Gram staining showing negativ and positiv bacteria

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Alexander
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Joined: Fri Jun 11, 2021 7:10 pm

Gram staining showing negativ and positiv bacteria

#1 Post by Alexander » Wed Aug 17, 2022 10:01 am

An example of grams staining showing gram-negative as well as gram-positive bacteria in close proximity. Specimen was taken from human sputum.
Sputum_Gram_2.jpg
Sputum_Gram_2.jpg (106.9 KiB) Viewed 881 times

Sure Squintsalot
Posts: 393
Joined: Mon May 16, 2022 3:44 pm

Re: Gram staining showing negativ and positiv bacteria

#2 Post by Sure Squintsalot » Wed Aug 17, 2022 10:41 pm

That's very cool....but:

What is "gram staining"?
How did you, specifically, do it?
What are its limitations?
Can I easily do it in the field?
What else might you see with this technique?

Alexander
Posts: 402
Joined: Fri Jun 11, 2021 7:10 pm

Re: Gram staining showing negativ and positiv bacteria

#3 Post by Alexander » Thu Aug 18, 2022 7:11 am

Sure Squintsalot wrote:
Wed Aug 17, 2022 10:41 pm
What is "gram staining"?
https://en.wikipedia.org/wiki/Gram_stain
How did you, specifically, do it?
I used a Gram staining kit consisting of 4 solutions.

solute the sputum for some time in ringer's solution
smooth out on a slide
let dry
heat fixing

1. crystal violet 1 minute
2. iodine 1 minute
rinse with water
3. decolorizer (a mixture of ethanol and acetone) 1 minute
rinse with water
4. safranine 1 minutes
rinse with water
let dry
dehydrate with isopropanol
cover
What are its limitations?
Not all bacteria react to it. An example are the mycobacteria causing tuberculosis.
Can I easily do it in the field?
You can do it in the field - but "easily"?
What else might you see with this technique?
Most animal and human cells react gram-negative as you may recognize in the picture shown. The big red spots are most probably the remainders of leukocytes for example.

Sure Squintsalot
Posts: 393
Joined: Mon May 16, 2022 3:44 pm

Re: Gram staining showing negativ and positiv bacteria

#4 Post by Sure Squintsalot » Thu Aug 18, 2022 10:58 pm

Thanks, Alexander, for taking the time for a detailed reply!

I'm trying to build a field microscopy and sampling kit. I'd like to be able to go off into the woods, find bacteria, and look at it under a microscope.
Unfortunately, I know next to nothing about sampling and prep techniques. I've been checking out kits, such as these: https://www.carolina.com/biological-med ... stain+kits:

Image

....but have a sneaking suspicion that they may a little ambitious for a first timer. In the woods.
Should I stick with something a little simpler?
Image

Alexander
Posts: 402
Joined: Fri Jun 11, 2021 7:10 pm

Re: Gram staining showing negativ and positiv bacteria

#5 Post by Alexander » Fri Aug 19, 2022 7:51 am

Sure Squintsalot wrote:
Thu Aug 18, 2022 10:58 pm

I'm trying to build a field microscopy and sampling kit. I'd like to be able to go off into the woods, find bacteria, and look at it under a microscope.

....but have a sneaking suspicion that they may a little ambitious for a first timer. In the woods.
Should I stick with something a little simpler?
Do mean bacteria or any type of microbes?

Bacteria are quite small. To see them at all staining or phase contrast and high power objectives are a must have.

Gram's staining is special and a first step in identifying bacteria by dividing them into two large groups.

If you just want to make the bacteria visible methylene blue is a good and simple choice. All bacteria will be stained blue and be visible with good contrast. Preparation is easy. Put your dry sample in a little amount of Ringer's solution to spread the bacteria into the solution. After a while bring a drop onto a slide and let it dry. Fix it by putting it 5 seconds into methanol or by drawing the slide a few times through the flame of a candle. Put a drop of methylene blue onto it. After a minute rinse with distilled water. Let dry. If you own immersion objectives designed for use without cover-slip you now may put the slide under the microscope. If your objectives require the use of cover-slips you must dehydrate the sample and cover it.

Microbes bigger - and much more interesting than bacteria - can easily be inspected in vitro. No preparation at all. Just put a drop of water on the slide, cover it and put it under the microscope. Low or medium power objectives are used for that type of examination.

Sure Squintsalot
Posts: 393
Joined: Mon May 16, 2022 3:44 pm

Re: Gram staining showing negativ and positiv bacteria

#6 Post by Sure Squintsalot » Fri Aug 19, 2022 5:54 pm

Again....your detailed reply is most useful.

Looks like I should stick to something even simpler for my first time out!

It would be, at some future date, be interesting to me to actually look at bacteria in the field. What's on the underside of dead leaves? In the first 3mm of soil? That fresh deer poop? In the bark of that dying tree?

Maybe this is even beyond the "scope" of a Zeiss Standard KF. Gonna pick a very basic stain kit, and go from there.

As for microbes, I'm still researching sampling and concentration methods.

Does anyone know if permanent slide prep is feasible in the field or are samples best brought back home and fixed to slides in a better controlled space?

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