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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

Pure culture of Acinetobacter on MacConkey agar plate

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  • $\begingroup$ They don't look any different to me from your photo. Perhaps a closer photo might help. All I see is some difference in thickness (depth?) of the colony perhaps, so one appears lighter colour than the other. $\endgroup$
    – bob1
    Sep 28, 2023 at 19:17
  • $\begingroup$ I will try to find a more clear example and I will add more essential quoted text to the background section about "Phenotypic Variants of Bacterial Colonies" $\endgroup$ Sep 28, 2023 at 21:46

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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.

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  • $\begingroup$ "There may also be variation in morphologies of colonies of the same strain. Hence categorization if isolates based on specific colony characteristics should be broad and based on several aspects of morphology rather than on only one or two characteristics. " What could be the variable characteristics that one should not always rely on? From my example color seems to be one. $\endgroup$ Sep 29, 2023 at 7:43
  • $\begingroup$ @FreezingSoul Categorization based on colony characteristics is necessarily broad. It is usually the very first step in ID, followed by more specific tests to discriminate between species or groups of types. If you were using a dichotomous key, it would be one of the first steps in ID it's that fundamental. $\endgroup$
    – bob1
    Sep 29, 2023 at 9:04
  • $\begingroup$ @FreezingSoul There's no one variable because it changes with each organism. An equivalent (at the risk of sounding racist; no racist intention here) might be comparing races of people - Asiatic peoples tend to have straight dark hair, so it's not a good characteristic to separate them internally into regions, but is useful to separate them from Caucasians and Africans (=genus from colony in bacteria), but in Caucasians hair might be useful for separating out groups internally (Germanic = blonde; Hispanic = dark, curly; Hibernian = red, equivalent to species within a genus for the bacteria) $\endgroup$
    – bob1
    Sep 29, 2023 at 9:17
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    $\begingroup$ Yeah, maybe i need to use this approach. $\endgroup$ Oct 1, 2023 at 13:10
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    $\begingroup$ @FreezingSoul You are overthinking it. In the medical training axiom: "If you hear hooves, think those are horses not those are zebras". Yes, some pathogens might be missed for rare cases, but that's where further investigation (repeat samples etc.) comes in. Overlap can be a problem, but the problem species is usually the most abundant species in the sample. Experience and training will tell you when this isn't the case! $\endgroup$
    – bob1
    Oct 1, 2023 at 20:51

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