[R-sig-ME] [R] Fwd: Re: ANOVA and Pseudoreplication in R

Ben Ward benjamin.ward at bathspa.org
Sun Feb 27 18:12:39 CET 2011

On 27/02/2011 05:46, Robert A LaBudde wrote:
> I think you are over-concerned with the term "pseudo-replication". All 
> this means is that the error source is nested, and not a full replicate.
> What you haven't done, and need to do, is to describe your experiment 
> in terms of the real variables and error sources. Then code them as 
> "fixed" or "random" and write the model form.
> You description is very complex and confusing. You need to identify:
> 1. Exactly what culture is used in each trial.
> 2. All subcultures: NE vs EV, which chemical, etc.
> 3. Which plate.
> 4. Which circle on which plate.
> 5. Which measurement on which circle.
> After you have identified all descriptors that identify each 
> individual measurement, put them in a file with columns for factors 
> and the outcome.
> At this point it should be clear that you have a number of random 
> factors: plate, circle, measurement, plus any culture replicates, if 
> you have them.
> One you successfully do this, you can write the model equation with 
> the proper nesting.
> The time to do all this is BEFORE you conduct the experiment.
I think then, number 1 mute, because it's all E.coli culture. NE and EV 
I think is a fixed effect, it should affect the mean, rather than the 
error/variance, as it the the bit of the experiment I'm manipulating and 
want to evaluate. Which plate is mute, because I put 3 circles on each 
plate, so there are no more than one plate for each treatment - because 
that's how the methodology is done in all books on the subject, and 
there wouldn't be enough plates to go around. Circle could be random 
because it could alter the variance/errors, I don't expect it to alter 
the mean. The measurements on the cirlces - there are 4 for each circle, 
are my (y) value, what I'm measuring, so I'm unsure on this one, I took 
4 measurements from each circle, and I would have thought they are all 
very similar in which case I think they may be a source of a little 
error, but this is also the thing I'm measuring so I'm unusre on what to 
do about this. As I said, my first port for advise told me It wouldn't 
be horrificly detrimental to my project at this level if I took means of 
the 4 values of each circles and worked with those instead, but if I can 
solve this with a mixed effect model, it would be better.
The reason this was not worked out before the experiment, was that it 
was always thought a straight worward ANOVA was going to be sufficient, 
because this project has not been done in our uni before and new 
territory even for my supervisor, were both discovering things about 
this project, so it's been a little experimental in places, in having to 
adapt the methodology if a certain issue of practicality arose. But it's 
nearly done now.

Ben W.
> At 10:44 AM 2/26/2011, Ben Ward wrote:
>> On 25/02/2011 21:22, Ben Ward wrote:
>>> -------- Original Message --------
>>> Subject:        Re: [R] ANOVA and Pseudoreplication in R
>>> Date:   Fri, 25 Feb 2011 12:10:14 -0800
>>> From:   Bert Gunter<gunter.berton at gene.com>
>>> To:     Ben Ward<benjamin.ward at bathspa.org>
>>> CC:     r-help<r-help at r-project.org>
>>> I can hopefully save bandwidth here by suggesting that this belongs on
>>> the R-sig-mixed-models list.
>>> -- Bert
>>> As an aside, shouldn't you be figuring this out yourself or seeking 
>>> local consulting expertise?
>> I did consult with the lecturer at university that knows most about 
>> stats, and he advised me:
>> "Pseudo replication is really about a lack of independence between 
>> measurements, So you need to work backwards and see where you are 
>> building in a known lack of independence.  And where that is the case 
>> you need to use means of all the values."
>> And I have done this and came to the conclusion I mentioned as to 
>> where I thought Pseudoreplicaton was comming from, however, I do not 
>> know about the one other 'potential' source as it really is for me at 
>> least, a grey area.
>> I've consulted a few forums that deal with the theory more and await 
>> any response. Until then I'll have to try and get as many opinions on 
>> it as possible.
>> -Ben W.
>>> On Fri, Feb 25, 2011 at 9:08 AM, Ben 
>>> Ward<benjamin.ward at bathspa.org>   wrote:
>>>>   Hi, As part of my dissertation, I'm going to be doing an Anova, 
>>>> comparing
>>>>   the "dead zone" diameters on plates of microbial growth with 
>>>> little paper
>>>>   disks "loaded" with antimicrobial, a clear zone appears where 
>>>> death occurs,
>>>>   the size depending on the strength and succeptibility. So it's 
>>>> basically 4
>>>>   different treatments, and I'm comparing the diameters (in mm) of 
>>>> circles.
>>>>   I'm concerned however, about Pseudoreplication and how to deal 
>>>> with it in R,
>>>>   (I thought of using the Error() term.
>>>>   I have four levels of one factor(called "Treatment"): NE.Dettol, 
>>>> EV.Dettol,
>>>>   NE.Garlic, EV.Garlic.   ("NE.Dettol" is E.coli not evolved to 
>>>> dettol,
>>>>   exposed to dettol to get "dead zones". And the same for 
>>>> NE.Garlic, but with
>>>>   garlic, not dettol. "EV.Dettol" is E.coli that has been evolved 
>>>> against
>>>>   dettol, and then tested afterwards against dettol to get the 
>>>> "dead zones".
>>>>   Same applies for "EV.Garlic" but with garlic).  You see from the 
>>>> four levels
>>>>   (or treatments) there are two chemicals involved. So my first 
>>>> concern is
>>>>   whether they should be analysed using two seperate ANOVA's.
>>>>   NE.Dettol and NE.Garlic are both the same organism - a lab stock 
>>>> E.coli,
>>>>   just exposed to two different chemicals.
>>>>   EV.Dettol and EV.Garlic, are in principle, likely to be two 
>>>> different forms
>>>>   of the organism after the many experimental doses of their 
>>>> respective
>>>>   chemical.
>>>>   For NE.Garlic and NE.Dettol I have 5, what I've called 
>>>> "Lineages", basically
>>>>   seperate bottles of them (10 in total).
>>>>   Then I have 5 Bottles (Lineages) of EV.Dettol, and 5 of 
>>>> EV.Garlic. - This
>>>>   was done because there was the possiblity that, whilst I'm 
>>>> expecting them
>>>>   all to respond in a similar manner, there are many evolutionary 
>>>> paths to the
>>>>   same result, and previous research and reading shows that 
>>>> occasionally one
>>>>   or two react differently to the rest through random chance.
>>>>   The point I observed above ("NE.Dettol and NE.Garlic are both the 
>>>> same
>>>>   organism...") is also applicable to the 5 bottles: The 5 bottles 
>>>> each of
>>>>   NE.Garlic and NE.Dettol are supposed to be all the same organism 
>>>> - from a
>>>>   stock one kept in store in the lab.
>>>>   There is potential though for the 5 of EV.Garlic, to be different 
>>>> from one
>>>>   another, and potential for the 5 EV.Dettol to be different from 
>>>> one another.
>>>>   The Lineage (bottle) is also a factor then, with 5 levels 
>>>> (1,2,3,4,5).
>>>>   Because they may be different.
>>>>   To get the measurements of the diamter of the zones. I take out a 
>>>> small
>>>>   amount from a tube and spread it on a plate, then take three 
>>>> paper disks,
>>>>   soaked in their respective chemical, either Dettol or Garlic. and 
>>>> press them
>>>>   and and incubate them.
>>>>   Then when the zones have appeared after a day or 2. I take 4 
>>>> diameter
>>>>   measurements from each zone, across the zone at different angles, 
>>>> to take
>>>>   account for the fact, that there may be a weird shape, or not quite
>>>>   circular.
>>>>   I'm concerned about pseudoreplication, such as the multiple 
>>>> readings from
>>>>   one disk, and the 5 lineages - which might be different from one 
>>>> another in
>>>>   each of the Two "EV." treatments, but not with "NE." treatments.
>>>>   I read that I can remove pseudoreplication from  the multiple 
>>>> readings from
>>>>   each disk, by using the 4 readings on each disk, to produce a 
>>>> mean for the
>>>>   disks, and analyse those means - Exerciseing caution where there 
>>>> are extreme
>>>>   values. I think the 3 disks for each lineage themselves are not
>>>>   pseudoreplication, because they are genuinley 3 disks on a plate: 
>>>> the "Disk
>>>>   Diffusion Test" replicated 3 times - but the multiple readings 
>>>> from one disk
>>>>   if eel, is pseudoreplication. I've also read about including 
>>>> Error() terms
>>>>   in a formula.
>>>>   I'm unsure of the two NE. Treatments comming from the same 
>>>> culture does not
>>>>   introduce pseudoreplications at Treatment Factor Level, because 
>>>> of the two
>>>>   different antimicrobials used have two different effects.
>>>>   I was hoping for a more expert opinion on whether I have identified
>>>>   pseudoreplication correctly or if there is indeed 
>>>> pseudoreplication in the 5
>>>>   Lineages or anywhere else I haven't seen. And how best this is 
>>>> dealt with in
>>>>   R. At the minute my solution to the multiple readings from one 
>>>> disk is to
>>>>   simply make a new factor, with the means on and do Anova from 
>>>> that, or even
>>>>   take the means before I even load the dataset into R. I'm 
>>>> wondering if an
>>>>   Error() term would be correct.
>>>>   Thanks,
>>>>   Ben W.
>>>>   ______________________________________________
>>>>   R-help at r-project.org mailing list
>>>>   https://stat.ethz.ch/mailman/listinfo/r-help
>>>>   PLEASE do read the posting guide 
>>>> http://www.R-project.org/posting-guide.html
>>>>   and provide commented, minimal, self-contained, reproducible code.
>> ______________________________________________
>> R-help at r-project.org mailing list
>> https://stat.ethz.ch/mailman/listinfo/r-help
>> PLEASE do read the posting guide 
>> http://www.R-project.org/posting-guide.html
>> and provide commented, minimal, self-contained, reproducible code.
> ================================================================
> Robert A. LaBudde, PhD, PAS, Dpl. ACAFS  e-mail: ral at lcfltd.com
> Least Cost Formulations, Ltd.            URL: http://lcfltd.com/
> 824 Timberlake Drive                     Tel: 757-467-0954
> Virginia Beach, VA 23464-3239            Fax: 757-467-2947
> "Vere scire est per causas scire"
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