STAINING TECHNIQUES AND TYPES PROCEDURE.

STAINING TECHNIQUES AND TYPES PROCEDURE, EXAMPLES ,RESULT.

1. Padmashree Institute Of M anagement A nd S ciences Submitted by : SHYLESH MURTHY I A M.Sc BT, 1 st sem Dept. of Biotechnology, PIMS TOPIC: STAINING TECHNIQUES

2. STAINING TECHNIQUES

3. INTRODUCTION Staining is an auxiliary technique used in microscopy. Stains and dyes are frequently used in biology and medicine to highlight structure in biological tissues. Stains may be used to define and examine bulk tissues, cell populations or organelles with individual cells. Bacteria are microscopic organisms. They are mostly colorless and to visualize them to study their structure, shape and other structural characteristics.

4. TYPES OF STAINS ACIDIC Negatively charged acid radicals imparts color in eosin, acid fuchsine, malachite green, I ndian ink. BASIC Positively charged basic radicals combines with negatively charged particles in cytoplasm and gives color. e.g., Haematoxillin , Methylene blue, Crystal violet. NEURTAL Both positively and negatively charged imparts different colors to different components. e.g., Geimsa’s stain and Leishman’s stain.

5. STAINING TECHNIQUES POSITIVE STAINING Where the actual cells are themselves colored and appear in a clear background. Simple staining: a stain which provides color contrast but gives same color to all bacteria an cells. e.g., Malachite green and Crystal violet. Differential staining: a stain which imparts different colors to different bacteria is called differential stain (which contains more than one stain). e.g., Gram’s stain, Acid fast staining and Endospore staining.

6. NEGATIVE STAINING : Where the cells remain clear (uncolored) & the background is colored to create a contrast to aid in the better visualization of the image. Nigrosin Indian ink

7. BACTERIAL SMEAR PREPARATION SMEAR : is a distribution of bacterial cells on a slide for the purpose of viewing them under the microscope. METHOD : Aseptically a small sample of the culture is spread over a slide surface. This is then allowed to air dry. The next step is heat fixation to help the cells adhere to the surface. The smear is now ready for staining.

8. SMEARING TECHNIQUES HEAT FIXATION Pass air dried smears through a flame two or three times. Do not over heat. Allow slide to cool before staining. METHANOL FIXATION Place air dried smears in a coplin jar with methanol for one minute. Alternatively, flood smear with methanol for one minute. Drain slides and allow to dry before staining.

9. SIMPLE STAINING LOEFFLER’S METHYLENE BLUE: It is generally the most useful, it shows the characterstics morphology of polymorphs, lymphocytes and other cells more clearly than do stronger stains such as gram stain or dilute carbol fuchsin . POLYCHROME METHYLENE BLUE: This is made by allowing Loeffler’s methylene blue to ‘ripen’ slowly. The slow oxidation of the methylene blue forms a violet compound that gives the stain its polychrome properties . The ripening takes 12 months or more to complete, or it may be ripened quickly by the addition of 1.0% potassium carbonate to the stain.

10. In contrast to the blue staining of most structures by methylene blue, the violet component stains acidic cell structures red-purple. DILUTE CARBOL FUCHSIN Made by diluting Ziehl-Neelsen’s stain with 10-20 times its volume of water. Stain for 10-25 seconds and wash well with water. Over staining must be avoided, as this is an intense stain, and prolonged application colors the cell protoplasm in addition to nuclei and bacteria.

11. REQUIREMENTS Loefflers methylene blue Dilute Carbol Fuchsin Distilled water Compound microscope Cedar wood oil Fixed smear

12. PROCEDURE Make a thin smear on a slide. Heat fixes the smear by passing the slide 2-3 times gently over the Bunsen flame with the smear side up. Pour Loeffler’s Methylene blue over the smear and allow it to stand for 3 minutes. Wash the stained smear with water and air dry it. Observe the smear first under low power (10X) objective, and then under oil immersion (100X) objective. Observe the presence of organisms and also the cellular content of sample.

13. GRAM STAINING Gram staining is most widely used differential staining in microbiology. It differentiates the bacteria into two groups: Gram positive and Gram negative. Gram Positive B acteria : They have a thick cell wall of peptidoglycan and other polymers Peptidoglycan consists of interweaving filaments made up of alternating N- acetylmuramic acid and N- acetylglucosamine monomers.

14. Gram Negative Bacteria : They have an outer membrane of phospholipid and bacterial lipopolysaccharides outside of their thin peptidoglycan layer. The outer membrane protects gram negative bacteria againsts penicillin and lysozymes.

15. Procedure of Gram staining It consists of four steps: Primary staining: the smear is covered Crystal Violet, for 1 minute and washed with water. Mordanting : it is then covered with Gram’s Iodine, kept for 1 minute, and washed with water. Decolorization : the smear is covered with alcohol and is washed with water immediately. Counter staining: the smear is then covered with safarnin , kept for 30 seconds and washed with water. Observe under microscope.

16. RESULT Bacteria that manage to keep the original purple dye have only got a cell wall-they are called Gram-positive. Bacteria that lose the original purple dye and can therefore take up the second red dye have got both cell wall and cell membrane-they are called Gram-negative.

17. ACID-FAST STAINING The acid fast staining is a modification of Ehrlich’s (1882) method also known as Ziehl-Neelsen stain. It is used for bacilli belonging to the genus Mycobacterium especially Mycobacterium tuberculosis , Mycobacterium laprae and also for Nocardia . Acid fastness of the acid-fast bacilli is attributed to the presence of unsaponifiable wax fraction called mycolic acid in their cell wall. Basic requirements: Primary and mordant staining with strong carbol fuchsin (red). Decolorization with acid alcohol. Counterstain with methylene blue.

18. PROCEDURE Make a smear. Air dry. Hear fix. Flood smear with Carbol-Fuchsin stain. Steam for 5 minutes. Add more of the stain as needed. Cool slide for 5 minutes. Wash with distilled water. Flood with acid alcohol (leave for 15 seconds). Tilt the slide 45⁰ over the sink and add acid alcohol drop wise until the red color stops streaming from the smear. Rinse with distilled water. Add Loeffler’s methylene blue stain (Counter stain). Leave the stain on smear for 15-20 seconds. Rinse the slide and let it dry. Use oil immersion objective to view.

19. RESULT The stained smear are contains pink colored slender rod shaped structures are seen with curved ends. The smear is positive for acid fast bacilli.

20. CAPSULE STAINING The capsules serves as protective material by slowing down or preventing penetration of chemicals and body juices. PRINCIPLE Chemically, the capsular material is a polysaccharide, a glycoprotein or a polypeptide. Capsule staining is more difficult then other types of differential staining procedures because he capsular material are water soluble and may be dislodged and removed with vigorous washing. The capsule is non-ionic, so that the dyes commonly used will not bind to it. Two dyes, one acidic and one basic, are used to stain the background and the cell wall, respectively.

21. PROCEDURE For positive staining of smears: Make a smear from colony of S.pneumoniae on a clean grease free glass slide, and allow it to air dry. Flood the smear with Crystal Violet and allow it to stain for 5-7 minutes. Wash the smear with 20% copper sulphate solution and dry it. Observe the smear first under low power(10X) objective, and then under oil immersion (100X) objective. In the culture smear, the capsule is seen as a light blue in contrast to the deep purple color of the cell.

22. For negative staining of smears: Take a clean grease free glass slide. Put a large loopful of undiluted Indian ink on the slide. Then add a small loopful of liquid bacterial culture to the Indian ink and emulsify. Take a clean, grease free cover slip and place on the ink drop and press it down, so that the flim becomes very thin and thus pale in color. Observe the wet flim under high power (40X) objective. The capsule in negative staining method is seen as clear refractile around the organism against a black background.

23. ENDOSPORE STAINING Spores are highly resistant inactive forms. The morphology of bacterial endospores is best observed in unstained wet flims under the phase contrast microscope, where they appear as large , refractile , oval or spherical bodies within the bacterial mother cells or else free from the bacteria. Different staining are available for staining of spores.

24. PROCEDURE Flims are dried and fixed with minimal flaming. Place the slide over a beaker of boiling water, resting it on the rim with the bacterial flim uppermost. When, within several seconds, large droplets have condensed on the under side of slide, flood it with 5% aqueous solution of Malachite green and leave it for 1 minute while the water continues to boil. Wash in cold water. Treat with 0.5% safranin and 0.05% basic fuchsin for 30 seconds. Wash and dry. This method colors the spores green and vegetative bacilli red. Lipid granules are unstained.

25. SUMMARY Staining is technique used in microscopy to enhance contrast in the microscopic image. Stain is substance that adheres to a cell, giving the cell color. Stains are classified as Simple stain, Differential stain and special stain. Gram staining is used to differentiate bacterial species as Gram-Positive and Gram-Negative based on the chemical and physical properties of cell wall. Acid fast staining technique or Ziehl - Neelsen stain divides bacteria into acid-fast and non-acid-fast and this is used in diagnosis of tuberculosis and Leprosy.

26. CONCLUSION Every bacterial cell is individually colorless and hence NOT VISIBLE under the light microscope. So, to enable the person to visualize its physical features – shape, size, arrangement, etc the bacterial cells are stained with specific dyes (stains). In bacteriology (or Microbiology), we make use of various staining procedures each having specific set of stains (dyes) – Gram’s staining – C rystal violet, Iodine and Safranin Capsule staining – Nigrosin and Indian ink Spore staining- Malachite green and Safranin.

27. REFERNCE Prescott L.M, Harely T.P and Klein D.A, Microbiology, 7 th edition P:30-52, Brown Publishers. www.biotechnika.com www.studymode.com