Acid-Fast Staining

Acid-fast staining, a differential staining procedure, is also known as Ziehl-Neelsen method as it was first developed by Franz Ziehl in 1882 and later modified by Friedrich Neelsen in 1883. Some bacteria are surrounded by a covering of fatty and waxy substances and are difficult to stain. But once they are stained, they can’t be easily decolorized even after the treatment with alcohol containing acid. Such organisms, because of their resistance to decolorization with acid alcohol are known as acid-fast. A few species particularly those in the genus Mycobacterium are acid-fast like Mycobacterium tuberculosis (causing tuberculosis) and Mycobacterium leprae (causative agent of leprosy).

In this staining procedure, cells are first heated with a primary stain i.e. Carbol fuchsin. This will stain every cell as red. Then they are destained with an acid-alcohol. Acid-fast cells have high lipid content in particular mycolic acid-a group of branched chain hydroxyl lipids so they can’t be easily decolorized even by treatment with alcohol and hence stains red. Non-acid fast bacteria lack the thick, waxy lipid layer like that of acid-fast bacteria and hence they lose the primary stain and get destained by acid wash. Finally, counter stain i.e. methylene blue is applied. Therefore acid-fast bacteria appears red in color and non acid-fast bacteria appears blue in color when viewed under the microscope.

Mordant

In microbiology, mordant is defined as a substance having a strong chemical affinity for the dye and the substrate i.e. microorganism. It basically helps the dye to fix on or in a cell. They are frequently used in staining process as they increases the interaction between the dye and cell, as a result of which, cells can be stained more strongly and can be easily visualized under microscope. Some commonly used mordants are – potassium alum, iodine, tannic acid etc.

In Gram staining procedure, iodine, used as a mordant, is an oxidizing agent. It causes the substance to oxidize more acid character thereby increases their affinity towards basic dye (like crystal violet). Iodine (I) along with crystal violet (CV) forms an insoluble complex CV-I which results in a dark purple color.

Negative Staining

In negative staining, actual specimen is left untouched and background is stained. This is the opposite of normal staining procedures in which actual specimens are stained. This technique is used to study cell morphological shape, size and arrangement, and also specific bacterial structures like the presence of diffuse capsules around many bacteria. In this staining procedure, bacteria are mixed with acidic dye like Indian ink or Nigrosin dye and then spread out on a slide as a thin film. It is then air dried and observed under microscope.

After giving up the hydrogen ion, the chromophore of the acidic dye becomes negatively charged. Since the bacterial surface is also negatively charged, it repels the dye and stains the background. As a result, the bacteria will show up as a clear spot against the dark background. This technique is also useful to stain those cells that are too sensitive to be heat fixed.

Gram Staining & Its Principle

Gram staining, a differential staining procedure, that divides bacteria into two groups i.e. Gram positive and Gram negative, is developed in the year 1884 by the Danish physician Hans Christian Gram. It is the one of the most widely employed staining technique in bacteriology.

In Gram staining procedure, the smear is first stained with a primary stain i.e. Crystal Violet (a basic dye). This is followed by the treatment with a mordant i.e. Iodine solution. Iodine helps the cell to stain more strongly by increasing the interaction between the two i.e. the dye and the cell. After this, the smear is decolorized by washing with acetone or ethanol and then it is counterstained with Safranin (a basic dye).

In this staining procedure, some organisms retain the color of primary stain (blue/purple) whereas others get decolorized and take up the color of counterstain (red/pink). Those organisms that retain the primary stain are known as Gram positive organisms and those that take the Safranin stain are known as Gram negative organisms. Thus, Gram reaction helps in classifying organisms into either of the two groups.

Some example of Gram positive bacteria are Staphylococcus aureus, Streptococcus pneumonia etc. Escherichia coli, Neisseria meningitides etc. are some examples of Gram negative bacteria.

Principle Of Gram Staining:

The basic principle of Gram staining is due to the difference in the cell walls of Gram positive and Gram negative bacteria. Gram positive bacteria have 20-30 nm thick cell wall that contains 70-80% of peptidoglycan and also it has very low lipid content. On the contrary, Gram negative bacteria have only 8-12 nm thick cell wall which contains only 10-20% of peptidoglycan. Lipid content is higher in Gram negative ones as compare to Gram positive. Also Gram negative bacteria have outer membrane which is absent in Gram positive ones.

        

In the Gram staining procedure when decolorizer (mixture of acetone and ethanol) is added, Gram positive cell walls dehydrate causing the closure of pores in the cell wall which results in the retention of crystal violet stain. On the other hand, the lipid content and thin peptidoglycan layer of Gram negative bacteria gets dissolved by the decolorizer as a result of which the dye gets washed off leaving the unstained bacteria which then takes up the counterstain.

Simple & Differential Staining

SIMPLE STAINING:

Staining is the process by which microorganisms are colored by stain or dye.  Simple staining is the one that contains only a single staining agent i.e. a single dye dissolved in a solvent. It is important because of its simplicity and ease of use. This procedure involves applying stain or dye to microorganisms in single application only. In this staining process, smear is covered with stain for a definite period of time, then the excess stain is washed off and blots the slide dry. It helps to study the shape, size and arrangement of bacteria. Mostly basic dyes are used in simple staining. Some common examples are Methylene blue, Crystal violet etc. 

DIFFERENTIAL STAINING: 

Differential staining is one that contains more than one dye i.e. two or more stains. Depending on the staining technique, the dyes can be applied separately or they can be mixed and applied in one solution. This staining technique differentiates bacterial cells or parts of cells visible. Gram staining and acid fast staining are two differential staining that separates microorganisms into groups. Gram staining divides bacteria into Gram positive and Gram negative whereas acid fast staining divides bacteria into acid fast and non acid fast. This technique is also used to visualize structures like spore, flagella, nucleus etc.

Fixation

Fixation is defined as the process of preserving and fixing the internal and external stucture of the cells into position. As a rule of fixation, a microorganism is killed & firmly attached to microscopic slide. The process of fixation basically inactivates the enzymes that might disrupt the cell morpholgy and thereby toughens the cell structures so that they do not change during staining & observation procedures. This process helps stained cells to resemble living cells as closely as possible.

There are basically two types of fixation – Heat fixation and Chemical fixation.

Heat fixation helps in preserving the overall morphology of the cell but not the internal structures. It is done by passing the slide, containing bacterial smear, 3 to 4 times quickly through upper portion of the flame.

Chemical fixative generally penetrates the cell and reacts with the cellular components like lipids and proteins thereby making them inoluble, inactive and immobile. Chemical fixation is generally used to protect fine cellular structures & the morphology of larger and more delicate microbes. Some common examples of chemical fixatives are the mixture of acetic acid, formaldehyde, ethanol, mercuric chloride and glutaraldehyde.