14.
How many bacteria were there in the initial 1 ml sample based on the dilution series
shown below? The labels below the tube show the amount of liquid before the transfer.
Assume no cell death, and assume that all bacteria successfully form colonies when
plated.
a. 6.5 x 108
b. 6.5 x 109
c. 6.5 x 1010
d. 6.5 x 1011
Introduction
In microbiology, questions like “How many bacteria were there in the initial 1 mL sample based on the dilution series and colony count?” test understanding of serial dilution, dilution factors and colony-forming unit (CFU) calculations.
Correctly interpreting volumes transferred at each step and the final plated volume is essential for solving such CSIR NET–style problems quickly and accurately.
Step-by-step solution of the dilution series
From the figure, the following transfers occur:
Tube 1
Add 1 mL of original sample to 9 mL diluent → total 10 mL.
Dilution factor in tube 1 = 1/10 = 10-1.
Tube 2
Transfer 0.5 mL from tube 1 into 4.5 mL diluent → total 5 mL.
Additional dilution = 0.5/5 = 1/10 = 10-1.
Cumulative dilution after tube 2 = 10-1 × 10-1 = 10-2.
Tube 3
Transfer 3 mL from tube 2 into 27 mL diluent → total 30 mL.
Additional dilution = 3/30 = 1/10 = 10-1.
Cumulative dilution after tube 3 = 10-2 × 10-1 = 10-3.
Tube 4
Transfer 1 mL from tube 3 into 99 mL diluent → total 100 mL.
Additional dilution = 1/100 = 10-2.
Cumulative dilution after tube 4 = 10-3 × 10-2 = 10-5.
Tube 5
Transfer 1 mL from tube 4 into 9 mL diluent → total 10 mL.
Additional dilution = 1/10 = 10-1.
Cumulative dilution after tube 5 = 10-5 × 10-1 = 10-6.
Tube 6
Transfer 2 mL from tube 5 into 18 mL diluent → total 20 mL.
Additional dilution = 2/20 = 1/10 = 10-1.
Cumulative dilution after tube 6 = 10-6 × 10-1 = 10-7.
Plating step
Finally, 10 μL (0.01 mL) from tube 6 is plated and produces 65 colonies.
Dilution due to plating 0.01 mL instead of 1 mL = 0.01/1 = 10-2.
Overall dilution of the plated sample relative to the original = 10-7 × 10-2 = 10-9.
So the dilution factor (inverse of dilution) from original sample to what is effectively counted on the plate per mL is: dilution factor = 109.
CFU calculation
Step 1: CFU per mL in tube 6:
CFU/mL in tube 6 = 65 colonies / 0.01 mL = 6500 CFU/mL.
Step 2: Convert to original sample:
Because tube 6 is 10-7 of the original:
CFU/mL original = 6500 × 107 = 6.5 × 1010 CFU/mL.
Thus, there were 6.5 × 1010 bacteria in the initial 1 mL sample, matching option c.
Why each option is right or wrong
Option a: 6.5 × 108
This value is 100-fold lower than the correct answer and would result from mistakenly treating the total dilution as 10-7 instead of 10-9, effectively forgetting the extra 10 μL plating dilution.
Many students stop at the tube-dilution calculation and ignore that only 0.01 mL was plated, so this option reflects that common conceptual error.
Option b: 6.5 × 109
This is 10-fold lower than the correct value and corresponds to assuming a total dilution of 10-8.
Such an answer typically arises if one miscalculates one of the intermediate steps (for example, thinking the 1 mL into 99 mL step is only a 10-fold dilution instead of 100-fold), showing partial understanding but incorrect handling of one dilution factor.
Option c: 6.5 × 1010 (correct)
This option correctly accounts for all seven serial dilution steps up to tube 6, giving a net dilution of 10-7.
It also includes the additional 100-fold dilution from plating 10 μL rather than 1 mL (final effective dilution 10-9), and converts 65 colonies from 0.01 mL to 6500 CFU/mL in tube 6 before back-calculating to 6.5 × 1010 CFU/mL in the original sample.
Option d: 6.5 × 1011
This is 10-fold higher than the correct answer and would result from assuming a total dilution of only 10-8 while also mishandling the volume-plated factor in the opposite direction.
It overestimates the original bacterial load and demonstrates an error in combining the volume and tube dilutions.
Key takeaways for exam preparation
- Always compute dilution per step as “volume transferred / total volume after transfer”, then multiply sequentially.
- At the plate, adjust CFU for the actual plated volume to obtain CFU/mL in that tube.
- Finally, multiply by the inverse of the cumulative dilution to return to the original sample’s concentration.
This systematic approach to a bacterial serial dilution “how many bacteria in initial 1 mL sample” question ensures fast and accurate solutions in CSIR NET and other microbiology exams.
