Quantitative microbiology
~ 1 ~
Microbiology quantitative tests
List safety rules associated with the laboratory
Always wash hands upon entering and before leaving the lab
Wash table top upon entering and before leaving the lab
Place all coats, books and purses at coat rack of under table
Keep the lab and especially the wash basins clean
Keep all unauthorized people out of the lab
Wear a lab coat, old shirt or apron to protect clothing
Keep your work area neat and organized
Do not begin work until you have received instructions
Report all spilled cultures, immediately
Discard all material used in the lab properly
Develop good personal aseptic technique
Report accidents immediately
Plate methods
Pour Plate Method
Test sample is pipetted into a sterile empty Petri plate. Next melted agar is added and plate is gently
swirled to mix content.
From each sample or dilution prepare 2 Petri plates
Precision of test could be increased by usage of more then 2 plates
Using aseptic technique pipette 1 mL of sample dilution into the Petri plate. (Use separate pipette tip to
each dilution)
Pour liquid agar medium cooled down to 45 - 50ºC into the Petri plate to achieve 3 – 4 mm layer. Mix
plate content carefully.
Cover agar with 4 mL of same medium to protect against bacteria growth on the surface
Time of whole procedure can not exceed 15 minutes
Spread Plate Method
To Peteri plate pour liquid agar medium to achieve 2 mm layer.
Prior to spread dry medium by opening Petri plate (removing condensed water)
Add sample dilution and spread bacteria using sterile glass bend rod.
Incubation
Reverse plates bottom up and incubate in apropriate temperature
Do not overload incubator
Keep distance between plates and incubator walls
Store no more then 6 plates in a column
Reading results
Count colonies on plates immediately after incubation. In special cases plates could stored at 5°C but no longer then
48 h.
Choose plates with colony count ranged between 30 and 300 and 2 following dilution
Use special counters on magnifying glasses
Discard plates with irregular bacteria growth
BACTERIA COUNTING
Pour Plate Method
Count bacteria number (L) in 1 mL or 1 g of product according equation:
L =
C
(N + 0.1 N ) d
1
2
x
Where:
C — number colonies on plates chosen for counting
N
1
— number of plates from the first dilution
N
2
— number of plates from the second dilution
Quantitative microbiology
~ 2 ~
d — dilution coefficient corresponding the first (lower) dilution
Counted number round to 2 essential numbers. Result express as number between 1.0 a 9.9 multiplied
by
10
n
, where
n
means appropriate power
In a case of lower count of colonies then 10, express result in given ways:
- less then 10 • 10
l
bacteria in 1 mL o cfu/1mL
- mniej niż 10 drobnoustrojów w l ml lub jtk/ml,
I a case of lack of bacteria express result in given ways:
- absent in 1 mL
- absent in 1 g
In a case when number of colonies ecxeeds 300 result should be given as approximation: over 300 • l/d
bacteria in l mL or l g or cfu/mL
Where d — means dilution coefficient e. g. 10
6
Spread Plate Method
a) Liczba kolonii na płytkach w granicach 10 + 150. Liczbę drobnoustrojów (Z.) w l ml lub l g próbki obli-
czvc wg wzoru
L =
C
(N + 0.1 N ) d a
1
2
x
x
Where:
C — number colonies on plates chosen for counting
N
1
— number of plates from the first dilution
N
2
— number of plates from the second dilution
d — dilution coefficient corresponding the first (lower) dilution
a — volume of spreaded sample on one plate (e. g. 0,1 ml, 1,0 ml).
Counted number round to 2 essential numbers. Result express as number between 1.0 a 9.9 multiplied
by
10
n
, where
n
means appropriate power
Most Probable Number
Statistically based method
Appropriate to low bacteria counts – les then 100 in l g or 10 in l ml.
Defined sample volume (normally 1 mL) add to 3 parallel test tubes with culture medium and incubate.
In a case of liquid products add 10 ml and 1 ml of sample and 1 ml of dilutions: 10
-1
, 10
-2
, 10
-3
.
Use separate pipettes of pipette tips to each dilution
Level of probability is expressed in 4 categories:
Category 1 The highest probability corresponds real results. In case of testing 3 samples form batch to category
I sequences from 3 following dilutions are included as follow: 1,0,0; 2,1,0; 3,0,0; 3,1,1 (compare tab. 1).
Category 2 Less probability. In case of testing 3 samples form batch to category 2 sequences from 3 following
dilutions are included as follow: 1,0,1; 1.2,0; 2,0,1 (compare tab. 1).
Category 3. Less probability then category 2. In case of testing 3 samples form batch to category 3 sequences
from 3 following dilutions are included as follow: 0,2,0; 2,3,0; 3,0,2 (compare tab. 1).
Category 0. Probability of achieving results corresponding to real result is minimal. In case of testing 3
samples form batch to category 3 sequences from 3 following dilutions are included as follow: 0,3,0; 2,1,2;
3,1,3; (compare tab. 1).
If tests are based only on MPN only categories 1 and 2 should be chosen.