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Thread: What techniques are used to idnetify microorganisms?

  1. #1
    Registered Senior Member Cat_with_no_eyes's Avatar
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    Smile What techniques are used to idnetify microorganisms?

    For example what are the possible techniques which can be used in a medical setting such as a biomedical lab/microbiology lab, to identify microorganisms, example: bacteria?


  2. #2
    I would use a microscope.

  3. #3
    Registered Senior Member Cat_with_no_eyes's Avatar
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    i'm talking about techniques used as far as getting fast and reliable identifications, not ancient techniques spidergoat :P

  4. #4
    I think the OP is looking for Techniques.

    I can only think of combining and separating microbes by engineering.

    ie. adding and removing from water to see how the substance reacts. sunlight?

  5. #5
    Valued Senior Member
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    i'm talking about techniques used as far as getting fast and reliable identifications
    Fast, reliable, affordable - - pick one.

    Culturing on various media, staining with various stains and observation under various light, observing growth habit and shape, checking certain DNA/RNA sequences, I've seen done.

    In the near future, micro assay chips preloaded with DNA/RNA (or characteristic protein, etc) recognition sites.

    Identifying stuff is underestimated, in my experience - people seem to take for granted that somebody, somewhere, just automatically knows what anything is.

    I've seen estimates of the percentage of bacterial types, kinds, species, or whatever that have never been named - that cannot be "identified", because they've never been described carefully in the first place - as high as 99%.

  6. #6
    voltage gated ion channel Hercules Rockefeller's Avatar
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    Quote Originally Posted by Cat_with_no_eyes View Post
    i'm talking about techniques used as far as getting fast and reliable identifications, not ancient techniques spidergoat :P

    AFAIK, diagnostic microbiology is an area in which the "ancient" techniques are still relied upon. The traditional ways of identifying bacteria are staining and microscopy (for a visual identification) in conjunction with culturing samples in a variety of different media and conditions (for confirmatory indications of metabolism and behaviour). In hospital pathology labs and microbiological diagnostic units they still perform these tests just as microbiologists did a century ago.

    Of course, these days there are also a widely utilised suite of molecular tools that enable us to look at the molecules that are present in a sample – DNA/RNA, proteins and carbohydrates. These are also diagnostic and can be somewhat quicker than culturing which can sometimes take days to perform. Depending on the precise questions being asked, microorganism identification is a combination of old-school and new-school technologies.

  7. #7
    Registered Senior Member Skeptical's Avatar
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    Traditional techniques such as culturing, staining etc., are still widely used and very important. In medical microbiology, diagnostics could not do without them. However, none of those methods can be called 100% reliable.

    Enter the world of nucleic acid sequencing. Suddenly exact identifications can be made. A whole lot more species suddenly appear.
    For example : traditionally the fungus Trichoderma viride was thought to be a single and separate species. Always looked the same under the microscope, and responded the same way to stains. Cultured the same. etc. Then, lo and behold, after introducing 18S ribosomal RNA sequencing techniques, it turns out that this 'species' is at least 100 different species.

    Bacteriologists these days frequently sample substrate (soil, water, other deposits) and carry out a DNA analysis of the whole sample. Thousands of new species of bacteria have been found in this way. It turns out that most bacterial species do not actually grow on traditional culture media. But they can be detected by their DNA.

    The future, as iceaura previously mentioned, will be nucleic acid probes. Hand held devices that will carry out a partial sequencing while you wait, and tell you what the dominant microorganisms are in the sample you are testing.

  8. #8
    voltage gated ion channel Hercules Rockefeller's Avatar
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    The first step in 'old-school' microbiological identification....

    http://en.wikipedia.org/wiki/Gram_staining

  9. #9
    Valued Senior Member WillNever's Avatar
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    Guys: "culturing" isn't a technique for identifying the microorganism. That is just the name for helping the organism proliferate in a habitable environment. For example, when a wound specimen (or a sputum specimen, or a urine specimen, or...) is taken and sent to the lab, they "culture" it so that it can be grow. Sometimes, people refer to "culturing the organism" as a sort of umbrella term that includes this process and then the testing that follows as well. However, that isn't technically correct.

    The testing begins after you have the culture. For bacteria, the lab will do a gram stain. All that is, is a staining of the culture. If the organism is stained, then it means it is "gram positive" which means that its cell membrane is very permeable and will be vulnerable to more antibiotics. If the stain doesn't stick, then the organism is called "gram negative" and it usually has a more resistant cell membrane, so not as many types of antibiotics will work on it. Also, some antibiotics work on only gram positive or only gram negative bacteria. Some work on both.

    This is called sensitivity testing. That's when they try out different antibiotics on the culture or expose it to different environments to see what will effectively kill the organism.

    As for identifying the actual species of microorganism, they still do that with a microscope. It happens that microbiologists are trained with the knowledge and reference material that helps them identify a microorganism by the way its structure and appearance and how it reacts to stains.
    Last edited by WillNever; 09-06-10 at 12:17 PM.

  10. #10
    voltage gated ion channel Hercules Rockefeller's Avatar
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    Quote Originally Posted by WillNever View Post
    Guys: "culturing" isn't a technique for identifying the microorganism. That is just the name for helping the organism proliferate in a habitable environment.

    Willnever,

    You are correct in your description of what “culturing” means, but you are totally incorrect when you suggest that this isn’t the means by which a microbiologist identifies a species of bacterium. Take it from someone who has actually performed diagnostic microbiology. (Albeit as an undergrad, but at least I have some actual experience in the subject at hand.)

    The mere act of culturing (ie. growing) bacteria in a series of successive different types of medium enables a microbiologist to identify the vast majority of clinically relevant bacteria simply from the growth results and the physiological properties that the bacteria exhibit as a result of the components of the medium. This time-honoured process can often identify a bacterium to the sub-species level without having to resort to modern molecular techniques (although a modern clinical micro lab will utilise both traditional microbiological techniques as well as modern molecular analysis techniques).

    There is a huge variety of different growth media that the clinical microbiologist can use to identify a species of bacterium – aerobic vs anaerobic growth, the shape, appearance and colour of colonies on indicator media, what temperature/pH/salts produces the best growth, whether the bacteria can utilise various compounds as sources of carbon (eg. acetate), whether the bacteria are capable of haemolysis, to what extent metal ions are toxic to the bacteria, how they metabolise phosphates, how they metabolise glutamate, whether they can metabolise thiol compounds, what classes of antibiotics they are sensitive to, and so on and so on and so on.

    Here is an example of the huge variety of media that is used in a micro diagnostic unit to culture bacterial samples for the purpose of identification:
    http://www.microbiol.unimelb.edu.au/...atalogue2.html
    http://www.microbiol.unimelb.edu.au/...atalogue3.html

  11. #11
    Valued Senior Member WillNever's Avatar
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    Hercules, introducing the organism to agar plates containing different antibiotics and wacky pHs (which is what is contained in those links) would be sensitivity testing. It just overlaps with growing them in a rather horrid culture, in those cases. Very generally, you would want the organism to proliferate enough so that you can introduce it to those different environments... without your whole sample being killed by one plate.

    You are interpreting the word "culturing" in an unnatural way. The growing of the bacteria doesn't help you identify them. The environment in which they are exposed to does.

  12. #12
    voltage gated ion channel Hercules Rockefeller's Avatar
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    Quote Originally Posted by WillNever View Post
    You are interpreting the word "culturing" in an unnatural way.
    That’s your opinion. The time I spent in an MDU argues that I know the meaning of the word ‘culturing’.


    Quote Originally Posted by WillNever View Post
    The growing of the bacteria doesn't help you identify them.
    It most certainly does.


    Quote Originally Posted by WillNever View Post
    The environment in which they are exposed to does.
    You’re playing childish semantic word games. That’s like saying it’s not bullets that kill people, it’s the loss of blood. Such semantic word games are a waste of time.

  13. #13
    Valued Senior Member WillNever's Avatar
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    Quote Originally Posted by Hercules Rockefeller View Post
    That’s your opinion. The time I spent in an MDU argues that I know the meaning of the word ‘culturing’.
    Hercules, I have taken microbiology and I've taken a ton of wound specimens to the lab here, where I know most of the people in there and what they do. Perhaps in the country where you live, they interpret the word somewhat differently.
    You’re playing childish semantic word games.
    Actually, I was thinking that about you. Everything I said is pretty much spot-on accurate, so you should probably cease to pursue this further, lest you accidently fall prey to the need to feel and sound "authoritative" to others.
    Last edited by WillNever; 09-07-10 at 07:39 AM.

  14. #14
    What the hell, man? Idle Mind's Avatar
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    WillNever, would you agree that the nutrient agar on which the bacteria are cultured is referred to as their environment?

  15. #15
    uniquely dreadful S.A.M.'s Avatar
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    Quote Originally Posted by WillNever View Post
    Hercules, I have taken microbiology and I've taken a ton of wound specimens to the lab here, where I know most of the people in there and what they do. Perhaps in the country where you live, they interpret the word somewhat differently.

    Actually, I was thinking that about you. Everything I said is pretty much spot-on accurate, so you should probably cease to pursue this further, lest you accidently fall prey to the need to feel and sound "authoritative" to others.
    Culturing is the very first step in microbial identification studies. This immediately tells you if the microbe grows in the broth or agar, if there is turbidity or gas formation and in the proper culture medium, one can detect species or genus specific antigens or nucleotide sequences in the specimen, culture medium or the cell culture system. In order to identify the bacteria, one needs to know its growth pattern by culturing, it is also used to isolate colonies by growth pattern, a necessary step in identification, plus the morphology of the colony on the culture media as well as by microscopic examination. The selection of the tests itself will depend on the kind and number of microorganisms present - in the case of bacteria, whether they are aerobic or anaerobic, gran positive or gram negative, bacillus or cocci - for filamentous fungi, identification is based almost entirely on morphology, gram positive bacteria that grow in the presence of air and carbon dioxide are easier to identify than gram negative bacteria which require more complex testing. Its only in the last decade or so that non-culture methods of identification have become available ie RT-PCR and microarrays.

    I spent six months wading through poop to identify probiotic microorganisms in humans and believe me you, its not easy

    Here is a nice short paper on new techniques for the OP:

    http://www.labautomation.org/journal/JALASneakPeek.pdf

    Some older methods explained:
    http://www.microbeid.com/Methods/methods.html

    A research paper on the topic:

    Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA
    http://onlinelibrary.wiley.com/doi/1...005.06.041/pdf
    Last edited by S.A.M.; 09-07-10 at 04:03 PM.

  16. #16
    Valued Senior Member WillNever's Avatar
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    Guys: you're just using different wording for something I already explained.

  17. #17
    uniquely dreadful S.A.M.'s Avatar
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    Really? What is the word you use for growing a specimen in broth or agar to determine morphology and growth pattern?

  18. #18
    voltage gated ion channel Hercules Rockefeller's Avatar
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    Quote Originally Posted by WillNever View Post
    Everything I said is pretty much spot-on accurate.....
    If you say so. (Nobody else is.)

    Quote Originally Posted by WillNever View Post
    ...so you should probably cease to pursue this further, lest you accidently fall prey to the need to feel and sound "authoritative" to others.
    You made me smile. I’m more than happy to let people draw their conclusions. I’m pretty sure what 99% of them will conclude.

    Quote Originally Posted by WillNever View Post
    Guys: you're just using different wording for something I already explained.
    Yeah, way to back down.

  19. #19
    Registered Senior Member Cat_with_no_eyes's Avatar
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    Quote Originally Posted by WillNever View Post
    Guys: "culturing" isn't a technique for identifying the microorganism. That is just the name for helping the organism proliferate in a habitable environment. For example, when a wound specimen (or a sputum specimen, or a urine specimen, or...) is taken and sent to the lab, they "culture" it so that it can be grow. Sometimes, people refer to "culturing the organism" as a sort of umbrella term that includes this process and then the testing that follows as well. However, that isn't technically correct.

    The testing begins after you have the culture. For bacteria, the lab will do a gram stain. All that is, is a staining of the culture. If the organism is stained, then it means it is "gram positive" which means that its cell membrane is very permeable and will be vulnerable to more antibiotics. If the stain doesn't stick, then the organism is called "gram negative" and it usually has a more resistant cell membrane, so not as many types of antibiotics will work on it. Also, some antibiotics work on only gram positive or only gram negative bacteria. Some work on both.

    This is called sensitivity testing. That's when they try out different antibiotics on the culture or expose it to different environments to see what will effectively kill the organism.

    As for identifying the actual species of microorganism, they still do that with a microscope. It happens that microbiologists are trained with the knowledge and reference material that helps them identify a microorganism by the way its structure and appearance and how it reacts to stains.
    Yeah, I have to say I agree with you on that one!

  20. #20
    Valued Senior Member WillNever's Avatar
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    Thanks. A lot of people tend to conflate the terms "culturing" and "sensitivity testing" in a way that is colloquial and common... but on a technical level, they are still two different (although interrelated) things.

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