A negative stain may be used  similar to a basic simple stain or as a simple capsule stain. We will only use it as a simple capsule stain.  A capsule is an extracellular structure that some species make that is destroyed by normal staining procedures (especially heat fixing and washing).  Capsule staining is a gentle staining procedure that does not destroy the bacterial capsule.  This procedure stains the background but does not stain the bacterial capsule or the bacterial cell.  (Note, some other capsule stains use a second stain as a counter-stain which does stain the cell or even the capsule with another color.)

Purpose: to make the cells show up better (by adding higher contrast to the background) and to allow for viewing the capsule.




Procedure for a Simple Capsule Stains (or a Wet Mount using a Negative Stain):
➤ Do not start with a bacterial smear!  Set up your bacteria, two to four slides, two cover slips, a Bunsen burner, and your loop.  It is also a good idea to make a third slide containing a positive control (a bacterial strain that has a capsule) if one is available so that you may view a positive result.  (One may find bacteria with capsules by gently inserting a clean toothpick under your gumline next to a tooth.)
➤ Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on your slides.  Congo Red is easier to see, but it does not work well with some strains.  India Ink generally works but it has tiny particles that display Brownian motion that must be differentiated from your bacteria.  Nigrosin may need to be kept very thin or diluted. Sterilize your loop and add an almost invisible amount of bacteria to one slide, smearing it in the dye.  You may spread your drop with the loop, or with a second slide that is brought into the stain drop and is then spread across the first slide.  It is recommended that you make more than one slide of a specimen, slightly increasing the amount of bacteria added to the second.
➤ Reflame your loop. 
➤ Add cover slips at a 45 degree angle to each slide.  You are essentially making a wet mount using a negative stain instead of water.
➤ First observe the slide with the greater number of bacteria, it is probably too many to work with, but look at it first to more easily observe your bacteria, look for an area of clearing caused by a  capsule around the cells.  Otherwise the dye should come right up to the cell.  When you think you know what you are seeing, switch to the slide with the fewer bacteria that hopefully will contain only a single layer of cells to be certain of your conclusions.
➤ If your bacteria are very motile, you may need to wait for them to die under the light before making your observations about a capsule.  You can turn up the light to speed this.
➤ Remember that a slide stained with a capsule stain contains live bacteria that should be handled with care and disposed of properly.



Procedure for a Dried Negative Stain:
We will not perform this procedure, it is detailed here just for your knowledge benefit.  Essentially, it is the similar to a Simple Basic Stain, except it uses a Negative Stain, and there are no washing and blotting steps.  Recall that negative stains are simple stains that do not stain the cell, they stain the background.

• Obtain a clean slide that has no fingerprints or dust on it (your instructor will inform you how to clean it if necessary.)
• Obtain a simple Negative Stain (India Ink, Congo Red, Nigrosin, or Eosin).  
• Place a small drop of a Negative Stain on one end of your slide.  
• Add a small amount of your bacterial culture to the drop and mix evenly without spreading.
• Now, bring a second slide held at a 45 degree angle into the stain drop and with this slide, spread the drop across the first slide.
• Dispose of the second spreader slide in disinfectant.
• Allow the slide to air dry.  Do not heat fix or wash.  View the slide under the microscope, a coverslip may be added if desired when using oil.
• Note, this procedure should not be used to look for capsules as the stain may shrink away from the cell during drying.

Microbiology is the study of small living things. Generally this means living things that are too small to see without the use of a microscope. These life forms are called microorganisms or microbes. Microorganisms include bacteria, archaea (a type of prokaryote a bit like bacteria but they have a distinct evolutionary origin), viruses, protozoa (single-cell eukaryotes like amoeba), microscopic fungi and yeasts, and microscopic algae (plant-like organisms). Microorganisms were discovered over three hundred years ago and it is thought that many new microbes have yet to be discovered. Microbiology is a wide area of science that includes bacteriology, virology, mycology, phycology, parasitology, and other branches of biology.

Most living things can be classified into prokaryotes or eukaryotes depending on whether their nuclear material (for example DNA) is surrounded by a membrane or not. Archaea and bacteria are prokaryotes. Most animals (including humans) and plants are eukaryotes. Protozoa, fungi, yeasts, and algae are eukaryotes. Viruses are a little different. Traditional classification systems do not classify viruses as living organisms. However in practise they are considered microorganisms. The study of viruses is called virology.

Microbiology therefore includes the study of both prokaryotic and eukaryotic microorganisms. In practise the majority of microbiology is concerned with bacteria and/or viruses although eukaryotic microbiology is also a very important branch of microbiology. Many diseases of animals (including humans) and plants are caused by bacteria, viruses, amoeba, and fungi. Bacteria are important in probiotics, they are used in food production (e.g. yoghurt and cheese) and biotechnology. Yeasts and fungi are important in food and drink production (e.g. wine, beer, bread) and are also used to produce important pharmaceuticals (e.g. antibiotics).

Microbiology science resource at Microbiology. Probiotics science resource at Probiotics and current topics on microbiology research at the Microbiology Blog.

genetically related to one another. This group of organisms includes several that cause
primary infections of the human gastrointestinal tract. Members of this family are major
causes of opportunistic infection (including septicemia, pneumonia, meningitis and urinary
tract infections). Examples of genera that cause opportunistic infections are: Citrobacter,
Enterobacter, Escherichia, Shigella, Salmonella, Yersinia, Klebsiella, Proteus, Hafnia, Morganella, Providencia and Serratia.

The term peptidoglycan was derived from the peptides and the sugars (glycan) that make a molecule; it is also called ‘Murein’. It is found only in bacterial cell walls, thus, its synthesis can be targeted by antibiotics. Peptidoglycan, the polymer of sugars and amino acids,  is a complex, interwoven network that surrounds the entire bacterial cell.




Peptidoglycan consists of carbohydrate backbone (glycan chain) composed of alternating units of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) molecules.  Glycan chains are connected by short peptides.Attached to each of the muramic acid molecules are a tetrapeptide consisting of both D- and L- amino acids, the precise composition of which differs between bacteria. Teichoic acid and lipoteichoic acid which are polymers of a sugar alcohol (ribitol or glycerol) are embedded in it.


Special Amino Acids Found in Peptidoglycan Layer:

➧ Diaminopimelic acid: Unique to bacterial cells.
➧ D- alanine: Involved in the cross links between tetrapeptides and in the action of penicillin.


Functions of Peptidoglycan Layer

• It provides rigid support to bacterial cells and maintains the characteristic shape of the cell.
• Allows bacterial cell to withstand media of low osmotic pressure, such as water.

Medical Importance of Peptidoglycan Layer