Staining Techniques

for under graduate students

1. STAINING TECHNIQUES HMB- 5111 CHRS, JAGDALPUR

2. STAINING To study Microbiological structures and to divide them into different groups, staining is used, in light microscopy. Numerous staining techniques are available for visualization, differentiation, and separation of bacteria in terms of morphological characteristics and cellular structures.

3. STAIN/ DYE A stain (dye) may be defined as an organic compound containing a benzene ring plus a chromophore and an auxochrome group. Used to stain/colour the micro organism for morphological and cell study.

4. Electrical charge on stain The ability of stain to bind with macromolecules such as protein and nucleic acid depends upon; Electrical charge on Chromogen Cellular components to be stained

5. Acidic stain Acidic Stains are anionic, which means that, on ionization of the stain, the Chromogen portion exhibits a negative charge and therefore has a strong affinity for the positive constituents of the cell such as Proteins. Picric acid is an example of acidic stain .

6. Basic stain Basic stains are cationic, because on ionization the chromogen portion exhibits a positive charge and therefore has a strong affinity for the negative constituents of the such as Nucleic acids. Methylene blue is a basic stain. Basic stains are commonly used for bacterial staining.

7. Fixed, stained smears Fixed stained preparations are most frequently used for the observation or the morphological characteristics of bacteria. The advantages of this procedure are that 1) The cells are made more clearly visible after they are colored, and 2) Differences between the cells of different species and within the same species can be demonstrated by use of appropriate staining solutions. The essential steps in the preparation of a fixed, stained smear 1) preparation of the film or smear 2) fixation and 3) application of one or more staining solutions.

8. Types of staining techniques There are two types of staining techniques; Simple Staining ▪ Use of Single stain Differential staining ▪ Use of two contrasting stains

9. Simple staining For visualization of morphological shape ( Cocci , bacilli, and spirilli ) Arrangement (chains, clusters, pairs, and tetrads) The coloration of bacteria by applying a single solution of stain to a fixed smear is termed simple staining. The fixed smear is flooded with a dye solution for a specified period of time, after which this solution is washed off with water and the slide is blotted dried. The cells usually stain uniformly. However, with some organisms, particularly when methylene blue is used, some granules in the interior of the cell may appear more deeply stained than the rest of the cell, indicating a different type of chemical substance.

10. Differential staining Staining procedures that make visible the differences between bacterial cells or parts of a bacterial cell are termed differential staining techniques . They are slightly more elaborate than the simple staining technique in that the cells may be exposed to more than one dye solution or staining reagent.

11. Gram staining The Gram staining technique is the most important and widely used microbiological differential staining technique. It was developed by Dr. Christian Gram in 1884, and categorizes bacteria according to their Gram character (Gram positive or Gram negative). In addition this stain also allows determination of cell morphology, size, and arrangement. It is typically the first differential test run on a specimen brought into the laboratory for identification. In some cases, a rapid, presumptive identification of the organism or elimination of a particular organism is possible.

12. PRINCIPLE OF GRAM STAINING The structure of the organism’s cell wall determines whether the organism is gram positive or negative. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary stain by resisting decolonization while others get decolorized by a decolorizer. Those bacteria which retain the primary stain are called Gram positive and those bacteria which gets decolorized and then get counterstained are called Gram negative .

13. REQUIREMENTS 1.CRYSTAL VIOLET • Primary stain. •Violet colored, stains all micro-organism. 2.GRAM IODINE • Mordant. • Forms Crystal violet iodine complexes. 3.DECOLORIZER • Acetone + Methanol •Removes Crystal violet iodine complex from thin peptidoglycan layers •Dissolves outer layer of Gram negative organism.

14. 4.GRAM SAFRANINE • Counter stain •Red colored •Stains thin walled Gram negative organism

15. PROCEDURE OF GRAM STAINING Step 1-Prepare a Smear Suspend some of the material to be stained in a drop of water on a microscope slide, spread the drop to about the size of a nickel. Fix by Heat or Allow to air dry. Step 2-Apply the Primary Stain Flood the Smear with Crystal Violet. Allow to stand for 1 min. Rinse with water to remove excess stain.

16. Step 3-Apply the mordant Flood the smear with iodine solution. Allow to stand 2 minute. Rinse with water to remove excess iodine. Step 4 -Decolorize Drip Decolorizer (80% Methanol +20% Acetone) across the slide about 5 sec. The slide should appear clear. Rinse with water to remove decolorizer.

17. Step 5-Counter stain Flood the slide with Safranin solution. Let stand for 2 minutes. Step 6 -Rinse, Dry and Observe Rinse with water to remove excess stain. Blot dry. Observe under Oil Immersion.

18. Examples of Gram Positive Organisms Bacillus, Nocardia , Clostridium, Propionibacterium , Actinomyces , Enterococcus , Cornyebacterium , Listria , Lactobacillus, Gardnerella , Mycoplasma , Staphylococcus, Streptomyces , Streptococcus etc

19. Examples of Gram Negative Organisms Escherichia, Helicobcater , Hemophilus , Neisseria , Klebsiella , Enterobacter , Chlamydia, Vibrio , Pseudomonas, Salmonella, Shigella

20. There are numerous other staining techniques designed to identify some particular feature of cell structure or composition. These techniques are summarized below. Sl. No Name of the staining technique Application 1 Acid fast stain Distinguishes acid fast bacteria such as Mycobacterium Spp. from non-acid fast bacteria. 2 Endospore stain Demonstrates spore structure in bacteria as well as free spores. 3 Capsule stain Demonstrates presence of capsules surrounding cells 4 Flagella stain Demonstrates presence and arrangement of flagella. 5 Cytoplasmic inclusion stains. Identifies intracellular deposits of starch, glycogen, poly phosphates, hydroxyl butyrate and other substances. 6 Giemsa stain Particularly applicable for staining ricketsia and someprotozoa .