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HELP! Why do I keep getting a mixed culture in my gramstain?

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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HELP! Why do I keep getting a mixed culture in my gramstain?

Postby SweetSarah » Mon Feb 20, 2012 2:00 am

Hi everyone I'm new here. I need help in doing one of the more earlier steps with my unknown-the gram staining part. I am not sure but I think I'm doing mine wrong. I hope you all could help explain to me why since my teacher doesn't really allow us to ask her, we can only ask eachother and no one I asked understood so I'm hoping someone here atleast can help me out! Thanks.

For my microbiology lab, we were to do our unknowns. I was given a mixed culture and have to identify the 2 unknowns. However, I did the gram stain after I did the streaking which I had a hard time with and that took me 2 days to do. So, I just did the gram staining Thursday and for some reason mine aren't coming out in one color (ex-pink or purple) but both. I get a mixture of colors, so that means I have a mixed culture right? I don't get then why it's not pure and keeps coming out as a mixed culture. what could I be doing wrong?? :(

After class, I stayed and kept doing the gram stain over and over but keep getting the same results. Sometimes the stain comes out more of one color than the other. For example for my blood agar plate, I did the gram stain and it was mostly stained pink BUT there were some purple. Should I just take what it mostly stains as or do it again?? Please help me. I'm afraid I may fail this class because I can't seem to do this right. I'd appreciate all help. My gram stains also keep varying like the first time I did it the blood agar it was mostly stained pink but next time it be purple (or gram positive) so that confuses me more. :(

So what could be the reasons for this?? And How can I get my culture to be a pure culture?? :/

Thanks so much in advance!
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Postby canalon » Mon Feb 20, 2012 3:23 am

Repeat streking of single and well isolated colonies would be the answer to insure that you have pure cultures.
As for your problem with colours: either you are not doing your staining properly, or you are doing it properly on high,y mixed populations with a different ratio of your 2 cultures every time.
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Re: HELP! Why do I keep getting a mixed culture in my gramstain?

Postby michimichi » Mon Feb 20, 2012 3:44 am

my plate count agar (Pour plating from a potable water) got green pigmentation that didnt occur usually.maybe it was pseudomonas,. i think
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Postby biohazard » Mon Feb 20, 2012 9:22 am

Like Canalon said, getting pure cultures is probably the key factor. Repeat the streaking several times if needed and be careful to pick isolated colonies and use sterile instruments. You should get well-isolated colonies with one or two well-streaked plates (take care to not take too much of the sample, because it easily overpopulates the plate and you cannot pick an isolated colony for the following plate - too much sample is a way more common problem that too little).

Have you prepared the staining solutions yourself? If one of the solutions is not properly done, it can cause wonky staining patterns. Also, if one of the staining steps goes wrong, you may end up having part pink and part purple slides. For example, if one of the solutions does not cover the whole area of the sample, the sample may partially retain the purple colour even if it was gram negative.

People new to Gram staining end up having mixed staining all the time because of the latter. Some bacteria also stain bit oddly by nature, usually this happens so that gram positive bacteria look negative on the slide. Other way round is rare and usually requires mistakes when performing the staining.

It is also important to be able to recognize real mixed culture from staining artefacts: in mixed cultures the bacterial cells are typically more or less mixed among one another (depending on their ratios and how well the bacteria are mixed when putting it on the slide). If there is problem with the staining, you usually end up having larger areas of purple and pink or pale and strong staining depending on what areas your reagents have covered, or you may have all-pink slides because of too long acetone treatment etc.

Long story short: first identify your problem, is it a mixed sample or failed staining and then take steps to fix the issue. Hope you can get your stains right!
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Postby Squirrell » Mon Feb 20, 2012 8:50 pm

I assume you're doing dilution streaks, not streak plates? That may be your problem if not.
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Postby SweetSarah » Sun Feb 26, 2012 5:41 am

I just want to say thank you to everyone who took the time to help me out and answer this. I think my main problem was with the gram staining as many of you mentioned and I did it again to make sure and finally got pure culture for my gram positive and negative. I was so happy mine came out all purple gram positive clusters so I'm assuming now it's a Staph and did some tests for that so far. For my gram negative it appears to be pink rods but they're really tiny!
I did redo the streaking in case before but got the same result/problem so realized it was probably my gram staining. Again thanks, I'm really glad I got some answers here since this was my first time posting and it makes me feel really welcome here.
I hope you all can help me along the way if I have more questions. Again thanks :)
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Re:

Postby SweetSarah » Sun Feb 26, 2012 5:42 am

Squirrell wrote:I assume you're doing dilution streaks, not streak plates? That may be your problem if not.


Hey actually I am doing streaking, Quadrant streaking to be exact. I probably should've mentioned that but just assumed that was the only kind of streaking. well it's the only kind we've done in my micro lab so far and only type I learned. so yeah but thats what I meant..
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Re:

Postby SweetSarah » Sun Feb 26, 2012 5:47 am

biohazard wrote:Like Canalon said, getting pure cultures is probably the key factor. Repeat the streaking several times if needed and be careful to pick isolated colonies and use sterile instruments. You should get well-isolated colonies with one or two well-streaked plates (take care to not take too much of the sample, because it easily overpopulates the plate and you cannot pick an isolated colony for the following plate - too much sample is a way more common problem that too little).

Have you prepared the staining solutions yourself? If one of the solutions is not properly done, it can cause wonky staining patterns. Also, if one of the staining steps goes wrong, you may end up having part pink and part purple slides. For example, if one of the solutions does not cover the whole area of the sample, the sample may partially retain the purple colour even if it was gram negative.

People new to Gram staining end up having mixed staining all the time because of the latter. Some bacteria also stain bit oddly by nature, usually this happens so that gram positive bacteria look negative on the slide. Other way round is rare and usually requires mistakes when performing the staining.

It is also important to be able to recognize real mixed culture from staining artefacts: in mixed cultures the bacterial cells are typically more or less mixed among one another (depending on their ratios and how well the bacteria are mixed when putting it on the slide). If there is problem with the staining, you usually end up having larger areas of purple and pink or pale and strong staining depending on what areas your reagents have covered, or you may have all-pink slides because of too long acetone treatment etc.

Long story short: first identify your problem, is it a mixed sample or failed staining and then take steps to fix the issue. Hope you can get your stains right!


@biohazard: Thank you so much! Your advice was so much in detail and really appreciated. You were right it was my gram staining that caused the problem. I think it was just too much decolorization/alcohol step I added that was why there were some pink in my gram positives. But I redid it several times and finally got them separate so am happy about that. The thing is though there is very little pink on my gram positive but like on the corner if you look on m slide but pretty much I can tell it should be gram positive because most of it is purple. it came out in tiny purple clusters by the way so I'm assuming I got a Staph organism and I just did the mannitol test so I know I don't got Staph aureus but am still waiting on other tests now to see which I can narrow it down to. Do you think I should still redo it even though am pretty sure it's gram positive?
My gram negative came out perfect though. they came out in tiny pink rods. Super tiny that I probably couldn't see if I didn't have my glasses on lol. Anyways thanks for all this. I did restreak too but i think it's mainly my stianing that caused me so much confusion. Thanks again :D
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Re: HELP! Why do I keep getting a mixed culture in my gramstain?

Postby SweetSarah » Sun Feb 26, 2012 5:55 am

michimichi wrote:my plate count agar (Pour plating from a potable water) got green pigmentation that didnt occur usually.maybe it was pseudomonas,. i think

You know what you could be right. I actually got mine to be pure culture after redoing the gram stianing part so that's why I had that problem earlier. but I got more far with my gram neg than my positive with my tests. I've done it so far and since my glucose test came out negative I got to narrow it down to a Pseudomonas. Now what I'm wondering is how in the world u knew that before I did! lol

Like I had to do all these tests just to find that out. wow you must be smart. You really seem on to something. btw u know what kind of pseduomonas it could possibly be? Mine came out glucose negative and my TSI slant has a red (alkaline) butt but has black on top which i'm assuming is for H2S (hydrogen sulfide) production but I don't know hwat type of Pseudomonas would produce H2s? do u know by any chance?? u just seem really smart and since u knew all this I was just hoping you could help me out with this. I tried looking this up but coudn't find anything. I hope i didn't do the tests wrong :/
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Re: Re:

Postby biohazard » Tue Feb 28, 2012 7:24 am

SweetSarah wrote:
@biohazard: Thank you so much! Your advice was so much in detail and really appreciated. You were right it was my gram staining that caused the problem. I think it was just too much decolorization/alcohol step I added that was why there were some pink in my gram positives. But I redid it several times and finally got them separate so am happy about that. The thing is though there is very little pink on my gram positive but like on the corner if you look on m slide but pretty much I can tell it should be gram positive because most of it is purple. it came out in tiny purple clusters by the way so I'm assuming I got a Staph organism and I just did the mannitol test so I know I don't got Staph aureus but am still waiting on other tests now to see which I can narrow it down to. Do you think I should still redo it even though am pretty sure it's gram positive?
My gram negative came out perfect though. they came out in tiny pink rods. Super tiny that I probably couldn't see if I didn't have my glasses on lol. Anyways thanks for all this. I did restreak too but i think it's mainly my stianing that caused me so much confusion. Thanks again :D


Well, if nothing else, doing a few more Gram stains helps you learn to understand the staining patterns and possible artefacts that may occur on the slides. You should keep doing staining with different bacteria so long that you are sure you can say if it is gram+ or -, at least in vast majority of cases.
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Postby SweetSarah » Sun Mar 04, 2012 6:57 pm

thanks everyone I finally got the gram staining! :)
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Postby michimichi » Tue Mar 20, 2012 2:21 am

hi sweetsarah, sorry for the late reply, i have been busy at work,
regarding the Pseudomonas, ...
since im into water testing, the most common is the Pseudomonas aeruginosa.
did u do all necessary test to determine if its pseudomonas?
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