Microbiology Spring 2007

akspudus · Apr 13, 2007

JDLAStew I just sent you a bunch of stuff. Hope your not on dial-up....its a rather large email...Lol.

If you find it useful, pass it on to anybody else that can use it.

I wish there was a way we could post files to this forum.

akspudus

peacelovestar · Apr 13, 2007

How many possible unknowns are you given?

Ours is 2 of a possible 23 and my head hurts just thinking about it. It's a mix meaning it has two different unknowns in one culture.

Cheers to everyone for the rest of the semester.

Jenny67 · Apr 13, 2007

We were told the test tubes that we chose had 2-5 unknowns and we had to isolate 2. "IF" I have done it correctly I have isolated my two unknowns and believe they are Streptococcus fecalis and Bacillus megaterium. Because I have 4 weeks until my report and slides must be turned in for the 'fun' of it I am going to start my processes over to make sure.

Good Luck to everyone! I am looking forward to this semester being over..

-Jenny

akspudus · Apr 14, 2007

Jenny67...sounds like you need to do a 4-way streak to isolate some colonies, then go from there. Sounds like you guys had more fun tasking for your unknowns. We were given two slant cultures and told to figure out what they were. You had a feeling one was going to be gram pos, the other gram neg.

Hope everyone does well!!

akspudus

Jenny67 · Apr 14, 2007

Ohhh..a 4 way streak is a good idea!

I think I would've prefered your Professor and the slant cultures!! :)

np_wannabe · Apr 14, 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... :uhoh3:

akspudus · Apr 15, 2007

We were taught to not let the alcohol rest on the smear. What we were taught to do is to hold the slide at an angle and drip the decolorizing agent (alcohol) uphill of the smear. This allows the alcohol to run over the smear, drop by drop. No time frame for how long....just long enough for the run off to come off clear. I know when I did this it was way longer than 5 seconds...more like 30 seconds...drop...drop...drop...etc just until the run off had no more crystal violet in it.

I wouldn't worry about leaving the alcohol on for 30 seconds. After the mordant (gram's iodine) is added, the crystal violet is pretty much locked into the gram positive bacteria. Leaving the alcohol on for too long will have more of a desiccating effect, damaging the cells...but I would expect that to take far longer than 30 seconds.

Don't sweat your Gram's Staining too much. As long as you get the steps in the right order, it is pretty hard to mess up. Smear preparation and fixing is more important. Too thick of a smear and your going to have unreliable results. Some of my classmates would make there smears so thick, you could see them "apparent" stain results from across the room. Make sure you only place a very small amount of inoculum on your slide, mix with one drop of water, spread it out on the slide....LET IT AIR DRY...then heat fix. The amount of inoculum I uses is about the size of the period at the end of this sentence. Only about 1-2 million cells....then spread out over 1-2 square cm. It should barely even look cloudy.

Be careful with your heat fixing. 2-3 quick swipes over a flame is enough. I usually hold the slide with my fingers when I do this....and those swipes over the flame are quick enough to not burn my fingers...or melt my gloves.

After smearing and fixing....looking through the slide should still look pretty much clear, maybe a little smudge...but clear. Stain away and be amazed at how many bugs are there in a nice even single layer. Easily visible to appreciate morphology and arrangement.

If you want, I could try and cut and paste...or email my step by step notes for smear and gram stain.

Anyhoo....hope this helps.

akspudus

np_wannabe · Apr 15, 2007

We were taught to not let the alcohol rest on the smear. What we were taught to do is to hold the slide at an angle and drip the decolorizing agent (alcohol) uphill of the smear. This allows the alcohol to run over the smear, drop by drop. No time frame for how long....just long enough for the run off to come off clear. I know when I did this it was way longer than 5 seconds...more like 30 seconds...drop...drop...drop...etc just until the run off had no more crystal violet in it.
I wouldn't worry about leaving the alcohol on for 30 seconds. After the mordant (gram's iodine) is added, the crystal violet is pretty much locked into the gram positive bacteria. Leaving the alcohol on for too long will have more of a desiccating effect, damaging the cells...but I would expect that to take far longer than 30 seconds.
Don't sweat your Gram's Staining too much. As long as you get the steps in the right order, it is pretty hard to mess up. Smear preparation and fixing is more important. Too thick of a smear and your going to have unreliable results. Some of my classmates would make there smears so thick, you could see them "apparent" stain results from across the room. Make sure you only place a very small amount of inoculum on your slide, mix with one drop of water, spread it out on the slide....LET IT AIR DRY...then heat fix. The amount of inoculum I uses is about the size of the period at the end of this sentence. Only about 1-2 million cells....then spread out over 1-2 square cm. It should barely even look cloudy.
Be careful with your heat fixing. 2-3 quick swipes over a flame is enough. I usually hold the slide with my fingers when I do this....and those swipes over the flame are quick enough to not burn my fingers...or melt my gloves.
After smearing and fixing....looking through the slide should still look pretty much clear, maybe a little smudge...but clear. Stain away and be amazed at how many bugs are there in a nice even single layer. Easily visible to appreciate morphology and arrangement.
If you want, I could try and cut and paste...or email my step by step notes for smear and gram stain.
Anyhoo....hope this helps.
akspudus

Thank you so much akspudus! I actually nail the gram stain every time--it's my favorite, but for some reason, I got distracted and thought I was using iodine (which you leave on for one minute), and not ethanol. by the time i came to my senses and realized i was not using iodine, it had been about 30 seconds. Unless my gram stain gave me a false reading, I have tiny G- rods...maybe E. coli?

I'm reading my media tomorrow to see what else i can find out about it...this is just too cool (I'm starting to think I'm a nerd at heart!)

Thanks again!

akspudus · Apr 15, 2007

Yes, if you did what you did. As in switch the gram's iodine for ethanol, you would get a gram VERY unreliable gram stain. Basically you would have a simple stain and not a differential stain. What you got to remember is that the gram iodine bonds with the crystal violet molecules in the periplasmic space of the cell wall. This makes the molecule to large to be washed away by the ethanol. Without the iodine, the crystal violet molecules are small enough to wash out and even gram positive bacteria WILL decolorize and appear to be gram negative.

akspudus

np_wannabe · Apr 15, 2007

Yes, if you did what you did. As in switch the gram's iodine for ethanol, you would get a gram VERY unreliable gram stain. Basically you would have a simple stain and not a differential stain. What you got to remember is that the gram iodine bonds with the crystal violet molecules in the periplasmic space of the cell wall. This makes the molecule to large to be washed away by the ethanol. Without the iodine, the crystal violet molecules are small enough to wash out and even gram positive bacteria WILL decolorize and appear to be gram negative.
akspudus

Good Point.

I'm going to email my teacher now and see if I can re-do my gram stain when I go in tomorrow.

Thank you for the information....I've been nervous about ruining the stain and thinking about re-doing it. This just confirms my suspicion.

Thanks again!

JDLAStew · Apr 16, 2007

After reading all about all your unknowns and whatnot, I feel bad for complaining! I should be grateful. Ours in 1 of 9 and no mixed cultures. After going through my notes and Akspudus', I'm pretty sure it's B. cereus....but of course I won't know for sure until my tests are done. At this point, I'm just dreading the paper--it's a minimum of 18 pages with no internet sources. Ick. Since I'm starting to complain again, I'll quit and be grateful that mine isn't as near as hard as the ones you all have.

Good luck to everyone. Just a few more weeks left, right? :)

ally21 · Apr 16, 2007

18 pages?! Double spaced? Mine came out to 7 pages single spaced. My teacher didn't want us to go overboard with explaining the procedures. We just had to explain why we did each test. Gram staining knocked a great majority off the list, then I did a streak plate, then the catalase test which helped me get down to just 3 unknowns. I was able to narrow it down to my final unknown by doing the MRVP tests. My teacher likes to knock off lots of points for stupid things. If we did a test that wasn't appropriate from the research on our unknown bacteria she'd probably deduct points for it. I'm glad it's over with because it took me almost 8 hours to finish the paper and I handed it in today. We had to figure out our unknown out of a list of 36 possibilities.