1. 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).

2. 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.

3. 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 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

4. 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.

5. 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)

6. Exercise 21: Enumeration Work as two pairs per group (max 5 groups)
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 ml 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).

7. Determination of Growth by Optical density
Same original
culture
Optical density
Standard Plate Count
CFU/ml

8. 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

9. 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.

10. *Label the plates with the dilution factor!
Standard Plate Count
Fig 21.1
*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).

11. 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 ml pipettes
(use beads to spread bacteria on plate)
1ml pipettes