Colonial mass and spot checking: Improving microbial plate counting

Viable plate counts provide a standardized means to assess bioburden, to generate growth curves, to calculate the concentration of cells from the source from which the sample was plated, and to investigate the effect of various environments or growth conditions on bacterial cell survival.

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In this week's article we return to the topic of plate counting. A previous article (“Means, ranges and replicates: Improving microbial plate counting”) looked at rounding and averaging; significant figures; countable ranges and statistical error from low counts. This article focuses on the methods, as well as some points about plate reading, as part of best practices to improve reproducibility and reliability.

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For bioburden determination, a viable titer of a microbial suspension can be determined by counting the number of colonies arising from a defined volume dispensed onto or into a plate, or through filtration (i.e. spread plate, pour plate or membrane filtration techniques). This leads to an assessment of colony-forming units per volume (such as one?milliliter, leading to CFU/mL).

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Colony distribution

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As a sample is serially diluted, and plated out at each stage, the distribution of organisms tends to follow Poisson distribution (1).

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Poisson distribution is a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time if these events occur with a known constant mean rate and independently of the time since the last event (2). The best way to overcome this distribution is to count a significantly meaningful number of colonies (a challenge in itself where there is a low count to begin with; much research place the optimal countable range as between 25 and 250 colony forming units – or sometimes 30 to 300 CFU) (3). Counts below 10 allow only a very poor estimation of the actual number of bacteria.

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From this, two challenges arise. These can be described as ‘analyze bias’ (the variability introduced primarily by crowding and analyst counting errors) and ‘variance’ (sampling and dilution errors).

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What are the causes of plating and counting errors?

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The causes of plate counting have been well-documented for decades. In the 1930s, an assessment pinpointed the common causes of error in counting to be (4, 5):

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1.????? Failure to know what to count,

2.????? Failure to see the colonies,

3.????? Carelessness (including counting too quickly – one study showed 18 seconds to count 100 colonies was optimal).

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In addition, care needs to be paid with preparation (the plating techniques) (6-8):

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• If the culture is not mixed prior to removing an aliquot, not enough bacterial cells will be transferred.
• With techniques like pour plates, variable cooling rates can affect recovery (overcome by avoiding stack pouring)
• Also with temperature, the temperature of the inoculated organism is also an important determinant affecting recovery
• pH stability of the culture is also an important factor, where lowering pH can lead to cell dormancy
• Rate of drying.
• Cell clumping and inadequate mixing.
• Presence of slime or capsules.
• Microbial adherence to container walls.
• Inadequate separation of microorganisms from antimicrobial agents.

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Plating methods

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Spread plating

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Spread plate techniques are used to separate microorganisms contained within a small sample volume, which is spread over the surface of an agar plate. This results in the formation of discrete colonies distributed evenly across the agar surface when the appropriate concentration of cells is plated(9). Spread plates should be permitted to absorb the inocula for 10 ?to 20 minutes, after which time they are inverted and incubated as required.

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As well as assessing the total number of colony forming units on a single plate to be enumerated, spread-plating is also used in enrichment, selection, and screening. An enrichment process involves plating a mixed culture on a medium or incubating plates in environmental conditions that favor growth of those microorganisms within the sample that demonstrate the desired metabolic properties, growth characteristics, or behaviors. This strategy does not inhibit the growth of other organisms but results in an increase in the number of desired microorganisms relative to others in the culture.

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However, ineffective spread plating leads to colony clusters and overlapping colonies, which makes counting difficult or even impossible.

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The humidity of the agar plates is important for cell spreading. The agar surface has to be free of visible moisture and aseptic spreading must be carried out until the liquid is completely absorbed by the agar (freshly prepared plates do not work as well as dry plates as it takes longer for the inoculum to absorb into the agar). Another important consideration is avoiding over-drying of the plates, as this can affect the growth promoting properties (10) (such as arising from excessively long storage times) (11).

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Ideally, microbial spread plating results in randomly distributed colonies on the agar surface. Whether this is achieved can be seen as a probabilistic approximation of circle number π.

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Streak plating?

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Streak plating is related to spread plating, although with spread plating the objective is enumeration. With streak plating, this is designed to isolate pure cultures of bacteria, or colonies, from mixed populations by simple mechanical separation. With the streak-plate procedure, a mixture of cells is spread over the surface of a semi-solid, agar-based nutrient medium in a Petri dish such that fewer and fewer bacterial cells are deposited at widely separated points on the surface of the medium and, following incubation, develop into colonies. The quadrant method for isolating single colonies is optimal (12).

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If too many bacteria are plated, then overlap of cells may occur and increase the probability of two or more bacteria giving rise to what appears to be a single colony.

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A form of streak plating is the basis of Mossel’s econometric method. This method produces ever-decreasing numbers of colony-forming units per surface area, as in spiral plating. This procedure evaluates, in quantitative terms, the ability of media to support the formation of colonies by organisms that it was designed to grow and to resist colonization by organisms that it is expected to suppress (13).

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Pour plating

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The pour plate method is used to count the number of microorganisms in a mixed sample, which is added to a molten agar medium prior to its solidification. The process results in colonies uniformly distributed throughout the solid medium when the appropriate sample dilution is plated.

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This technique enables the total number of colony forming units within the agar and on surface of the agar on a single plate is enumerated.

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Pour plate vs spread plate

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Generally, different plating methods are comparable depending on the application (studies tend to show agar droplet technique, flooding technique and pour plates producing comparable data) (14). Other studies point to plate flooding (as opposed to the spreading of a smaller droplet) to be inferior (15). The coefficient of variation between the methods are broadly similar and invariably within 10% (16).

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The spread plate procedure is superior to the pour plate technique where end goal is to isolate colonies for further analysis since colonies grow accessibly on the agar surface whereas they become embedded in the agar with the pour plate procedure (17).

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Membrane filtration

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Membrane filtration is a technique that is especially useful for testing water samples. In this procedure, water is drawn through a special porous membrane designed to trap microorganisms larger than 0.45 μm. Hence, filtration largely depends on a mechanical sieving action that suspends and holds organisms that cannot pass through the pore size of the membrane filter. Some microorganisms may also be suspended or adsorbed to the surface of the filter as a result of some electrostatic forces; and this is because some membrane filters are highly adsorptive and thus can absorb particles suspended in a fluid via electrostatic action.

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Afterwards, the filter is applied to the surface of an agar plate and incubated, where nutrients diffuse through the filter from the growth medium and allow the development of bacterial colonies.

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Membrane filters are thin films made up of cellulose nitrate, cellulose acetate, nylon, polycarbonate or polyvinylidene materials. The technique dates back to the 1950s (18).

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With membrane filtration two problems can emerge:

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• Indistinct colonies: If growth covers the entire filtration area of the membrane or a portion of it, and colonies are not discrete, report the test results as “Confluent growth”.
• High colony density: If the total number of colonies exceeds 100 per membrane or the colonies are too indistinct for accurate counting, it is typical to report the results as “Too numerous to count” (TNTC).

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Overcoming counting errors

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However, the plate is prepared, after a period of incubation, colonies need to be accurately counted. As indicated above, counting errors can occur. What can improve the accuracy of plate counting?

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Light and magnification

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“Illumination can be secured from the two opposite sides and in which the lens is held at a certain distance from the plate seems to give the most satisfactory results.”

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More plates, less variation

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The greater the number of replicate plates used, then the mean of counts fluctuates less and the coefficients of variation of colony counts per plate decreases further (19). This is a phenomenon that is coincident to the central limit theorem (where, under appropriate conditions, the distribution of a normalized version of the sample mean converges to a standard normal distribution. This holds even if the original variables themselves are not normally distributed) (20).

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Automation

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Automatic plate counters have been covered in other articles in this series. Some reasons why these technologies are preferred is because manual enumeration processes are somewhat subjective, error prone, suffer from a low throughput; they can also be time-intensive, tedious, and laborious.

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Summary

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This article has looked at the long-established plate counting methods with a focus on some potential causes of error.

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References

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1.????? Fujikawa H. [The Validity of the Poisson Distribution to Analyze Microbial Colony Counts on Agar Plates for Food Samples]. Shokuhin Eiseigaku Zasshi. 2023;64(5):174-178. Japanese. doi: 10.3358/shokueishi.64.174

2.????? Ross, Sheldon M. (2014). Introduction to Probability Models (11th ed.). Academic Press

3.????? Diane M. Tomasiewicz, Donald K. Hotchkiss, George W. Reinbold, Ralston B. Read, Paul A. Hartman, The Most Suitable Number of Colonies on Plates for Counting, Journal of Food Protection, 1980, 43(4): 282-286,

4.????? Schacht FL, Robertson AH. Laboratory: Accuracy Of Counting An Agar Plate. Am J Public Health Nations Health. 1932, 22(1):80-3

5.????? Fruin JT, Idll TM, Clarke JB, Fowler JL, Guthertz LS. Accuracy and Speed in Counting Agar Plates, J Food Prot. 1977;40(9):596-599

6.????? Koburger JA. Stack-Pouring of Petri Plates: A Potential Source of Error .J Food Prot. 1980;43(7):561-56

7.????? Woolfrey BF, Gresser-Burns ME, Lally RT. Effect of temperature on inoculum as a potential source of error in agar dilution plate count bactericidal measurements. Antimicrob Agents Chemother. 1988, 32(4):513-7

8.????? Woolfrey BF, Lally RT, Ederer MN. Influence of technical factor variations during inoculum preparation on the agar dilution plate-count method for quantitation of Staphylococcus aureus oxacillin persisters. Antimicrob Agents Chemother. 1986 ;30(5):792-3

9.????? Jett, B. D., Hatter, K. L., Huycke, M. M., Gilmore, M. S. Simplified agar plate method for quantifying viable bacteria. BioTechniques. 23, 648-650 (1997).

10.? Schiffel F, Noll S. Approximation of circle number π by spread-plating Escherichia coli. Biochem Mol Biol Educ. 2023;51(3):312-315

11.? Kennedy JE Jr, Phillips PE, Oblinger JL. Effect of Stored Pre-Poured Plates on Microbial Enumeration Using the Surface Plate Method. J Food Prot. 1980 Aug;43(8):592-594

12.? Sanders, E. R. Aseptic Laboratory Techniques: Plating Methods. J. Vis. Exp. (63), e3064, doi:10.3791/3064 (2012).

13.? Mossel DA, Bonants-Van Laarhoven TM, Ligtenberg-Merkus AM, Werdler ME. Quality assurance of selective culture media for bacteria, moulds and yeasts: an attempt at standardization at the international level. J Appl Bacteriol. 1983;54(3):313-27

14.? Koller W, Jelinek JA. Die kulturelle Keimzahlbestimmung im Agartropfen [Viable bacterial counts by agar-droplet technique (author's transl)]. Zentralbl Bakteriol Orig A. 1976;235(4):527-53

15.? Olsen RL, Richardson GH. A Flooded Plate Loop Count Procedure 1. J Food Prot. 1980 Jul;43(7):534-535

16.? Treuhaft MW, Arden Jones MP. Comparison of methods for isolation and enumeration of thermophilic actinomycetes from dust. J Clin Microbiol. 1982;16(6):995-9

17.? Korsholm E, S?gaard H. Colony counts in drinking water bacteriology--importance of media and methods. Zentralbl Bakteriol Mikrobiol Hyg B Umwelthyg Krankenhaushyg Arbeitshyg Prav Med. 1987185(1-2):112-20

18.? Tsuneishi N, Goetz A. A method for the rapid cultivation of Desulfovibrio aestuarii on filter membranes. Appl Microbiol. 1958;6(1):42-4

19.? Fujikawa H, Tsubaki H. Characteristics of Microbial Colony Counts on Agar Plates for Food and Microbial Culture Samples. Shokuhin Eiseigaku Zasshi. 2019;60(4):88-95

20.? van der Vaart, A.W. (1998). Asymptotic statistics. New York, NY: Cambridge University Press.