Measurement of Bacterial Growth Curve Using Spot Plating Technique

Materials and Methods

I. Growth of Escherichia coli in Media Containing Different Carbohydrates

A. Preparation of Media and Sugar Solutions Containing Different Carbohydrates

• For the preparation of the Escherichia coli as a test organism, 50ml of minimal
medium (0.1g Peptone; 0.25gNaCl; 0.0015g K2HPO4: 50ml sterile H2O) was
prepared in 250 ml reagent bottle.
• For the establishment of growth curve, 2 sets of 300ml of minimal medium (0.6g
Peptone; 1.5g NaCl; 0.09g K2HPO4; 300ml sterile H2O) were prepared in 500ml
reagent bottles.
• For stock solutions of sugars, 25% glucose and 25% lactose were prepared in 250
ml reagent bottles.
• Maximum Recovery Diluent (1.5 g Peptone; 12.75 g NaCl; 1500 ml distilled water)
was prepared for the serial dilution of the samples to be spot-plated to estimate
the population density of bacteria. 9 ml of MRD was transferred into 112 test tubes
(25 ml) to be used for the dilution.
• For the solid media that will be used for spot plating, 300 mL nutrient agar (8.4 g
Nutrient agar and 300 ml distilled water) was prepared.
• Different pipette tips and petri plates to be used in the experiment were also
prepared.
• All the liquid and solid media, pipette tips, petri plates, diluent, and sugar solutions
were sterilized at 121°C at 15psi for 15 minutes.

B. Preparation of growth flasks, inoculum, and agar plates.

• A loopful of Escherichia coli was inoculated into a sterilized minimal medium and
was incubated overnight with continuous shaking.
• Growth bottles were prepared by aseptically adding sugar solutions to the different
reagent bottles with 300ml minimal medium to obtain a 2.5% concentration for
each sugar solution. Two different set-ups were prepared: (1) glucose and (2)
lactose.
• Agar plates were prepared by aseptically pouring the prepared nutrient media onto
the petri plates. The plates were then left to cool for the agar to solidify. Once agar
was set, the area of the petri plates was equally divided into 6 sections. The first
three sections were labeled with the dilutions 10−3 , 10−5 , 10−7 , and the other three
sections served as duplicates of each dilution.
C. Growth Measurement by Spot Plating Technique

• The prepared inoculum was equally divided and inoculated to the different reagent
bottles.
• Immediately after inoculation, the bottles were swirled to distribute the cells to the
medium.
• For the serial dilution, 4 test tubes containing 9ml of MRD were prepared and
labeled with their corresponding dilution factor (10−1, 10−3 , 10−5 , 10−7 ). Then, 1 ml
of the inoculated medium was aseptically transferred to test tube #1 (10−1 ) . The
test tube was covered with a cap and vortexed. 0.1 ml of sample from test tube #1
was then aseptically transferred to test tube #2 (10−3 ). It was then again closed
and vortexed before transferring 0.1 ml of sample to test tube #3 (10−5 ), and the
process was repeated until test tube #4 (10−7 ).
• To establish the growth for 0-hour incubation, 0.02 ml of sample from the dilutions
10−3 , 10−5 , 10−7 were aseptically spot plated into their designated sections in the
labeled petri plate.
• Right after spot plating for the 0-hour incubation, the growth bottles were placed in
a shaker set at 200 rpm.
• Samples from the different growth bottles were continuously diluted and spot-
plated every 30 minutes for the next 6.5 hours of incubation.
• After 6.5 hours of incubation, serial dilution, and spot plating, the plates were
incubated for 24 hours to observe growth.
• After incubation, the number of colonies formed in each plate was manually
counted to compute the CFU/ml.
• The computed CFUs/ml for each plate were plotted against incubation time using
MS Excel XY scatter plot with logarithmic scale to obtain the bacterial growth
curve.