Rapid Methods, MPN, Coliform Plate Count, and Virus Structure: 04-BS-13, May 2016, Q7

Question 6: Rapid Methods, MPN, Coliform Plate Count, and Virus Structure (20 marks)

Question text not reproduced: the examination questions are © Engineers and Geoscientists BC. Open the official past paper (linked at the top of this page) to read the question, then follow the worked solution below.

(a) Four rapid methods for microbiological analysis. (1) ATP bioluminescence — luciferin/luciferase reaction produces light proportional to microbial ATP, giving a result in minutes rather than the 24–48 h of plate counting. (2) Impedance/conductance microbiology — growth-driven changes in the electrical impedance of a growth medium are tracked continuously, and the time to a detectable impedance change correlates with initial cell density. (3) Flow cytometry — fluorescently labelled cells are counted and characterized individually as they pass a laser, giving total and viable counts in minutes. (4) Direct Epifluorescent Filter Technique (DEFT) / immunological methods (ELISA) / nucleic-acid methods (PCR, qPCR) — any of these (nucleic-acid amplification/hybridization or antibody-antigen binding) are also standard rapid alternatives to culture-based counting.

(b) Most Probable Number (MPN) principle. MPN is a statistical estimation method for organisms that are difficult or impossible to enumerate by direct plate count (e.g. coliforms in water, where cells may be too dilute or the medium selects for growth/gas production rather than visible colonies). A sample is serially diluted and multiple replicate tubes of selective/differential broth are inoculated at each dilution; each tube is scored simply as positive (growth/gas, indicating ≥1 viable organism was present in that aliquot) or negative. The pattern of positive tubes across the dilution series is compared against a standard MPN statistical table (based on the Poisson distribution — the probability that a given aliquot contains at least one organism follows a Poisson process) to read off the most probable number of organisms per unit volume of the original sample, along with a 95% confidence interval. It estimates a population density statistically from presence/absence data rather than by directly counting colonies.

(c) Coliform CFU/g by the weighted-dilution method.

Given.

Dilution	Plate I	Plate II
$10^{-2}$	250	228
$10^{-3}$	155	176
$10^{-4}$	10	5

Approach. Only plates with colony counts in the reliably countable range (conventionally 25–250 CFU/plate) are used; the standard weighted-mean formula then combines the two countable dilutions into a single estimate, rather than using either dilution alone.

Screen for countable plates. $10^{-2}$ (250, 228) and $10^{-3}$ (155, 176) both fall in the 25–250 countable range. $10^{-4}$ (10, 5) is below 25 CFU/plate — too few colonies for a statistically reliable count — and is excluded.
Apply the weighted-mean (Standard Methods) formula. $$N=\frac{\sum C}{(n_1+0.1n_2)\,V\,d}$$ where $\sum C$ = sum of counts on all plates used, $n_1,n_2$ = number of plates at the lowest and next dilution used, $V$ = volume plated (mL), $d$ = dilution factor of the lowest (n₁) dilution used. $$\sum C=250+228+155+176=809,\quad n_1=2,\ n_2=2,\quad V=0.1\ \text{mL},\quad d=10^{-2}.$$
Compute. $$N=\frac{809}{(2+0.1(2))(0.1)(10^{-2})}=\frac{809}{(2.2)(0.001)}=\frac{809}{0.0022}=\boxed{3.68\times10^{5}\ \text{CFU/g}}.$$

Quantity	Result
Plates used	$10^{-2}$, $10^{-3}$ (duplicates each)
$\sum C$	809
Coliform count	3.68 × 10⁵ CFU/g

(d) Enveloped vs. non-enveloped virus structure.

Figure 5. (i) Enveloped virus: nucleic acid core → protein capsid → host-derived lipid bilayer envelope studded with glycoprotein spikes (used for host-cell attachment/fusion). (ii) Non-enveloped (naked) virus: nucleic acid core directly enclosed by a protein capsid (often icosahedral) with no lipid membrane.

The envelope (when present) is acquired by budding through a host membrane and makes the virus generally more sensitive to desiccation, detergents, and disinfectants (the lipid bilayer is disrupted), whereas non-enveloped viruses rely on their protein capsid alone for environmental protection and tend to be hardier and more resistant to inactivation (e.g. by alcohol-based sanitizers).

Topic: Rapid microbiological methods; MPN statistical enumeration; plate-count weighted-mean calculation; viral structural classes.

Key relations: weighted-mean plate count $N=\sum C/[(n_1+0.1n_2)Vd]$; MPN via Poisson-based presence/absence statistics.

Why this works: restricting the plate count to the 25–250 CFU/plate window and weighting two adjacent countable dilutions (rather than reading a single plate) reduces the Poisson sampling error inherent in any small colony count, which is exactly analogous to why MPN needs multiple replicate tubes rather than a single presence/absence test.

Common pitfall: including the $10^{-4}$ counts (10, 5) in the average — counts below ~25 CFU/plate have disproportionately large relative sampling error and are excluded by convention, not averaged in alongside the reliable dilutions.

Source: Madigan et al., Brock Biology of Microorganisms, Ch. 5 (microbial enumeration methods, MPN, viral structure).

Question 7 of 9: Rapid Methods, MPN, Coliform Plate Count, and Virus Structure

Question 6: Rapid Methods, MPN, Coliform Plate Count, and Virus Structure (20 marks)