How do microbiologists decide whether two morphotypes on the same plate belong to the same strain?

Question

Background

Colony: A colony, in microbiology, is a visible cluster or mass of microorganisms (such as bacteria or fungi) that has grown and multiplied on a solid agar medium. Each colony represents a population of genetically identical microbial cells that originated from a single cell or a small group of cells. Colony formation is an essential step in the isolation and identification of microorganisms.

Colony morphology refers to the visible characteristics of a bacterial or fungal colony when grown on a solid agar medium. It includes features such as size, shape, color, texture, and edge appearance. Each type of microorganism may exhibit distinct colony morphologies, aiding in their identification and classification.

"It should be noted that when a microbiologist examines an agar plate and determines that mixed bacterial types are present, it is actually mixed bacterial colonial types (morphotypes) that are being observed. These visually distinctive colonies often represent different species (or even genera), but they may also represent phenotypic variants of a single genotypic organism."

Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th. Wolters Kluwer; 2017

Question

In diagnostic microbiology and in order to save time, how do microbiologists decide whether two morphotypes on the same plate belong to the same strain?

I'm not talking about great differences in morphologies as Lactose fermenters vs Non-Lactose fermenters on MacConkey agar, but ones like these: The center in Type A is purple while the center in Type B is pink.

Pure culture of Acinetobacter on MacConkey agar plate

Answer 1

You have to remember that when a microbiologist looks at a plate, they are looking to observe a particular something, which depends on exactly what they are doing, and they will have some idea of the range of morphotypes expected for those species and/or strains.

They also aren't doing this in isolation - they will use tests to further confirm what they see, often starting with simple tests like Gram staining and going on to other biochemical tests, genetic tests (sequencing possibly or PCR), or even MALDI-ToF.

Experience also plays a part - if this is something being done routinely, say in a clinical lab, then the investigator know what species to expect, and which ones are things to further investigate for that particular sample type. This means they will have some idea (it may not be formal knowledge, as in taught, just experience) of the range of potential morphotypes for a particular species and be able to use that information to assess a plate quickly and accurately. Exact identification of a strain and/or species is often impossible without further testing (e.g. you can't normally tell the difference between antibiotic resistant and non-antibiotic resistant strains without susceptibility testing, or even the difference between various Streptococcus species without testing), though pathogenic factors such as bacterial capsules in some strains of a species (e.g. capsules in *E. coli*), might be obvious from colony morphology immediately.

In the case of a pure strain streaked on a plate, as in your photo, changes in colony morphology might or might not indicate genetic changes reflected by phenotypic changes. An example might be loss of flagellae, means the bacteria are no longer motile, so the colony is less spread-out than ones with flagellae. Pigementation changes might also indicate a phenotypic and/or genetic change - perhaps processing a metabolite by a different pathway or able to do it only under certain conditions - no genetic change, just a phenotypic.