Microbiology Spring 2007

np_wannabe · Apr 16, 2007

We just inoculated our cultures today, and I'm worried about my gram stain. I left the ethanol on for about 30 seconds, instead of about 5. I keep reading places that say you can't leave it on too long because of XYZ, but does anyone know how long is too long? I have a G- rod, but I'm just not sure if i screwed it up or if that is reliable....anyone know how long is too long??? I'm going to see what I can find over the next couple of days, if I can't find anything that says I'm okay, I'm going to ask if I can do another stain.
I hope someone out there has some idea...

I read my media today--I definitely have E coli, so my G- stain was right! If anyone out there leaves there ethonal on for a little too long, it's okay...

me33 · Apr 18, 2007

I have a lab test tomorrow and was wondering if anyone knows what the substrate is for MRVP test. Is there one substrate for the MR and a different one for VP? I can not find the answer. He has these questions on a study guide. Any help would be greatly appreciated.

akspudus · Apr 18, 2007

Both the Methyl Red (MR) and Voges Proskauer (VP) tests use the same media. MR-VP media. Its same media for both tests. Different reagents obviously:

Methyl Red for MR Test

Barritt's reagent (Parts A and B) for VP Test

Hope this helps. Let me know if you need more. I think I have the actual MRVP recipe somewhere.

Akspudus

me33 · Apr 18, 2007

Thanks for your help. I did have those as reagents but he is also asking for substrate. What do you think because I can not find the answer for a substrate

JDLAStew · Apr 19, 2007

I thought I had this thing licked. Tonight, my gram stain looked Gram positive. At this point, all I have for sure are rods. My EMB turned out pinkish/purple with a yellow halo?!?!?! My MacConkey had a slight bit of pink growth were I started and then went to nothing at the bottom. I haven't completed my last plate which is Mannitol Salt. My nutrient agar growth is creamy white/buff, shiny, flat. And like I said rods. Does anyone know why my EMB turned out this way? It doesn't make sense with any of my previous results. Yep, there they are...there's the tears.

akspudus · Apr 20, 2007

Hmmm....You can rule out E. Coli. E. Coli forms a metallic green sheen on the surface of its colonies. A for growth in EMB, only gram-negative bacteria grow on EMB agar. Gram-positive bacteria are inhibited by the dyes eosin and methylene blue added to the agar. Enterobacter produces large, mucoid, pink to purple colonies with no metallic green sheen on EMB agar.

The yellow halo you see is an area where nutrients have been used and depleted from the agar.

So I would be thinking E. aerogenes.

E. aerogenes growth on MacConkey Agar is described as good to excellent with a pink to red mucoid apearance.

And Yup...its a rod shaped bacteria.

So....my money is on E. aerogenes. You need to do some carbohydrate fermentation tests...and Methyl Red (MR) and Voges Proskauer (VP) tests to be sure.

Hope this helps.

akspudus

JDLAStew · Apr 20, 2007

one problem, we don't have e. aerogenes. and we can only do one more plate, mannitol salt. we are only allowed to do nutrient agar, emb, ma, and msa and 2 gram stains..and a catalase test. i was catalase positive. the only microbes we received were 1 of the following 9:

bacillus

cereus

bacillus

subtilis

escherichia

coli

klebsiella

pneumoniae

micrococcus

luteus

micrococcus

roseus

proteus

vulgaris

serratia

marcescens

staphylococcus

epidermis

any ideas??

akspudus · Apr 20, 2007

Dang....no E. aerogenes...that kinda bights. Hmmm....its got to be Gram negative for it to grow on EMB. Gram Neg bacilli. E. Coli is already ruled out since you didn't get a metalic green color on your EMB. So that leaves K. Pneumonia, S. marcescens or P. vulgaris. Both K. pneumonia and S. marcescens have blueish purple color characteristics on EMB.

Your mannitol salt should end up showing no growth.

S. marcescens is aerobic - catalase positive.

P. vulgaris is aerobic - catalase positive.

K. Pneumonia is anaerobic - catalase negative.

So...

Most likely it is P. vulgaris based upon your observations.

Akspudus

JDLAStew · Apr 20, 2007

It doesn't look "swarming" yet the bacteria really grew in size on the plate. You can see my streaks then it really forms large colonies out from it. Very shiny and flat. It resembles b. cereus in the agar but with everything I do it looks like it could be something else. I'm about to start writing the paper on one of them and pray I get it right. My friend and I had a good breakdown cry tonight. She's stuck on hers but I would be money hers is B.subtilis--well with the except her EMB looked like mine. The purple-ish color with the yellow halo look. Her's is dry, mine is shiny. I'm sorry for explaining this junk over and over. My hubby has no idea what I'm talking about and this seems to be my only vent area. Other students will look over at what I have and say, no that's not what you have--I'm sure I have that and yours doesn't look like it. ACK! Do they think that helps? Anyway I'm off for a small weekend trip. Will be back Sunday. Have a good weekend and thank you Akspudus for ALL of your help. I mean it.

catzy5 · Apr 20, 2007

It doesn't look "swarming" yet the bacteria really grew in size on the plate. You can see my streaks then it really forms large colonies out from it. Very shiny and flat. It resembles b. cereus in the agar but with everything I do it looks like it could be something else. I'm about to start writing the paper on one of them and pray I get it right. My friend and I had a good breakdown cry tonight. She's stuck on hers but I would be money hers is B.subtilis--well with the except her EMB looked like mine. The purple-ish color with the yellow halo look. Her's is dry, mine is shiny. I'm sorry for explaining this junk over and over. My hubby has no idea what I'm talking about and this seems to be my only vent area. Other students will look over at what I have and say, no that's not what you have--I'm sure I have that and yours doesn't look like it. ACK! Do they think that helps? Anyway I'm off for a small weekend trip. Will be back Sunday. Have a good weekend and thank you Akspudus for ALL of your help. I mean it.

Are you kidding? don't apologise this is helping me! I can't help you I am not that far yet we just started our's I apprectiate hearing your tests and what you have.

I am frustrated with mine as wll, seems like my stainings weren't coming out very well yesterday I couldn't gram stain at all, My negitive came out ok and I am fairly certain we have no spores but thats about it.

catzy5 · Apr 25, 2007

So I am not sure if anyone can help me but the Large tube is my FTM What would you say the oxygen requirements are? Faculative aneorobe? We don't have any strict aneorobes. So why is there no growth at the top? What have I done??

keystudent · Apr 26, 2007

So I am not sure if anyone can help me but the Large tube is my FTM What would you say the oxygen requirements are? Faculative aneorobe? We don't have any strict aneorobes. So why is there no growth at the top? What have I done??

Don't get discourage. I don't think you've done anything wrong. Could it Aerotolerant anaerobe? :idea: They are microorganisms that can not use O2, but are no harmed by it either. So, there would be growth through out but not much, if any, at the top.

I hope this helps. We are finish with our unknowns and cleaning up lab. So, I remember the gussing game hoping I could get some help from somewhere. Well, I never thought to check here. I've been so busy, this is the first time in awhile I have actually been on here. Well, I am saying all of that to say this even though I didn't have the help I still did very well on my paper. It was 13 pages long and I got a 95 on it. The best advice I can give you is to look at all of the test that would help you to narrow it down. Ex: Indole (SIMS), the Phenylalanine Deamination, Oxidase, EMB, Endo, etc. I hope I was able to help. If you have anymore questions post a reply. God Speed to You!! You will get it.

PS, try to keep a good journal entry. What I mean by that is go ahead and explain exactly what the test shows. Not that it is just neg or pos. Go ahead and take the few extra minutes to write it in with the results. This way you would have a better understanding of it.

Night! :) :balloons: