Transcript Lab 18

Enumeration
(determine the numbers of bacteria in a sample)
Direct Measurement of Microbial Growth
• Microscopic count - the microbes in a measured volume of a
bacterial suspension are counted with the use of a specially
designed slide (Petroff-Houser chamber).
Enumeration
Direct Measurement of Microbial Growth
• Membrane filter method – used to test large volumes of sample
– In filtration, known volumes are filtered through membrane filter with pores
0.45 μm in diameter, bacteria are retained on the surface of a membrane filter
and then transferred to a culture medium to grow and subsequently be counted.
Enumeration
Direct Measurement of Microbial Growth
• A standard plate count
– Serial dilutions are prepared of a sample
– Aliquots of the dilutions are plated onto media
– The number of colonies are counted after 24-48 h of incubation
• The number of viable microbes and assumes that each bacterium grows
into a single colony;
•
– The number of bacteria in the original sample is determined by multiplying the number
of colonies by the dilution factor.
Plate counts are reported as number of colony-forming units (CFU).
• Only numbers between 30 and 300 CFU are considered statistically valid
Enumeration
Direct Measurement of Microbial Growth
• The most probable number (MPN) method can be used for
microbes that will grow in a liquid medium;
• The number of bacteria in a sample can be determined by the
relationship of some growth parameters to statistical probability.
Enumeration
Estimating Bacterial Numbers by Indirect Methods
• A spectrophotometer is used to determine turbidity (cloudiness or
haziness of a fluid caused by individual particles) by measuring the
amount of light that passes through a suspension of cells.
– Measures absorbance or optical density (OD)
– Relate OD (optical density) to actual numbers of bacteria determined by
a standard plate count (standard curve)
Lab 18 Goals and Objectives:
Exercise 21: Enumeration
Work as two pairs per group (max 5 groups)
Both pairs use same Escherichia coli culture
Use pre-poured plates: no molten agar pour plates
Divide work: One pair does Standard Plate Count, other does
Absorbance Readings
Plate count pair needs:
Absorbance pair needs:
3 – 99ml bottles sterile water
4 nutrient broth tubes
4 pre-poured plates
5 cuvettes
4 tubes of beads
10ml pipettes
(use beads to spread bacteria on plate)
1ml pipettes
For absorbance readings, make the required dilutions (1:2, 1:4 1:8, and
1:16) in the nutrient broth tubes and then transfer ~4ml of each to a
separate cuvette for reading in the machine. Cuvette #1 is for the
original undiluted culture (1:1).
Determination of Growth by Optical density
Same original
culture
Optical density
Standard Plate Count
CFU/ml
Fig 21.9
Determination of Growth by Optical density
1. Make dilutions in culture tubes
2. Put 4ml of
original culture in
a cuvette, label it
1:1
3. Transfer ~4ml
each dilution to a
cuvette labeled
with dilution (1:2,
1:4, 1:8, 1:16)
4. Read absorbance number for all 5 in spec beginning with most dilute
To save time:
1. 4ml culture into 1:2 culture tube with broth, mix with pipette
2. Suck up whole volume (8ml) with same pipette, dispense 4 ml into 1:4
broth culture tube and remaining 4 ml into 1:2 cuvette.
3. New pipette, mix 1:4 culture tube, suck up whole volume, dispense
4ml into 1:8 broth culture tube and remaining into 1:4 cuvette.
etc.
Fig 21.1
Standard Plate Count
*Label the plates with the dilution factor!
When you plate 1ml of some dilution, the dilution factor is that
dilution. When you plate only 1/10 of 1 ml (0.1ml), you have in
effect diluted it 10 fold more (so add another zero to the end).
• Plate count pair needs:
Absorbance pair needs:
3 – 99ml bottles sterile water
4 nutrient broth tubes
4 pre-poured plates
5 cuvettes
4 tubes of beads
10ml pipettes
(use beads to spread bacteria on plate)
1ml pipettes