 Lact. curvatus DSM° 20019
S. cerevisiae UCDVE $ 926 Lact. brevis DSM° 20054
S. bayanus DSM° 70412 Lact. plantarum DSM° 20174
S. bayanus DSM° 3774 Lact. hilgardii DSM° 20176
S. pastorianus DSM° 6580 Lact. paracasei
S. pastorianus DSM° 6581 Pediococcus pentosaceus
S. paradoxus DBVPG + 6411 DSM° 20336
Weissella kimkii
Kokuria kristinae DSM° 20032
Kokuria varians DSM° 20033
Leuconostoc cremoris DSM° 4196
Lactococcus lactis DSM° 2345
Oenococcus oeni DSM° 20252
Enterococcus hirae
*American Type Culture Collection, Manassas, Virginia, USA.
$ University of California Department of Viticolture and Enology, Davis,
USA.
°Deutsche Sammlung von Mikrorganismen und Zellkulturen, GmbH,
Braunschweig, Germany.
§ Department of Microbiology, Zagreb, Croatia.
+ Dipartimento di Biologia Vegetale, Perugia, Italia.
 Yeast pure colonies were cultured overnight at 30°C in
YPD medium and centrifuged at 6000 rpm for 10 min at
10°C. The pellet was washed with sterile, cold, deionised
water and subjected to the DNA extraction as described
by Manzano et al.9
Bacteria pure colonies were cultured overnight in MRS
broth and centrifuged at 6000 rpm for 10 min at 10°C.
The pellet was washed with sterile deionised water and
subjected to DNA extraction as described by Manzano et
al.8 To eliminate presence of RNAs, RNase (Sigma, Mi-
lan, Italy) was added to the DNA (incubation for 1 h at
37°C). Concentration of DNAs extracted from yeasts and
bacteria was measured using a SmartSpec TM 3000 spec-
trophotometer (Bio-Rad, Milan, Italy) and standardized at
~50 ng µL–1 before being used in the PCR assay. Samples
were stored at –20°C prior to subsequent analyses.
PCR amplification for a subsequent TGGE
analysis of yeast isolates
Primers Shaf + GC and Shar designed to amplify a
fragment of 207 bp between Internal Transcribed Spacers
(ITS) 1 and 2 were used: forward primer Schaf (5⬘-GTA-
GTGAGTGATACTCTT-3⬘) and reverse primer Schar
(5⬘-AGAACATGTTGCCTAGAC-3⬘) were designed and
annealing sites and expected PCR amplicon sizes were
checked with the “Amplify” (Madison, Wisconsin, USA)
program. Schaf primer anneals from 18 to 38 bp and Schar
primer from 210 to 229 bp giving a fragment length of
211 bp. In order to use the resulting amplicons for TGGE
a “GC-clamp” (5⬘-CGCCCGCCGCGCGCGCGGCGG-
GCGGGGCGGGGGCACCGCGCG-3⬘) was added to
the 5⬘ end of the Schaf primer 9.
PCR amplification was carried out in a 50 µL reaction
mixture according to the protocol proposed by Manzano
et al.9. A 5 µL aliquot of the product was separated elec-
trophoretically in a 2% agarose gel (Sigma, Milan, Italy)
stained with ethidium bromide (0.5 µg mL–1 ) in 0.5× TBE
buffer (0.045 M Tris borate, 0.001 M EDTA pH 8) and
compared with Molecular Weight Marker 100 bp (Sigma,
Milan, Italy).
TGGE analysis of yeasts
Yeast strains reported in Table I were used to optimize
the PCR-TGGE protocol used to differentiate different
strains obtained from beer fermentation processes.
The DCode Universal Mutation System (Bio-Rad Lab-
oratories, Richmond, USA) was used for the sequence
specific separation of the PCR products. Electrophoresis
was performed in a 0.8-mm polyacrylamide gel (8% w/v)
acrylamide-bisacrylamide (37:5:1) (Fluka, Germany) con-
taining 7 mol L–1 urea (Fluka). The gels were subjected to
a constant voltage of 120 V for 4.5 h from 56.5 to 60.5°C
with 0.8°C/h temperature ramp for yeasts. After electro-
phoresis gels were stained for 20 min in a 1.25× TAE
(0.04 M Tris acetate, 0.001 M EDTA pH 8) containing 1×
(final concentration) SYBR Green (Sigma, Milan, Italy),
visualized under UV light and filed using the GeneSnap
Syngene Software (Cambridge, UK).
PCR conditions for bacteria
Amplification was performed with a Peltier Thermal
Cycler, PTC 220 DNA engine Dyad (MJ Research, Wal-
tham, Massachusetts, USA) in a final volume of 50 µL
using the primers P1 5⬘-CGCGCGTGCCTAATACA-
TGC-3⬘ with a GC clamp and primer P2 5⬘-TTCCCC-
ACGGCTTACTCACC-3⬘ which targeted the V1 region
of the 16S rRNA according to the protocol proposed by
Cocolin et al.3 PCR fragments were analyzed by electro-
phoresis in a 2% agarose gel as reported above.
DGGE analysis of bacteria
The Dcode universal mutation detection System (Bio-
Rad Laboratories, Richmond, USA) was used for a
DGGE analysis of the PCR products obtained from single
colonies. Electrophoresis was performed in a 0.8-mm
polyacrylamide gel (8% (w/v) acrylamide-bisacrylamide
(37.5:1)) using the denaturant gradient from 40% to 60%
increasing in the direction of the electrophoresis. The gels
were subjected to a constant voltage of 130 V for 3.5 h at
60°C, and after electrophoresis they were stained as re-
ported for TGGE.
RESULTS AND DISCUSSION
Classical microbiological methods
The dry yeasts b1 and b2 showed a CFU /g of 2.8 ×
1010 and 5.5 × 109 respectively while the values obtained
for samples a and c analyzed before being inoculated in
the wort were respectively 5.0 × 107 and 5.3 × 109 CFU /g.
The viabilities of the b1 and b2 dry yeasts were 67% and
73% respectively. No non-Saccharomyces wild yeasts that
could grow on lysine media were present in the starters as
indicated by the absence of growth.
On the basis of the appearance of the typical S. cere-
visiae morphological characteristics, 44 S. cerevisiae
strains were picked up, purified and tested. Of the 44 iso-
lated and tested S. cerevisiae strains, 38 were able to fer-
ment galactose.
In spite of the same LAB contamination level shown
by two dry yeast starters (b1 and b2) before their utiliza-
Fig. 1. TGGE of colonies isolated from brewery B on WLN
media. Lane 1: b1F1.2; lane 2: b1F1.1; lane 3: b2F1.3; lane 4:
b2F1. 2; lane 5: b2F1.1; lane 6: b2.3; lane 7: b2.2; lane 8: b2.1;
lane 9: b1.2; lane 10: b1.1; lane 12: Saccharomyces cerevisiae
ATCC 36024.
VOL. 111, NO. 2, 2005 205
 A B
Fig. 2. A. TGGE samples isolated from brewery A; lane 1: Saccharomyces cerevisiae ATCC 36024; lane 3: Saccharo-
myces cerevisiae 926; lane 4: Saccharomyces cerevisiae 812; lane 5: a.1; lane 6: a.2; lane 7: a.3; lane 8: aF1.1; lane 9:
aF1.2; lane 10: aF2.1; lane 11: aF2. 2; lane 12: aF3.1; lane 13: aF3. 2; lane 14: aF4.1; lane 15: aF4.2; lane 16: aF4.3.
B. TGGE samples isolated from brewery C; lane 1: Saccharomyces cerevisiae ATCC 36024; lane 2: c.1; lane 3: c.2;
lane 4: c.3; lane 5: c.4; lane 6: yeast cF1.1; lane 7: cF1.2; lane 8: cF2.1; lane 9: cF2.2; lane 10: cF2.3; lane 11: cF3.1;
lane 12: yeast cF3.2.

Fig. 3. Standard LAB DGGE; primer P1/P2. Lane 1: Lb. plantarum DSM 20174; Lane 2: Lb. sakei DSM 6333; Lane 3:
Lb. curvatus DSM 20019; l Lane 4: Lb. brevis DSM 20054; Lane 5: Lb. casei DSM 20011; Lane 6: Lb. paracasei; Lane
7: Pediococcus parvulus DSM; Lane 8: Pediococcus pentosaceus DSM; Lane 9: Enterococcus hiirae; Lane 10: Oeno-
coccus oeni; Lane 11: Lb. hilgardii DSM 20176; Lane 12: Lc. lactis DSM 2345; Lanes 13–14: Kokuria kristinae DSM
20032; Lane 15: Kokuria varians DSM 20033; Lane 16: Leuconostoc cremoris DSM 4196; Lane 17: Weissella kimkii.

206 JOURNAL OF THE INSTITUTE OF BREWING

tion (9 × 10 CFU /g), different levels were observed after
the fermentations. The b1 reached 1 × 102 CFU /g while
b2 reached 2.7 × 106 CFU /g. Different hygienic practices
applied to the brewery process influenced the bacterial
charge of the wort. Brewery C showed the higher LAB
levels detected during the analyses, from 1.7–1.8 × 107
(samples cF1 and cF2) to 3 × 107 CFU /g (sample cF3). In
brewery A the values of the LAB were under 1 × 102
CFU /g for the slurry yeast before being used in the fer-
mentation process and from 1.3 × 102 to 3.3 × 102 after
the four fermentations (aF1, aF2, aF3 and aF4) while the
values increased from 3.5 × 104 to the maximum level
achieved by the sample aF7, 5.1 × 106, during the last
fermentations, from aF5 to aF8.
Molecular methods
The single DNA fragments obtained by PCR for two
colonies morphologically different (b1.1 and b1.2) iso-
lated from WL agar plating in the yeast b1 (brewery B),
migrated to the same position in TGGE (Figure 1 lane 10
and 9) suggesting the same sequence for both PCR prod-
ucts. The amplification products obtained by PCR for
three colonies (b2.1, b2.2, b2.3) isolated from WL agar on
the basis of the different morphologies exhibited by the
yeast b2 showed the same profile in TGGE (Figure 1 lanes
6, 7 and 8). The b1 amplicons (Figure 1 lanes 10 and 9)
migrated to different positions in the gel in respect with
the b2 amplicons (Figure 1 lanes 8, 7 and 6) allowing us
to differentiate the two yeasts employed as starters in beer
production. After the fermentation (F1) with the starter
b1, two colonies (b1F1.1 and b1F1.2) isolated from WL
agar were subjected to PCR-TGGE analyses. The ampli-
con obtained, b1F1.2 (Figure 1 lane 1), migrated to the
same level of the amplicon obtained by the starter (Figure
1 lanes 9 and 10), on the contrary the fragment b1F1.1
(Figure 1 lane 2) migrated to a different level indicating
that the two colonies isolated after fermentation (b1F1.2
and b1F1.1) belong to different strains. In the case of the
starter b2, the colonies isolated from WL agar (b2F1.1,
b2F1.2, b2F1.3), obtained by plating the sample after the
fermentation process, showed different TGGE profiles
(Figure 1 lanes 5, 4 and lane 3). Two PCR products ob-
tained from the samples b1F1.1 (yeast b1) and b2F1.3
(yeast b2) migrated to the same levels demonstrating the
same sequence. Since they were isolated from two differ-
ent fermentation processes it is reasonable to assume that
the same yeast was present during the two fermentations,
probably because it was present on the raw material used
for the brewing or from the environment and not from the
starters used.
All strains isolated from brewery A belong to the same
strain of S. cerevisiae used as starter; the TGGE profiles
are identical for all samples (Figure 2A). Similar results
were obtained for brewery C (Figure 2B). No correlation
was found between galactose fermentation results and
strain TGGE differentiation, in fact DNA fragments
showed migration profiles typical of S. cerevisiae behav-
iours independent from the negative galactose fermenta-
tion obtained.
The use of specific primers for the Saccharomyces
sensu stricto group is a reliable and fast system to identify
and to compare the different Saccharomyces cerevisiae
strains used as starters in beer production. The TGGE
technique (Figure 3) applied in this work allowed the
intraspecific differentiation among the strains tested and it
was possible to differentiate starters on the basis of the
size of their amplicons and to detect a “wild” S. cerevisiae
strains not belonging to any of the two starters used in the
brewery B. This culture-independent method allows sepa-
ration of DNA molecules that differ by single bases, char-
acterizing analysed strains in a short time. Identification
results are available within 8 h after cell growth onto the
isolation media used. Moreover the method is useful in
the direct detection of microbes contaminating foods or
beverages without the need of plating or, as reported by
Manzano et al.10 in the monitoring of industrial or craft
fermentation processes. The PCR-TGGE or PCR-DGGE
methods can be used in identification of contaminants or
in strain differentiation in a short time without employing
complicated biochemical tests.
A total of 44 Gram-positive, catalase negative rod or
cocccus-shaped strains isolated from MRS with the addi-
tion of Delvocid were identified by PCR-DGGE (Table
II). On the basis of different positions reached by the PCR
products in the DGGE the bacteria were identified as
Lactobacillus brevis, the unique species in the brewery A
and the main species detected in breweries (70%), in ac-
cordance with the data reported by Juvonen and Satokari 6
and Satokari et al.14 Kokuria, Lactobacillus brevis, Lb. sa-
kei and Lactococcus lactis were detected in the brewery B.
Lactobacillus brevis, Lb. hilgardii and Pediococcus par-
vulus were recovered in the brewery C 5. No Gram nega-
tive catalase negative strains were isolated from aF1 and
aF2 samples demonstrating only lactic acid bacteria were
detected in beer samples using microaerophilic sampling
conditions.
Most hazardous for the brewing industry are the LAB
belonging to the genera Lactobacillus and Pediococcus,
because they can produce haze or rope and cause unpleas-
Table II. DGGE identification of LAB strains isolated before and after
brewing.
Brewery Samples DGGE identification /total isolated
A* a 3 Lactobacillus brevis /3
aAF 3 3 Lactobacillus brevis /3
aF 4 3 Lactobacillus brevis /3
aF 5 3 Lactobacillus brevis /3
aF 6 3 Lactobacillus brevis /3
aF 7 3 Lactobacillus brevis /3
aF 8 3 Lactobacillus brevis /3
B B1 3 Kokuria kristinae /3
b1F1 3 Lactobacillus brevis /3
b2 1 Lactococcus lactis /3
2 Lactobacillus sakei /3
b2F1 3 Lactobacillus brevis /3
C c 2 Lactobacillus hilgardii /2*
cF 1 3 Lactobacillus brevis /3
cF 2 2 Pediococcus parvulus /3
1 Lactobacillus brevis /3
cF3 3 Lactobacillus brevis /3
*From samples aF1 and aF2 no LAB were isolated; from sample c only
2 LAB strains were isolated
VOL. 111, NO. 2, 2005 207
ant flavour changes such as sourness and atypical odour 13.
Various contamination levels were present among the
breweries analyzed due to the hygiene practices applied.
REFERENCES
1. Buiatti, S., Birra. In: Chimica degli alimenti, Piccin: Padova,
Italy, 2004, pp. 537–587.
2. Cavazza, A., Grando, M.S. and Zini, C., Rivelazione della flora
microbica di mosti e vini. Vignevini, 1992, 9, 17–20.
3. Cocolin, L., Manzano, M., Cantoni, C. and Comi, G., Denatur-
ing gradient gel electrophoresis analysis of the 16S rRNA gene
V1 region to monitor dynamic changes in the bacterial popula-
tion during fermentation of Italian sausages. Applied and Envi-
ronmental Microbiology, 2001, 67(11), 5113–5121.
4. Enari, T.-M., Beer. In: From Beer to Molecular Biology: The
Evolution of Industrial Biotechnology. Fachverlag Hans Carl,
Nurnberg, 1999, pp. 11–32.
5. Jespersen, L. and Jackobsen, M., Specific spoilage organisms in
breweries and laboratory media for their detection. Int. J. Food
Microbiol., 1996, 33, 139–155.
6. Juvonen, R. and Satokari, R., Detection of spoilage bacteria in
beer by polymerase chain reaction J. Am. Soc. Brew. Chem.,
1999, 57, 99–103.
7. Lopez, V., Fernandez-Espinar, M.T., Barrio, E., Ramon, D. and
Querol, A., A new PCR-based method for monitoring inoculated
wine fermentations. Int. J. Food Microbiol., 2002, 81, 63–71.
8. Manzano, M., Giusto, C., Iacumin, L., Cantoni, C. and Comi, G.,
A molecular method to detect Bacillus cereus from a coffee
concentrate sample used in industrial preparations. Journal of
Applied Microbiology, 2003, 95, 1361–1366.
208 JOURNAL OF THE INSTITUTE OF BREWING
9. Manzano, M., Cocolin, L., Longo, B. and Comi., G., PCR-
DGGE differentiation of Saccharomyces sensu stricto strains.
Antonie van Leeuwenhoeck, 2004, 85, 23–27.
10. Manzano, M., Cocolin, L., Iacumin, L., Cantoni, C., Comi, G.,
A PCR-TGGE (Temperature Gradient Gel Electrophoresis) tech-
nique to asses differentiation among enological Saccharomyces
cerevisiae strains. Int. J. Food Microbiol., 2005, 101(3), 333–339.
11. Noble, S., Practical aspects of fermentation management. The
Guild Review Paper N.9 The Brewer, 1997, 253–261.
12. O’Connor-Cox, E., Mochaba, F.M., Lodolo, E.J., Majara, M.
and Axcell, B., Methylene blue staining: use at your own risk.
Tech. Q. Master Brew. Assoc. Am., 1997, 34, 306–312.
13. Sakamoto, K. and Konings, W.N., Beer spoilage bacteria and
hop resistance. Int. J. Food Microbiol., 2003, 89, 105–124.
14. Satokari R., Juvonen, R., Mallison, K., von Wright, A. and
Haikara, A., Detection of beer spoilage bacteria Megasphaera
and Pectinatus by polymerase chain reaction and colorimetric
microplate hybridisation. Int. J. Food Microbiol., 1998, 45(2),
119–127.
15. Smart K., Oxford Symposium on Food and Cookery, Oxford
Brookes University, Oxford, September 3–4, 2004.
16. Tompkins, T.A., Stewart, R., Savard, L., Russell, I. and Dow-
hanick, T.M., RAPD-PCR Characterization of brewery yeast
and beer spoilage bacteria. J. Am. Soc. Brew. Chem., 1996, 54,
91–95.
17. Tsuchiya, Y., Kaneda, H., Kano, Y. and Koshino, S., Detection of
beer spoilage organisms by polymerase chain reaction technol-
ogy. J. Am. Soc. Brew. Chem., 1992, 50, 64–66.
18. Van der Aa Kuhle, A. and Jespersen L., Detection and identifi-
cation of wild yeasts in lager breweries. Int. J. Food Microbiol.,
1998, 43, 205–213.
(Manuscript accepted for publication April 2005)