Can Gram Staining be done from a Selective Media?

Hi microgupta,

  Gram staining can be done on almost anything - tissue, sputum,
blood,urine, liquid media, solid agar media, etc.! Selective media
simply inhibits growth of organisms that you *don't* want!

  I don't know exactly what kind of selective media you are speaking
of...but I often Gram stained organisms from Thayer Martin, PEA, HE,
and TSA slants, all selective media. What might be difficult to do
would be to subculture an organism from selective media to another
media...say a broth. You might take inhibiting substances from the
selective media onto the new media.

 An important step when selecting a colony on the selective media for
Gram stain, is to use your loop, or a wooden stick to select the
center of the colony. You want to avoid scooping up agar that can, for
a novice, be confusing when examining the slide. (You will quickly
learn the appearance of agar artifacts under the 'scope however!) You
also want to make a *thin* smear, being careful not to damage cells
and organisms while doing so.

Below, I have found a few sites that describe making/staining a smear
with Gram stain. Many use organisms from selective media.
  

http://www2.truman.edu/~jherrera/Microbiology/unknown-instructions.pdf

http://66.102.7.104/search?q=cache:iYLR9QY39mkJ:biology.ucok.edu/PersonalPages/Meeks/Study%2520for%2520Lab%2520Test%25201.ppt+gram+stain+++selective+media&hl=en

http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/StreakingTechnique.htm

http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/GramStain.htm

Hope this clears things up for you! Please request an Answer
Clarification, before rating, if this answer is not clear.

Regards,
crabcakes

Search Terms
Knowledge of topic
Gram Stain + selective media

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Request for Answer Clarification by microgupta-ga on 05 Nov 2004 11:43 PST

Dear Crabcake
Thanks for the response. I am talking about staining form media like
Pseudosel for Pseudomonas or Mannitol salt agar for Staphylcoccus.
Whether I can Gram stain from these media , as in one article I have
read not to use selective media for gram stain as it makes gram
positive organisms to look like Gram negative coccoi. Though I never
faced such problem anytime still I am want to know the exact method.
Please refer the following link for reference (Trouble shooting -
Smear Preperation):
http://www.biolog.com/Microlog%20Minutes_1.pdf 
Regards
microgupta

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Clarification of Answer by crabcakes-ga on 05 Nov 2004 15:14 PST

Hi microgupta, Thank you for clarifying exactly which kind of
selective media you are using.

 I believe you are referring to this statement:

"The medium from which the colonies are taken is important. Often, if
you take colonies from liquid media or from selective media, cells
will appear Gram-negative and more coccoid.
Sol u t i o n s : Take sample from fresh colonies from nutrient agar medium."



This does not mean you *can't* Gram stain a colony from Pseudocel
media or mannitol salt. I have done it with great success. It's
possible that the organisms that were closer to the agar may appear
more coccoid,  but pseudomonas does stain Gram negative, and they are
short curved rods. No sarcasm intended here either, but by the time
you have a pure culture on selective media, one is reasonably and
presumptively sure what the organism should look like!  :-)
http://reaannecy.free.fr/Documents/infectio/pseudomonas.jpg


 Even if you get a few organisms that look *off* in morphology and
stain color, the majority of the organisms should stain just fine. As
I mentioned in the previous answer, you want to select your sample
from the center of the colony.  This will give *cleaner* organisms,
and avoind scraping agar along with the organisms.

"Pseudomonas is a Gram negative, rod shaped bacteria that possesses
polar flagella. Soil samples from various locations were reservoirs
for obtaining the bacteria and innoculated in broth. Successful
isolation of this bacteria produces small and smooth colonies on
Pseudosel plates that appear yellow, opaque, and round. Gram stain,
wet mount, catalase, and oxidase tests confirmed the presence of a
Pseudomonas species. Further experiments using oil plates showed no
visible signs of growth or oil degradation."
http://www.wam.umd.edu/~asmith/longjoyce/homepage.html


I'm not sure what exact procedure you are seeking. Other than making a
smear that is not too thick, using a fresh log-phase colony, and
selecting the middle of the colony, the staining procedure would be
the same. I want to remind you the types I listed in my answer, Thayer
Martin, PEA, HE, and TSA slants are also selective media. I have found
no documentation, other than your link, regarding problems in Gram
stains made from selective media. (In a way, all media is selective
media - not all organisms grow on any one media!)

The closest documentation I could find is this ststement from a lab
exercise, where the student is instructed to make Gram stain smears
from mannitol salt agar. "Examine the Mannitol Salt Agar and Blood
Agar restreak plates. Are the colonies the same
and the same as the originals? Gram stain each. After observing the
Gram stains, choose one  (either the skin or throat isolate) and
streak a BHI slant. Record your results in the blank
protions of the Results pages above."
http://www.fiu.edu/~makemson/MCB2000Lab/Ex8HumanSymBact.pdf


Just for fun, here's a photo of a plate growing P.aeruginosa
http://www.cat.cc.md.us/courses/bio141/labmanua/lab13/psa.html

If you need further clarification, and can tell me what "Exact method"
you are wanting, I will be glad to see if I can find this for you.

regards,
crabcakes

Thanks a lot. The answer helped me a lot.

If you have a sufficient quantity of cells, you can wash away the
selective media in some 'neutral' solution (e.g. phosphate buffered
saline) before the gram stain.  this would probably change the
staining problems.  it wouldn't change the morphology changes however.