Bioprocess Technology : SCBT 32113

Practical 3

1) Measuring Bacterial Growth

2) Cultivation of obligate anaerobes

G. Prabhakaran
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 Measuring Bacterial Growth
(Growth of Microbes : increase in number of cells, not cell size)

Cell Number vs Cell Mass

Ways of monitoring and measuring growth:

1. Direct methods – count individual cells

Example : Plate Counts, Filtration, Direct Microscopic Count

2. Indirect Methods – measure effects of

bacterial growth
Example : Turbidity, Metabolic activity, Wet weight, Dry weight

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Direct method – Colony count

Cell Viability Measurement: Spread Plate

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Optimal number to count is 30-300 colonies
 Colony count at different time intervals
Fig. i7.6

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Standard Growth Curve

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Indirect Method

Measurement of Turbidity

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 Bacterial growth
• The time taken for a microbial population to double in number is
called the doubling time (DT). The time taken for a single cell to
divide is called the generation time

2N

1N

N DT

time

• The mean generation time of a population is equal to the doubling time.

• Doubling time is a measure of growth rate

a short doubling time implies a fast growth rate.
Mean Generation Time and Growth Rate

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Plotting growth on graphs

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Use logarithmic graphing for bacterial growth

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Figure 6.13
 The slope  net is constant.
During the exponential (or logarathmic) growth phase, a bacterial culture
mimics a first-order chemical reaction, i.e. the rate of increase of cells is
proportional to the number of bacteria present at that time. The constant of
proportionality, µ, is an index of the growth rate and is called the growth rate
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constant:
Specific growth rate is defined as the increase in cell
mass per unit time, e.g., grams cells (g) per gram cells (g)
per hour:

The specific growth rate is commonly given by the symbol,

µ (mu), and the most common units are in reciprocal hours
(h−1);

Specific growth rate constant is a way of measuring

how fast the cells are dividing in a culture. It is defined
on the basis of doubling rate, and mathematically it
can be explained
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 Semilog Plot using natural logarithms
100
Natural log Cell biomass

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Natural
Gradient =  Log (ln)

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0 1 2 3 4 5 6 7 8 9 10
Time (Hrs)
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 Semilog Plot using natural logarithms
100
y2  y1
Natural log Cell biomass

Y2

x2  x1
10 Y1
Natural
Gradient =  Log (ln)

X1 X2
1
0 1 2 3 4 5 6 7 8 9 10
Time (Hrs)
µ, is an index of thegrowth rate and is  Specific growth rate 16
called the growth rate constant:
The doubling time is inversely proportional to the specific growth rate
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 Estimating Bacterial Numbers by Indirect Methods

• Metabolic Activity
-measure product as a function of time

Example: Bacterial culture produces acid as a

metabolic product, therefore you can monitor
growth by monitoring change in pH over time.

• Dry weight-filter all cells, dry in desiccator, and

weigh on scale
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Metabolic Activity

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of fungal mass

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Measuring Bacterial Growth - Practical
1) Dry weight- time consuming and not very sensitive

2) Turbidimetric measures- quick, easy and sensitive

optical density  the

number of microbes

E. coli, 1 OD600 = ~ 8 x 108

cells/ml
TO DETERMINE THE SPECIFIC GROWTH RATE OF THE BACTERIAL CULTURE

1. Over night shake flask culture 10 ml ( incubated 10 to 12 hours,

250 rpm at 37 C)

2. 1 ml inoculum for 9 ml medium in test tubes. 10 tubes. Incubated

10 to 12 hours, 250 rpm at 37 C

3. Every two hours take 1 tubes and measure OD at 600 nm.

4. Measure OD for 0 hr, 2 hr, 4hr, 8hr, 10hr and 12 hr

5. Plot Time Vs OD graph

6. Calculate the generation time

7. Calculate µ for the bacterial culture

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TO DETERMINE THE WET WEIGHT OF THE BACTERIAL CULTURE

1. Over night shake flask culture 10 ml ( incubated 10 to 12 hours, 250

rpm at 37 C)

2. 10 ml inoculum for 90 ml medium in conical flask. Incubated 10 to 12

hours, 250 rpm at 37 C

3. Weigh a empty falcon tube and record it.

4. After 10 hours, take the culture broth in the pre-weighed falcon tube.
Spin the broth at 4500 rpm for 15 minutes

5. Discard the broth. Weigh the falcon tube and record the difference in
weight.

6. The difference in weight of the tube gives wet weight of culture.

7. It forms the bases for the medium optimization.

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Cultivation of obligate anaerobes

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 Oxygen Requirements

• Obligate aerobes – require O2

• Facultative anaerobes – can use O2

but also grow without it

• Obligate anaerobes – die in the

presence of O2
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 Candle jar

• Provides
low O2,
high CO2

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ANAEROBIC JAR

Fig. 7.10b

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 Anaerobic indicators change color as
anaerobic atmosphere is attained.
Methylene blue OR Resazurin indicator
turns pink with an oxygen
concentration of 0.1% or less.
After 2-3 hours the Anaerobic
Indicator will change from pink to
white. This will give a visual
indication of anaerobiosis.

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70 % ETHANOL USED AS ANAEROBIC JAR CLEANING
Anaerobic Studies Chamber

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1) What is the function of the GasPak Anaerobic jar?

Create an anaerobic, microaerophilic, or CO2 enriched

environment

methylene blue strip: blue oxidized

methylene blue strip: colorless/white reduced

Function of palladium : catalyze a reaction between

hydrogen and free oxygen in the jar to produce water

2) What does sodium bicarbonate and sodium borohydride

react with and what does it produce - water; hydrogen and
carbon dioxide gases 30