yeast-6.pdf

(32 KB) Pobierz
Microbiology
Yeast–6
Page 1 of 1
YEAST VIABILITY BY SLIDE CULTURE
The viability of culture yeast is of utmost importance.
It may be determined by the following slide-culture
technique employing a medium containing malt extract,
yeast extract, glucose, and peptone supplemented with
maltose and zinc (supplemented MYGP medium) (1).
This medium is superior to wort medium supplemented
with zinc in that it is easily duplicated from laboratory to
laboratory, yields higher viable cell counts, and is free of
the troublesome particulate matter found in wort media.
Medium
Malt extract (Difco or equivalent)
Yeast extract (Difco or equivalent)
Glucose (Fisher Scientific
dextrose anhydrous or equivalent)
Peptone (Difco or equivalent)
Maltose (Sigma Grade II or equivalent)
Agar (BBL granulated or equivalent)
Zinc sulfate (1.5 g ZnSO
4
·7H
2
O
dissolved in 100 mL water)
Water
0.3 g
0.3 g
1.0 g
0.5 g
6.0 g
1.5 g
1.0 mL
100 mL
suspension (about 1 × 10
6
cells/mL) onto the surface of
the agar. Place drops at each end of the slide, so that a
cover slip can be easily placed on each. Do not apply
pressure to cover slips. Check the preparation at a
magnification of 200–250× to make sure that cells are
not overcrowded.
Replace the Petri dish lid, label, and incubate at 25°C
for approximately 12–16 h, but for no longer than 18 h.
After incubation, examine the slide cultures at a
magnification of 200–250×. Cells that give rise to
microcolonies are counted as viable. Single cells not
giving rise to microcolonies are considered dead.
Enumerate a combined total of at least 500 micro-
colonies and cells.
Calculation
% Viable cells
=
No. microcolonies
×
100
Total No. cells and microcolonies
Autoclave at 1 bar (15 lb/in.
2
), 121°C, for 20 min.
Apparatus
(a)
Forceps.
(b)
Bunsen burner.
(c)
Slides,
glass, 230- × 20-mm.
(d)
Cover slips,
glass.
(e)
Pipette,
1-mL.
(f)
Micropipette,
capable of measuring 100
μL.
(g)
Microscope,
(200–250× magnification).
(h)
Incubator,
25 ± 0.5°C.
(i)
Petri dish,
sterile (100- × 15-mm).
Method
With forceps, gently flame a glass slide over a Bunsen
burner and place into a sterile Petri dish. Using a pipette,
spread approximately 1 mL of molten supplemented
MYGP medium evenly over the slide.
After the agar has solidified, place two drops of yeast
Notes
1. Microcolonies near the edge of the cover slip often
grow together and are not suitable for counting. There-
fore, if possible, count colonies in the center of the slide
where there is better distribution.
2. Collaborative testing indicated that cultivating
yeast on supplemented MYGP agar produced more
consistent and reliable results than wort agar or standard
MYGP agar. However, extensive evaluation of media
types was not performed. It may be possible to obtain
adequate results using alternative standard laboratory
media for culturing brewing yeast, such as YPD agar.
Reference
1. American Society of Brewing Chemists. Report of Subcommit-
tee on Microbiology.
Journal
42:132, 1984.
1984, rev. 2011
doi: 10.1094/ASBCMOA-Yeast-6

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika:

Zgłoś jeśli naruszono regulamin