A bacterium is a single-celled (unicellular) microorganism that does not have a nucleus or any other membrane-bound organelles. Bacteria are sometimes called 'prokaryotes.' In Greek, 'prokaryote' literally means 'before the nut' (where 'the nut' is the nucleus.)

Bacteria adapt to become well-suited to their environments, and therefore come in many shapes and forms. However, they all have a few parts in common.

BACTERIA

Classification
Because bacteria are so diverse in both form and habitat, biologists have struggled with their classification, (also called their 'taxonomy.') For many years, bacteria were called 'monerans,' and placed in a kingdom of the same name. Although you still might hear bacteria referred to as monerans, it's not a term currently accepted among biologists.
Life is now classified into three domains; a domain is a taxonomic level that is higher than a kingdom, and based on an organism's DNA. Bacteria is one of these domains, while the others are eukarya, composed of organisms that have nucleated cells, and archaea, composed of unicellular prokaryotes, many of which have evolved to exploit extreme environments. Organisms in the archaea domain were originally considered a type of bacteria - known as archaebacteria - but scientists have since reclassified them. In this image, groups of archaea are indicated by green lines, and bacteria are in blue. Red lines indicate eukaryotes.



How we group bacteria
Scientists try to make sense of the sheer number and diversity of bacteria by grouping them in various ways. Here are just a few ways that are accepted in the scientific and medical communities:
Shape: Bacteria are usually one of three shapes. Cocci are round; bacilli are rod shaped; spirilla (also called spirochetes) are spiral.

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.

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

Amaç, B grubu Streptococcusların oluşturduğu CAMP (Christie, Atkins, Munch-Peterson) faktörünü tespit etmektir. Bu testi bulan bilim adamlarının ilk harfleri alınarak CAMP adı oluşturulmuştur.



Testin yapılış Tekniği;

➤ Bazı Staphylococcusların (Staphylococcus aureus) var olduğu düşünülen örnekten,
özeyle alınarak %5‟lik koyun kanlı agarın ortasına boydan boya kalın bir çizgi
şeklinde ekilir.
➤ Bu ekim çizgilerine dik doğrultuda, Staphylococcus ekim çizgisine dokundurmamaya
dikkat ederek incelenecek mikroorganizmadan 2-3cm uzunlukta ekim yapılır.
➤ Plaklar etüvde, 37°C'de 18-24 saat inkübe edilir.
➤ İnkübasyon sonunda, Streptococcus ve Staphylococcusların üreme yapıları incelenir.
Birbirlerine yakın oldukları yerde ok başı şeklinde hemoliz meydana gelirse
CAMP testi pozitif olarak değerlendirilir.

Principle of CAMP Test

CAMP test is done to detect the production by group B but not other Streptococci, of a diffusible extracellular protein (CAMP factor) that acts synergistically with the β-lysin produced by Staphylococcus aureus to cause enhanced lysis of red blood cells on blood agar.

1. Touch a well isolated colony with a sterile straight wire.
2. Inoculate TSI by first stabbing through the centre of the medium to the bottom of the tube and then streak the surface of the slant.
3. Leave the cap loose and incubate the tube at 35 in ambient air for 18 to 24 hours.
4. Observe the reaction