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Background

The spread plate method is a technique used in microbiology to enumerate and isolate bacteria or other microorganisms present in a sample. It involves evenly spreading a liquid sample containing microorganisms onto the surface of a solid agar medium in a Petri dish.

Inoculum Density: Inoculum density refers to the concentration or density of microorganisms (bacteria, fungi, etc.) present in the sample that is being plated onto the agar surface. It is typically measured as the number of colony-forming units (CFUs) per unit volume of the sample.

Inoculum Volume: Inoculum volume refers to the volume of the sample that is spread or plated onto the surface of the agar medium during the spread plate method. It is typically measured in microliters (µL) or milliliters (mL).

Question

Does inoculum volume affect spread plate outcome? If I pipetted 0.1ml 0.5 McFarland of bacteria into the agar plate before spreading, would it differ from pipetting 0.5ml of the same density?

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2 Answers 2

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Theoretically, for CFU determination the volume of the inoculum shouldn't matter as long as you plate a dilution series and find the plate with a countable number of colonies. It also shouldn't matter if you use the spread-plate method or the pour plate method or something else (like drop plates or spiral plates). But all of that assumes ideal conditions where you are able to get a perfect and uniform spread over the whole plate every time. In the real world there are practical limitations on the range of usable inoculum volumes when spreading liquid onto agar plates.

When making spread plates it's best to maintain continuous spreading motion until all (or almost all) of the liquid from the inoculum has been absorbed by the agar medium. If you don't, you can end up with unevenly distributed colonies, which can make otherwise countable dilutions difficult or impossible to count accurately. If the volume is too large, it may take an impractical amount of time for the liquid to absorb. On the flip side, the volume still needs to be large enough to cover the whole plate evenly. If it's too small (say 10µL) you may have trouble spreading it evenly across the entire surface.

There's also some potential for variation in consistency when dispensing different volumes. Most micropipettes are slightly more accurate at the high end of their range than the low end. So, IF you were using the same p1000 pipette, your replicates could be slightly more consistent with larger volumes than small ones. But, properly calibrated, the dispensed volume should vary by less than 10% even at the lowest setting of most pipettes, and if this were a concern, it could be remedied by switching to a more appropriately sized pipette.

Lastly, if you are trying to make a bacterial lawn, for something like a Kirby-Bauer test, changing the inoculum volume can affect the outcome of the test. McFarland standards aren't always perfectly calibrated for every organism, but overloading the plate with 5x the standard inoculum certainly has the potential of skewing the test results. The methods for these tests are standardized to improve repeatability between labs. Essentially, a denser inoculum will reduce the size of the inhibition zone, while a reduced inoculum will increase the inhibition zone. Since the inhibition zone determines whether a particular isolate is classified as resistant or susceptible to a given drug, inoculation protocols are standardized to make sure patients receive the appropriate care.

Source: EUCAST Disk Diffusion Method for Antimicrobial Susceptibility Testing - Version 9.0 (January 2021)

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  • $\begingroup$ Thanks for the great answer. I'm trying to do the last: bacterial lawn for Kirby-Bauer test. Could you reconsider that it won't affect kirby-bauer test? I'm saying this by gut feeling. It must have to do something with the McFarland standard concentration being fixed at 0.5 for the kirby bauer. $\endgroup$ Feb 23 at 17:32
  • $\begingroup$ @FreezingSoul I see. It's worth mentioning that AST protocols typically call for inoculating lawns with sterile cotton swab dipped in the bacterial suspension, rather than spreading a specific volume on a plate. I'm sure there's some variation in the actual volume transferred from the swab to the plate, but probably not on the order of the 5-fold difference specified in your question. See updates to my answer for more specifics on how inoculum density can impact the observed inhibition zones (I confess that I haven't evaluated those statements personally). $\endgroup$
    – MikeyC
    Feb 23 at 19:15
  • $\begingroup$ I tried to keep the question as abstract as possible so more people would benefit from it. But, my problem is that I think my agar soaks the 0.1ml so fast that I don't have time to spread it. Maybe because I dry them by incubating them for 24 hours after pouring, which I don't plan to give up, because it's primarily a sterility check. Is there any problem if I add my 0.1 inoculums to some 0.5ml saline before spreading and spread the whole 0.6ml, since i can't withdraw more inoculum volume? $\endgroup$ Feb 24 at 11:43
  • $\begingroup$ i think nevermind, the problem may be that i waited to too long for the spreader to cool, it says in a textbook that you got 15 seconds after pipetting the inoculum to spread otherwise the water will be soaked. $\endgroup$ Feb 24 at 11:48
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To add to the answer by @MikeyC . The spread plate technique for calculation of colony forming units has volume plated as a variable:

CFU/ml = (number of colonies x dilution factor) / volume of culture plated

This means that you account for the volume in the calculation performed after doing your counting.

Of course, as MikeyC mentioned, the volume matters to some extent as if you add too much of too high a density suspension, then you will have an uncountable plate. To compensate for this, what is usually done is a dilution series, where you choose which dilution is used for the calculation This also provides a secondary check on your count, as a plate below (lower dilution) the countable dilution should be TMTC and the one above should have approximately 1/(dilution factor) fewer colonies. For example, in a 10-fold dilution factor series, your countable plate might have 50 colonies, the one below should have ~500 (TMTC) and the one above should have about 5.

You should also note that the dilution factor is also compensated for in the equation.

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