Microbiology Spring 2007 - page 22

:welcome: Gonna try to stick this one!!! Heres to a wonderful semester for all us MBers :):welcome:... Read More

  1. by   JDLAStew
    I've googled various things....no my teacher never looked at any of our slides even before we started. Her whole deal is pretty much teach yourself. After searching I think I've got it down to either one of the 2 bacillus species. The picture of b. subtilis on wikipedia looks identical to my Gram stain--looks magenta and purple (maybe not so pink??) However, my agar growth looks more like cereus from this website http://www.bact.wisc.edu/Microtextbo...cle&art_id=123 in fact, my cereus looks identical to that! We are supposed to use Bergey's during the final too and I have no clue about how to use it and my teacher has never shown us. I have felt very lost all semester and it's across the board with my classmates. I know a lot of people enjoy micro but I've hated it...I've been frustrated and lost since day one! And we didn't make our own slants. She had slants numbered and we had to take it and do a gram stain from it and plate it in NA at 2 different temps. Then start from there. We can do MA, MSA, and EMB but we can't do them all unless we justify why we did them in our paper. And, did any of you have cereus look peach in agar before? When we grew it earlier this semester ours turned peach and I can't find a peach sample anywhere on the interent. I think ours was messed up then so that is throwing me off. Ack! Anyway I'm gonna try to get some sleep and NOT think about micro!
  2. by   akspudus
    What tests are you running on your unknown? Fermentation? Catalase? IMViC? I had both of those bacteria species as a possibility in my unknowns. I have all the test results from Bergey's compiled into a spreadsheet if you need them.
    B. Cereus was one of my unknowns. I had the following test results:
    Gram - Pos, Acid Fast - Neg, Endospore - Pos
    Lactose Fermentation - neg for acid and gas
    Dextrose Fermentation- pos for acid neg for gas
    Mannitol Fermentation- Neg for acid and gas
    Nitrate Reduction - Pos Citrate Utilization Pos
    Starch Hydrolysis - Pos Catalase Test Pos
    Methyl Red - Pos

    Cultural Characteristics
    Pigment - Beige to White Texture - Smooth Size - Large Spreading
    Form - Rhizoid Elevation - Flat to Raised Margin - Undulated to curled

    Hope this helps....let me know if you need a copy of those spread sheets or have any other questions.

    akspudus

    P.S. Make sure you take a look online at Gram staining techniques. The staining protocol isn't too hard, but there are some common places people make mistakes. Also, be sure your smear isn't to thick. You should barely be able to tell that there is something there. If you pile to many cells into the smear, then your staining will be unreliable.
    Last edit by akspudus on Apr 13, '07
  3. by   JDLAStew
    We get to do nutrient agar, Eosin Methylene Blue, MacConkey agar, and Mannitol Salt. I believe we get to do catalase. Our unknowns could be 1 of 9 and based on colony morphology and the Gram stain, I know I've got one of the 2. My agar plate looks identical to b.cereus, but my gram stain doesn't look like it--more like subtilis. I would appreciate any help. I was up til 5am googling, and checking on ask.com. I'll PM you my email address so you can send anything and everything you have! You can hold me to it, I will help you anyway possible that I can as we go through school! I'm really just upset and worried over this! (Can you tell I freak out, a lot? LOL)
  4. by   akspudus
    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
  5. by   peacelovestar
    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.
    Last edit by peacelovestar on Apr 13, '07
  6. by   Jenny67
    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
  7. by   akspudus
    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
  8. by   Jenny67
    Ohhh..a 4 way streak is a good idea!

    I think I would've prefered your Professor and the slant cultures!!
  9. by   np_wannabe
    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...
  10. by   akspudus
    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
    Last edit by akspudus on Apr 14, '07
  11. by   np_wannabe
    Quote from akspudus
    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!
  12. by   akspudus
    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
    Last edit by akspudus on Apr 15, '07
  13. by   np_wannabe
    Quote from akspudus
    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!

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