Basic Bacteria staining
Duncan Griffiths
If you have just read the microscopy article, now with your newfound microscope skills we can now attempt some staining of bacteria for viewing under the microscope in a very basic way, but nonetheless some may find it rewarding to do. Before we get started a word about the main man.

Bacteria fall into two basic forms Gram+ Positive and Gram-Negative. This specific type of “Gram” classification is derived from a process for staining bacteria. The process for staining bacteria for identification was discovered by a Danish man called?

You guess it Dr Hans Christian Joachim Gram.

Gram studies in botany and natural science earned him a BA from his school in Copenhagen in 1871, he
became an assistant in botany and zoology, his love and interest for botany eventually lead him to discover the foundations of pharmacology and the use of microscopes of his time. After developing an interest in medicine
he obtained his MI from the university off Copenhagen in 1878 he became a physician in the municipal hospital of Copenhagen And received a gold medal for his hematology work.

In 1883, by then Gram was a physician and was working with R Koch, discovered that bacteria fell into one of two categories when stained first with crystal violet followed by a sequential bath in an iodine solution then washed with a de-staining agent.
He found that some bacteria would resist the removal of the crystal violet when flushed with acetone or ethanol and would show up blue under a microscope, while other bacteria cells would be bleached, later bacterial samples would be further counter stained with Safranin and the remaining bacteria that where previously bleached would hold onto this red stain if the sample where not washed any further with a de-staining agent 

Under the microscope bacteria that resisted de-colorization would show up deep blue or purple and became known as Gram + Positive, where as bacteria which lost the blue stain and took up the red stain became know
as Gram – Negative.
Gram’s work on bacteria staining led to the idea of the Magic Bullet and in the end led Fleming to recognize the significance of penicillin and the age of chemotherapeutics was born.
Ironically although the staining technique carries his name, Gram never did develop the Safranine counter stain, that was the final element of his work and was completed by a German pathologist called Carl Weigert.

Contrary to what you may think the staining of bacteria for disease is possible by the amateur in a limited way,
you will show Gram positive and Gram negative bacteria and this will indicate what an infections composition is. This information will save you valuable time by applying, a more appropriate medication, I.E. you can choose an antibiotic that responds better to either gram + or gram – bacteria. However, to diagnose the exact bacterial strain; you would need to culture the bacteria in a laboratory, this technique is not recommended for the amateur.

Bacteria is basically one of two shapes cocci (round) or bacilli (rod shaped) however, of the bacteria that infect our fish you WILL need to stain, so as to distinguish between the two shapes.

Fish disease.
Gram negative Bacilli (staining red) such as pseudomonas or Aeromonas can cause sever disease in the form of ulcers and lesions and mouth rot and fin rot and necrosis of the anus and the gill. Material from such areas can be removed with a swab and be smeared on a clean slide and allowed to dry.
Post mortem samples taken from the kidney or liver etc can be smeared directly on the slide after blotting away excess blood.


Staining Technique.

Equipment and supplies You will need for your staining is, methyl violet, iodine solution and Safranin you will also need some isopropyl alcohol, all these items are very easy to get hold of, also they are very cheap from most good microscope stores. You will also need some gloves and a wire rack to wash and dry the stained slide over an
old disposable bowl

Take a smear from the site of an infection, this is done by applying a sterile swab or a wire loop, apply this sample to a clean slide smearing the slide very thinly and allow to dry.
Once the slide sample is dry heat fix the material by passing the slide {sample side upper most} over a Bunsen burner or similar small flame very quickly two or three times. The aim is that the slide should become warm in the hand but not to hot, if smoke appears to rise off the sample, start over, the sample is ruined.

1, Place the slide, again material side up on the wire rack and apply the methyl violet with a pipette or simple dropper for 30 seconds.

2, Tip off the methyl violet stain and apply iodine solution for a further 60 seconds.

3, Tip off the iodine solution and rinse with the isopropyl alcohol allowing the alcohol to wash the blue stain away, continue until no more stain appears to flow from the preparation, {acetone would do}

4, wash well now with plain water and allow to dry.

5, Apply the safranin stain to the slide for 3 minutes.

6, tip stain off, wash with plain water blot, and allow to dry thoroughly.
The preparation is now ready for viewing, at 400X at least.

The slide image will be a series of blue specks and red specks. The blue are obviously Gram positive and the red Gram negative. Although this technique is the main stay of all bacterial staining still in use today, a very small percentage of bacteria will stain incorrectly this is *normal* as no staining technique is 100%

In simple terms, if for instance we were looking at bacterial types and counts in bird droppings, evidence of Gram Positive bacteria (stained blue) would be considered normal {beneficial bacteria}, and evidence of Gram Negative bacteria (stained red) would be abnormal and would indicate an infection.

Certainly from a fish pathology point of view we are on the lookout for Gram negative most of the timein the form of pseudomonas and aeromonas. By comparing the concentration of red cells to blue we can get some idea of how far advanced the disease is and possibly how to start treating it, or if we need to call on expert advise.





Some examples of bacteria categories

Gram positive bacteria






Gram negative bacteria