How to count colonies: manually vs using a colony counter

Colony counter and rapid plating methods are used by more and more microbiologists in their daily routines as bacterial plating and counting procedures had become such a must in the microbiology laboratory workflow.

Microbiologists need to solve the tedious problem of counting colonies which, sometimes, are too small and can be masked by the specific color of the culture medium or the enumeration itself is slow and long. That is why these daily routines need a better and more efficient way to optimize the processes employed.  

Currently, different methods exist under the same purpose: determine the number of colonies (CFU) in a sample of food, drug, or water. In this way, using a known volume of the sample results simply extrapolating the number of CFUs per plate to CFU per ml of bacteria culture or grams of sample. The general formula to calculate CFU/ml is well known:

The number of microbes/ml= number of colonies x dilution of the sample.

 

Colony counter methods: the counting problems 

 

According to FDA BAM (Chapter 3), the suitable colony counting range is 25-250 colonies per plate. When plates have more than 250 CFUs they are considered too numerous to count or may inhibit the growth of some bacteria. 

On the other hand, plates with less than 25 colonies do not have enough colonies to consider the plate statistically representative of the analyzed sample. Therefore, to get optimum results from spread plating, before inoculating the sample on the plate we need to perform serial dilution and plate at least three plates. This range also varies according to the method used. 

After incubating the plate under appropriate conditions for the microorganism, the colonies are counted. For the spread, pour or drop methods, the colony counting is self-explanatory: to count each colony dot once. A marker can be used pointing each counted colony on the back of the Petri dish.

The grid used in the spiral plating is divided into 8 sections and divided into 4 concentric rings. Counting shall be restricted to the 3 and 4 ring sections of the counting grid for 100 mm Petri plates and 1, 2, 3, and 4 ring sectors in 150 mm plates. Each ring is furtherly divided into three rings (3c, 3b, 3a and 4c, 4b, 4a). 

The counting takes place in 1/8 sectors of the counting grid’s rings, it must start at the outermost ring of the grid and proceed inwards. When a count of 20 colonies is met, counting can be resumed until the rest of the colonies in this same ring sector are counted, it must then stop.

Next, colonies that are present in the same rings of the opposed sector must be counted. A table is provided with the Spiral Plater to know the volume dispensed in the ring where the count has been performed and the method employed. Therefore, following this formula we can quickly know the CFU/ml:

CFU/ml = (N’ + N’’) / (V / 4)

N’ = CFU count in the first 1/8 ring sector = CFU 

N” = CFU count in the opposite 1/8 ring sector = CFU

V = Volume of the corresponding space

 

 

The conventional methods: a comparison with spiral plate and Eddy Jet

So, when the three conventional methods (pour plate, surface spread plate, and drop count) are compared with the spiral plate method, we find some disadvantages. The main one lies in the repetitive nature of the work, so it is not surprising that microbiologists have long sought a means to mechanize viable count techniques.

On the one hand, this system permits the estimation of microbial concentrations over a range of about three orders of magnitude, for example, Eddy Jet with a single spiral can have a detection range between 4.10² and 4.10⁵ in the most useful and better sowing of 50µl, without recourse to serial dilution of the sample and using only a single agar plate for each sample.  

Therefore, the spiral plate method can replace advantageously any of the other methods for the quantitative estimation of viable microorganisms in foods. And we can increase the speed of colony count using an automatic colony counter. 

 

Solutions: The Dot and the SphereFlash®

IUL’s technology ease and quicken the process using LED lighting. The choice of a good illumination for a colony counter is key. The optimal orientation of the light reduces errors during the count, reducing artifacts, and increasing the discrimination of colonies:  

  • DOT: the use of a digital counter such as DOT can be useful for microbiology labs involved in colony enumeration, the operator can tick detected colonies with a specifically devised marker, meanwhile a digital display will increase the total counts. IUL’s DOT Colony Counter has a large 120 mm diameter lens with a DINx2 magnification power. The light can be adjusted to the needs of the user and accounts for surrounding light inside the laboratory.

 

  • SphereFlash: the SphereFlash is an automatic colony counter that meets all the requirements that may be required in a modern microbiology laboratory. With its Colony Lite software version, it performs colony counting simply and reliably thanks to its world-class software and its patented built-in lighting system. 

The lighting is done through LEDs that are oriented to the internal wall of a moving spheric chamber.

Its patented spheric chamber eliminates reflections in both colonies and Petri plate and provides uniform illumination for an improved image capture that will lead to better, more reliable, and consistent results.