Talk:Gram stain

Attention

The attention tag was added since the article looks to much of a procedure, and not an encyclopedic article. One solution would be to move the procedure to Wikibooks. Or it could be rewritten. -- Egil 09:12, 21 Apr 2005 (UTC)

There is little to say about Gram staining other than its uses and how it's done. Perhaps the procedure should be trimmed. JFW | T@lk 02:15, 5 Jun 2005 (UTC)

I added some clinical uses and Gram's original reference. I don't agree that the procedure should be omitted. JFW | T@lk 02:27, 5 Jun 2005 (UTC)

I'd like to see some more information on what happens to the bacteria at each step. More information on roles the cell wall structures have with gram-staining? Thanks.. -Anonymous, 2 August 2005 (ACST)

I think the procedure how gram staining is done is important enought to be mentioned in Wikipedia. Not in a Howto style; listing the basic steps should be enought. Besides the Picture is good, but a photo showing gram positive an d negative bacteria side by side would be much better. --Thewob 20:51, 4 November 2005 (UTC)

The procedure should definitely be kept in. It is important to understand how staining and counter-staining work, and the method should explain that Gram+ retain the original stain, while Gram- do not. The article itself is about the Gram Stain, so why would the steps involved in the Gram stain be left out?

As for the merge tag, is anyone going to discuss why they added it? G+ and G- should keep their own articles. There is enough information on each article to warrant their existence. This tag has been present since the 29th October 2005, if nobody replies to this post soon then I will remove the tags. Mushin^talk 01:46, 6 December 2005 (UTC)

On another note, "a photo showing gram positive an d negative bacteria side by side would be much better. "--Thewob. I agree, and will attempt to find a suitable photo of a mixed sample stain. I will also at some point add information on the use of fluorescence microscopy to reduce G-staining to a one-step procedure. (ref: Brock) Mushin^talk 01:52, 6 December 2005 (UTC)

I must oppose merging Gram-negative into Gram-staining. Gram-negative and Gram-Positive are more than just the results of tests to identify sickness. Based on those tests, the terms have been applied to two subkingdoms of Eubacteria. From a medical point of view, this may be insignificant, but from a biologist's point of view, it is not. It's like merging "Oaks" and "Walnuts" into "Nuts: edible or not?"

Don't forget to sign your posts with four tildes (4x~) even if you aren't a signed in user! Post above attributed to User:70.88.233.70. I agree, there is no good reason to merge the articles. Mushin^talk 20:35, 6 December 2005 (UTC)

I also oppose merging Gram Negative with Gram Staining. I arrived at Gram Negative from Proteobacteria and took that path because I was interested in the gram-negative properties of such bacteria. Gram Staining per se holds no interest to me. ~~Bacteria browser. 1st January 2007 (Happy New Year)

Gram stain v. Wright stain

The picture is wrong. This is NOT a picture of a Gram stain. This is a picture of a Wright stain - a stain meant to stain WBC's, not bacteria. If this was really a Gram stain, then the nucleus of the cells would be decolorized and would end up being pink, not purple. However, I don't know how to change the picture. Please, someone has to correct this picture.

Interesting--the CDC has it identified as a Gram stain: [1]. Are they being sloppy in their terminology, perhaps? TenOfAllTrades(talk) 18:34, 1 April 2006 (UTC)

Sloppy or not, it's just wrong. I work in a clinical lab. I work with Gram stains and Wright stains. Please find a correct picture.

Two things: 1) Please sign your posts on talk pages so we can follow the discussion. 2) While I have been able to locate a large number of public-domain G+ve/G-ve images, I have not been able to find a high-quality image of a mixed culture Gram stain. Do you have any suitable PD images to illustrate the differential staining? MarcoTolo 23:27, 1 April 2006 (UTC)

This really is a Wright's rather than Gram stain; the nuclear detail in the white cells is far too good for an underdecolorized Gram; this one slipped past the reviewers for the EID article, it would've taken a lab person to catch it. The second image is fine; but the CDC anthrax image needs to go away from this article. If more good Gram stain images are needed I can provide some for public-domain use.SCampb29 (talk) 21:22, 29 March 2012 (UTC)

'Most Useful' to 'Most Common'

Gram staining is useful, but I wouldn't consider it the most useful stain. It helps determine the nature of the cell wall, but that alone is not terribly useful when it comes to identifying an organism. I've changed it to 'most common' because it's the most basic, most taught stain for microbiology. --Sugarskane 04:36, 20 July 2006 (UTC)

The classification of gram negative and gram positive organisms is the essential first step in deciding the nature of an infection and the appropriate antibiotic coverage for an infection before a culture and sensitivity is available. It is the most common stain performed precisely because it is the most useful. - Nunh-huh 04:47, 20 July 2006 (UTC)

Discrepancies

• Under "Medical", I've never heard of gram staining for infection. Can anyone verify this? I'm almost positive this is incorrect -- you would have to culture in order to have enough bacteria to get a good stain. Additionally, I don't think hospitals require 24 hour staff just to do gram stains... I've removed this section, as I see nothing valid about it. --Sugarskane 04:41, 20 July 2006 (UTC)

You're wrong. Sputum, urine, CSF and wound drainage specimens are routinely gram stained. If lab personel are not on duty, resident housestaff provide this essential service. You're certainly right that not all hospitals staff their microbiology labs round the clock. -- Nunh-huh 04:45, 20 July 2006 (UTC)

Are you sure you aren't confusing a gram stain with a culture on a medium? What kind of a 'result' can be produced? I'm doubtful that there would be a significant amount of bacteria to get a good stain. --Sugarskane 00:36, 22 July 2006 (UTC)

Yep, he's sure. To take a random example, you can Gram stain CSF fresh from a lumbar puncture; this is routinely done to look for the presence of bacteria when bacterial meningitis is suspected. (Our article has a picture of a Gram – or maybe a Wright – stain of CSF showing anthrax bacilli.) All of the fluids listed above can be centrifuged to concentrate bacteria and other solidish things that shouldn't be there in those liquidish samples, the concentrated stuff can be stained readily. Certainly, those samples can also be cultured to detect lower levels of bacteria as well. TenOfAllTrades(talk) 01:01, 22 July 2006 (UTC)

Well, if you're sure then that's fine I guess -- it just seems like a simple gram stain wouldn't be descriptive enough to prove meningitis. Sounds good though.

The idea is to get fast feedback. Continuing with the meningitis example, suppose you suspect bacterial meningitis based on symptoms (headache, stiff neck, etc.). Performing a lumbar puncture and Gram stain of the CSF you immediately determine two important bits of information:

• Bacteria are present (or not)
• Bacteria are Gram-positive (or negative)

The morphology (appearance, shape) of the stained bacteria can give you what is sometimes a very good idea of the identity of the pathogen; just knowing whether the bacteria are Gram-positive or -negative will allow the selection of an appropriate antibiotic. Waiting for a culture in the case of bacterial meningitis might well kill your patient. TenOfAllTrades(talk) 01:41, 22 July 2006 (UTC)

Would bacteria be present in enough concentration to show up?

Often, yes. CSF doesn't usually contain any cells, so you can concentrate any that are there using a short spin in the centrifuge. Any contaminating yeast or bacteria, and the white blood cells that show up to fight infection, are going to be easily visible under the microscope once stained. This page indicates that 10⁵ colony forming units (CFUs) per mL of fluid will reliably give a positive Gram stain; this falls to about 25% chance of a positive stain when you're down to 10³ CFU/mL. In practice, about 10% of bacterial meningitis cases will not be detected on a Gram stain but will show up later on culture. TenOfAllTrades(talk) 02:35, 22 July 2006 (UTC)

Very, very cool! The centrifuge was the flaw in my entire thinking -- sorry for the removal of the section...you learn something new every day =) --Sugarskane 04:51, 22 July 2006 (UTC)

Name - gram staining vs. gram stain

Should this article be named gram staining or gram stain? All of the other articles in category:staining are of the "stain" type, but I see the usefulness in having this article named gram staining. I am undecided. However, I am strongly opposed to renaming the other articles in the category. -- Kjkolb 02:22, 7 May 2007 (UTC)

Original Purpose

I am not sure if this stain was originally "invented" to differentiate between those two bacteria mentioned in the article. I was under the impression that it was developed to isolate bacteria in animal tissue, where the bacteria is one colour and the tissue is another. I will attempt to produce documentation of this! :) Rikku (talk) 18:06, 17 May 2008 (UTC)

Section removed

This section was removed per WP:NOTHOWTO: -- œ^™ 04:48, 22 March 2010 (UTC)

How-To

• 1a. If Sample is from BROTH: Apply 1 or 2 loopfuls of broth to a clean slide. Air dry.
• 1b. If Sample is from PLATE: Place a drop of water onto a slide. Add a colony to the water and mix. SPREAD out the liquid as much as possible. Air .
• 2. Heat fix the slide. Allow the slide to cool.
• 3. Stain with Crystal Violet for 30-120 seconds by flooding the slide with stain.
• 4. Apply Iodine solution for 30-120 seconds by flooding the slide with iodine.
• 5. CAREFULLY, decolorize for 1-3 second(s) with Acetone, or as long as needed (10-60 seconds) with 70% IPA/ 30% Acetone. Immediately after, rinse with water.
• 6. Counterstain with Safranin for 30-60 seconds by flooding the slide with stain. Rinse with water.
• 7. Blot the slide with a disposable towel, gentle heat OK for the last of the moisture. Remove slide and view organisms using the oil immersion objective of your microscope.

see the results

Gram-indeterminate bacteria need expansion

Gram-indeterminate bacteria lacks of examples, a comprehensive list of non-gram staining bacteria or even its own wikipedia entry, that I think should be. I will add a link in 'See also' to 'Acid fast' that could be a substitute for now.